Patent Grant
10655140
The present application is a National Stage of International Patent Application No. PCT/CN2016/073182, filed Feb. 2, 2016, and claims the priority of Chinese Patent Application No. 201510078810.3, filed on Feb. 13, 2015, the disclosures of which are incorporated herein by reference in their entirety.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named 109127_000004_Sequence_Listing.txt and is 16 kilobytes in size.
The present invention relates to a herbicide-resistant protein, encoding gene and use thereof, especially a 2,4-D-resistant protein, encoding gene and use thereof.
Weeds can quickly run out of the valuable nutrients in the soil which are necessary for the growth of crops and other target plants. At present, there are many types of herbicides for weeds control, among which is a particularly popular herbicide, glyphosate. Glyphosate-resistant crops have been developed, such as corn, soybean, cotton, beet, wheat, and rice, and the like. Thus, it is possible to spray glyphosate in the fields planted with glyphosate-resistant crops to control weeds without significant damage to the crops.
Glyphosate has been widely used all over the world for more than 20 years, resulting in the overdependence on the technology of glyphosate and glyphosate-tolerant crops. In addition, high selection pressure has been forced to the naturally more glyphosate-tolerant plants among the wild weed species or the plants which have developed resistance to glyphosate activity. It has reported that a few weeds have developed resistance to glyphosate, including broad-leaved weeds and gram ineous weeds, such as Swiss ryegrass, Lolium multiflorum, Eleusine indica, Ambrosia artemisiifolia, Conyza canadensis, Conyza bonariensis and Plantago lanceolata. In addition, the weeds which are not the agricultural problem before the widespread use of glyphosate-tolerant crops also gradually prevailed, and are difficult to be controlled with glyphosate-tolerant crops. These weeds mainly exist along with (but not only with) broad-leaved weeds which are difficult to be controlled, such as species from Amaranthus, Chenopodium, Taraxacum and Commelinaceae.
In the area of glyphosate-resistant weeds or the weed species which are difficult to be controlled, growers can make up the weakness of the glyphosate through tank-mixing or using other herbicide which can control the omissive weeds. In most cases, a popular and effective tank-mixing partner used to control broad-leaved weeds is 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D has been used to control broad-spectrum broad-leaved weeds more than 65 years under agriculture and non-crop conditions, and is still one of the most widely used herbicides in the world. The limit for further use of 2, 4-D is that its selectivity in dicotyledonous plants (such as soybeans or cotton) is very low. Therefore, 2,4-D is generally not used on (and generally not close to) sensitive dicotyledonous plants. In addition, the use of 2,4-D on gramineous crops is limited to a certain extent by the properties of the potential crop damage. The combination of 2,4-D and glyphosate has already been used to provide a stronger sterilization process before planting the no-till soybeans and cotton. However, due to the sensitivity of these dicotyledonous species to 2,4-D, these sterilization processes must be carried out 14 to 30 days before planting.
Same as MCPA, 2-methyl-4-chloropropionic acid and 2,4-D propionic acid, 2,4-De is also a phenoxy alkanoic acid herbicide. 2,4-D is used to selectively control broad-leaved weeds in many monocotyledonous crops such as corn, wheat and rice, without serious damage to the target crops. 2,4-D is a synthetic auxin derivative of which the function is to disorder the normal cytohormone homeostasis and to hinder the balance of controlled growth.
2,4-D shows different levels of selectivity on certain plants (for example, dicotyledonous plants are more sensitive than gramineous plants). Different 2,4-D metabolisms in different plants are one explanation for the different levels of selectivity. Plants usually metabolize 2, 4-D slowly. Thus, different activities of targeted points are more likely to explain different responses to 2, 4-D of plants. Plant metabolism of 2, 4-D is usually achieved through two steps of metabolism, i.e. the conjugation with amino acids or glucose following the hydroxylation in general.
As time goes on, the microbial populations have gradually developed effective, alternative pathways to degrade this particular foreign substance, which result in the complete mineralization of 2,4-D. Continuous application of herbicides on microbes can be used to select the microorganisms which use herbicides as carbon sources so as to make a competitive advantage in the soil. For this reason, 2,4-D was currently formulated with a relatively short soil half-life period and without obvious legacy effect on the subsequent crops, which promotes the application of 2, 4-D herbicide.
Ralstonia eutropha is one organism of which the ability for degrading 2,4-D has been widely studied. The gene encoding the enzyme in the first enzymatic step of mineralization pathway is tfdA. TfdA catalyzes the conversion of 2,4-D acid into dichlorophenol (DCP) through α-oxoglutarate-dependent dioxygenase reaction. DCP hardly has herbicide activity compared with 2,4-D. TfdA is used to introduce 2,4-D resistance into dicotyledonous plants which are usually sensitive to 2, 4-D (such as cotton and tobacco) in transgenic plants.
A number of tfdA type genes have been identified which encode proteins capable of degrading 2,4-D in the environment. Many homologs are similar with tfdA (amino acid identity >85%) and have similar enzyme activity with tfdA. However, not all proteins having structures such as TauD which have the function of degrading 2,4-D, and a large number of homologs have significantly lower identity (25-50%) with tfdA while contain characteristic residues associated with α-oxoglutarate-dependent dioxygenase Fe2+ dioxygenases. Therefore, the substrate specificities of these different dioxygenases are indefinite. A unique instance which has low homology (28% amino acid identity) with tfdA is rdpA from Sphingobium herbicidovorans. It has been shown that this enzyme catalyzes the first step in the mineralization of (R)-2,4-D propionic acid (and other (R)-phenoxy propionic acids) and 2,4-D (phenoxyacetic acid).
With the emergence of glyphosate-resistant weeds and the expanded application of 2,4-D herbicide, it is necessary to introduce 2,4-D resistance into the target plants sensitive to 2,4-D. At present, no reports have been found about the expression levels of herbicide-resistant protein 24DT22 in plants and their herbicide tolerance.
The purpose of the present invention is to provide a herbicide-resistant protein, coding gene and use thereof. The present invention is intentioned to provide a new 24DT22 gene which has higher herbicide tolerance in plants.
In order to accomplish said purpose, the present invention provides a herbicide-resistant protein, comprising:
In order to accomplish said purpose, the present invention provides a herbicide-resistant gene, comprising:
The stringent conditions might be as follows: hybridization in 6×SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65° C. and followed by washing membrane one time using 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS, respectively.
In order to accomplish said purpose, the present invention also provides an expression cassette, comprising said herbicide-resistant gene under the regulation of operably linked regulatory sequence.
In order to accomplish said purpose, the present invention further provides a recombinant vector, comprising said herbicide-resistant gene or said expression cassette.
In order to accomplish said purpose, the present invention provides a method for producing a herbicide-resistant protein, comprising:
Furthermore, the transgenic host organism includes plants, animals, bacteria, yeast, baculovirus, nematode or algae.
Preferably, the plant is soybean, cotton, corn, rice, wheat, beet or sugar cane.
In order to accomplish said purpose, the present invention also provides a method for extending the target range of herbicides, comprising: co-expressing the nucleotide encoding the herbicide-resistant protein or the herbicide-resistant protein encoded by the expression cassette with at least one second nucleotide that is different from said protein or the protein encoding by said expression cassette.
Furthermore, the second nucleotide encodes glyphosate-resistant protein, glufosinate-ammonium-resistant protein, 4-hydroxyphenylpyruvic acid dioxygenase, acetolactate synthase, cytochrome protein or protoporphyrinogen oxidase.
In present invention, the herbicide-resistant protein 24DT22 is expressed in a transgenic plant accompanied by the expressions of one or more glufosinate-resistant protein and/or glufosinate-ammonium-resistant proteins. Such a co-expression of more than one kind of herbicide-resistance protein in a same transgenic plant can be achieved by transforming and expressing the genes of interest in plants through genetic engineering. In addition, herbicide-resistant protein 24DT22 can be expressed in one plant (Parent 1) through genetic engineering operations and glyphosate-resistant protein and/or glufosinate-ammonium-resistant protein can be expressed in a second plant (Parent 2) through genetic engineering operations. The progeny plants expressing all genes of Parent1 and Parent 2 can be obtained by crossing Parent1 and Parent 2.
In order to accomplish said purpose, the present invention also provides a method for selecting transformed plant cells, comprising the steps of transforming multiple plant cells with the herbicide-resistant gene or the expression cassette and cultivating said cells at a herbicide concentration which allows the growth of the transformed cells expressing the herbicide-resistant gene or the expression cassette while kills the un-transformed cells or inhibits the growth of the un-transformed cells, wherein the herbicide is a phenoxy auxin.
In order to accomplish said purpose, the present invention also provides a method for controlling weeds, comprising the step of applying an effective amount of one or more herbicides to the field planted with crops which comprises said herbicide-resistant gene, said expression cassette or said recombinant vector.
Preferably, the herbicide is a phenoxy auxin.
In order to accomplish said purpose, the present invention also provides a method for protecting plants from the damage caused by herbicides, comprising the step of introducing said herbicide-resistant gene, said expression cassette or said recombinant vector into plants, so as to make the resulted plants produce a certain quantity of herbicide-resistant protein sufficient to protect them from the damage caused by herbicides.
Preferably, the said herbicide is a phenoxy auxin or aryloxy phenoxy propionate and said plants are selected from the group consisting of soybean, cotton, corn, rice, wheat, beet and sugarcane.
In order to accomplish said purpose, the present invention also provides a method for controlling glyphosate-resistant weeds in a field planted with glyphosate-tolerant plants, comprising the step of applying an effective amount of herbicides to the field planted with glyphosate-tolerant plants, wherein said glyphosate-tolerant plants comprise said herbicide-resistant gene, said expression cassette or said recombinant vector.
Preferably, said herbicide is a phenoxy auxin and said glyphosate-tolerant plant is monocotyledon or dicotyledon.
In order to accomplish said purpose, the present invention also provides a method for conferring crops with resistance to 2,4-D herbicides, comprising the steps of introducing said herbicide-resistant gene, said expression cassette or said recombinant vector into plants.
Preferably, said plant is soybean, cotton, corn, rice, wheat, beet or sugarcane.
In order to accomplish said purpose, the present invention also provides the use of herbicide-resistant proteins tolerant to phenoxy auxin herbicides, comprising:
The herbicide-resistant gene, said expression cassette or said recombinant vector is introduced into plants. The conventional methods used in present invention to introduce foreign DNA into plant cells include but are not limited to Agrobacterium-mediated transformation, Particle Bombardment, direct intake of DNA into protoplast, electroporation or silicon-mediated DNA introduction.
The 2,4-D resistant genes and subsequent resistant crops according to present invention provide a good choice to control glyphosate-resistant (or high tolerance or succession) broad-leaved weed species in crops. 2,4-D is a broad-spectrum, relatively cheap and powerful broad-leaved herbicides. If stronger crop tolerance in both dicotyledons and monocots could be provided, good efficacies could be provided for growers. 2,4-D-tolerant transgenic dicotylenons also have a higher flexibility in application time and administration amount. Another use of the 2,4-D herbicide-tolerance trait is that it could be used to prevent damages to normal sensitive crops such as 2,4-D drift, volatilization, transformation (or other remote movement phenomenon), misuse, destruction and the like. Various mixtures of different phenoxy auxins have been widely used to treat specific weed spectrum and environmental conditions in different areas. Using 24DT22 gene in plants can provide protections against broader-spectrum phenoxy auxin herbicide so as to improve the flexibility and controllable weed spectrum and provide protections to the full range of commercially available phenoxy auxin drift or other long distance phenoxy herbicides damages.
Phenoxy auxin herbicides are usually formulated as active acids, but some commercialized preparations are formulated as one of several corresponding ester preparations. Since general plant esterases in plants can convert these esters into active acids, they are also considered to be the substrates of 24DT22 enzyme in plants. Similarly, they can also be the organic or inorganic salts of the corresponding acids. When expressing chiral propionic acid, propionic acid salt or propionic ester herbicides, even if different CAS numbers may correspond to an optically pure compound. When denominating the herbicides, we still consider that racemic (R, S) or optically pure (R or S) enantiomer is a same herbicide. The possible dosage ranges can be those treated alone or combined with other herbicides in the applications in crops or non-crops.
It has been identified that the 24DT22 gene possesses the characteristics to allow the application of phenoxy auxin herbicide in plants after expressing the genetically engineered 24DT22 in plants, of which the inherent tolerance does not exist or is not enough to allow the application of these herbicides. In addition, 24DT22 gene can provide protection on phenoxy auxin herbicides when the natural tolerance is not enough to allow selectivity in plants. One, two or several phenoxy auxin herbicides can be continuously or tank-mixedly combined with it to treat plants only comprising 24DT22 gene. Dosage range of each phenoxy auxin herbicide used to control the broad-spectrum of dicotyledonous weeds ranges from 25 to 4000 g ae/ha, more generally from 100 to 2000 g ae/ha. Combination of these herbicides belonging to different chemical classes and having different action modes in a same field (continuously or tank-mixedly) can control most potential weeds which are intentioned to be controlled by the herbicides.
Glyphosate is widely used because it controls very broad spectrum of broad-leaved and gram ineous weed species. However, the repeated use of glyphosate in the application of glyphosate-tolerant crops and non-crops has (and will continue to) selectively resulted in the succession of the weeds to species with more natural tolerance or glyphosate-resistant biotype. Most of the herbicide resistance management strategies recommend using effective amount of tank-mixed herbicide partners as a way to delay the appearance of resistant weeds. The herbicide partners provide the control of a same species but with different modes of action. The overlay of 24DT22 gene and glyphosate-tolerance trait (and/or other herbicide-tolerance traits) can provide the control of glyphosate-resistant weed species (broad-leaved weed species controlled by one or more phenoxy auxins) in glyphosate-tolerance crops by selectively applying glyphosate and phenoxy auxin (such as 2,4-D) on the same crops. Applications of these herbicides might be the individual use of single herbicide composition in a tank mixture containing two or more herbicides with different action models simultaneously or sequentially (e.g. before planting, before seedling emergence or after seedling emergence) (interval time ranged from 2 hours to 3 months). Alternatively, compositions of any number of herbicides representing every class of compound could be used at any time (from within 7 months after planting to the time of harvest (or, as to a single herbicide, it refers to preharvest interval in which the shortest one is selected)).
Flexibility is very important in the control of broad-leaved weeds, i.e. application time, dosage of a single herbicide and the ability to control stubborn or resistant weeds. The dosage of glyphosate which overlays with glyphosate-resistant gene/24DT22 gene can range from 250 to 2500 g ae/ha; the dose of (one or more) phenoxy auxin herbicides can range from 25 to 4000 g ae/ha. The optimum combination of the application time depends on the specific conditions, species and the environment.
Herbicide formulations (such as esters, acids or salt formulas or soluble concentrates, emulsified concentrates or soluble solutions) and additives of tank-mixture (such as adjuvant or compatilizer) can significantly affect the weed control of a given herbicide or a combination of one or more kinds of herbicides. Any chemical combinations of any of above herbicides are comprised in the scope of this invention.
As well-known by one skilled in the art, the benefits of the combination of two or more action modes in improving the controlled weed spectrum and/or natural species with more tolerance or resistance weed species can be extended to chemicals capable of producing other herbicide tolerances besides glyphosate-tolerance in crops through artificial means (transgenic or non-transgenic). In fact, the following resistance characteristics can be encoded alone or be multiply overlayed so as to provide the ability to effectively control or prevent weeds from succession to any category of the above-mentioned herbicide resistances: glyphosate resistance (such as resistant plant or bacteria, EPSPS, GOX, GAT), glufosinate-ammonium resistance (such as PAT, Bar), acetolactate synthase (ALS) inhibitory herbicide resistance (such as imidazolidinone, sulfonylurea, triazole pyrimidine, sulphonanilide, pyrimidine thiobenzoate and other chemical resistance genes such as AHAS, Csrl, SurA etc.), bromoxynil resistance (such as Bxn), resistance to inhibitor of HPPD (4-Hydroxyphenylpyruvate dioxygenase), resistance to inhibitor of phytoene desaturase (PDS), resistance to photosystem II inhibitory herbicide (such as psbA), resistance to photosystem I inhibitory herbicide, resistance to protoporphyrinogen oxidase IX (PPO) inhibitory herbicide (such as PPO-1), phenylurea herbicide resistance (such as CYP76B1), dicamba degrading enzyme etc.
As to other herbicides, some of other preferable ALS inhibitors include triazolopyrimidine benzenesulfonamide (cloransulam-methyl, diclosulam, flumetsulam, metosulam and pyrimidino triazoles sulfonamide), pyrimidine thiobenzoate and flucarbazone. Some preferable HPPD inhibitors include mesotrione, isoxaflutole and sulcotrione. Some preferable PPO inhibitors include flumioxazin, butafenacil, carfentrazone, sulfentrazone and diphenyl oxide (such as acifluorfen, fomesafen, Lactofen and oxyfluorfen).
In addition, 24DT22 gene can be overlayed alone with one or more other input (such as insect resistance, fungus resistance or stress tolerance or output (such as the increased yield, improved oil mass, improved fiber quality) traits, or overlayed with one or more other input (such as insect resistance, fungus resistance or stress tolerance) or output (such as the increased yield, improved oil mass, improved fiber quality) traits after overlaying with other herbicide-resistant crop characteristics. Therefore, this invention can provide the ability to flexibly and economically control any number of agronomy pests and a complete agronomy solution to improve crop quality.
24DT22 gene in this invention can degrade 2,4-D, which is the basis of important herbicide-resistant crops and of the possibility of selection markers.
Almost all the herbicide combinations for broad-leaved weeds could be controlled by the transgenic expression of 24DT22 gene. 24DT22 gene as an excellent herbicide tolerant crop trait can be overlayed with, for example, other herbicide-tolerant crop characteristics, such as glyphosate resistance, glufosinate-ammonium resistance, ALS inhibitor (such as imidazolidinone, sulfonylurea and triazolopyrimidine benzenesulfonamides) resistance, bromoxynil resistance, HPPD inhibitor resistance, PPO inhibitor resistance and the like) and insect resistance traits (Cry1Ab, Cry1F, Vip3, other Bacillus thuringiensis protein or insect-resistant protein derived from the non-bacillus). In addition, 24DT22 gene can be used as a selection marker to assist the selection of the primary transformant of plants genetically modified with another gene or genogroup.
Phenoxy alkanoate group can be used to introduce stable acid functional groups into herbicides. Acidic groups can import phloem activity by “acid capture” (the property required by herbicide effect) so as to be integrated into the new herbicides for activity purpose. There are many commercially available and experimental herbicides as substrates of 24DT22. Therefore, tolerances to other herbicides can be obtained by using present invention.
The crop herbicide-tolerance trait of this invention can be used in a new combination with other crop herbicide-tolerance traits (including but not limited to glyphosate tolerance). Because of the newly acquired resistance or inherent tolerance to herbicides (such as glyphosate), the combinations of these traits produce new methods to control weed species. Therefore, in addition to crop herbicide-tolerance traits, present invention also includes new methods for controlling weeds by using herbicides, in which the said herbicide-tolerance is obtained through the enzyme produced by the transgenic crops.
The present invention can be applied to a variety of plants, such as Arabidopsis, tobacco, soybean, cotton, rice, corn and brassica. The present invention can also be applied to a variety of other monocotyledonous (such as gramineous herbage or grassy carpet) and dicotyledonous crops (such as alfalfa, clover and tree species, etc.). Similarly, 2, 4-D (or other 24DT22 substrates) can be applied more actively to gramineous crops with moderate tolerance, and the resulted tolerance of which traits are raised will provide growers the possibility to use these herbicides with more effective dosage and broader administration time without the risk of crop injury.
The genomes of plants, plant tissues or plant cells described in this invention refer to any genetic materials in the plants, plant tissues or plant cells, and include the nucleus, plasmids and mitochondrial genomes.
The “resistance” described herein is heritable, and allows the plants to grow and reproduce under the case that effective treatment is applied to the given plants using common herbicide. As acknowledged by one skilled in the art, even if a certain damage of the plant caused by herbicides is obvious, the plant can still be considered “resistance”. The term “tolerance” described herein is broader than the term “resistance” and includes “resistance” and the improved ability of particular plant resistant to the various degree of damages induced by the herbicides which result generally in the damages of the wild type plants with the same genotypes under the same herbicide dosage.
As described herein, polynucleotides and/or nucleotides form a complete “gene” and encode proteins or polypeptides in the host cells of interest. It is easy for one skilled in the art to realize that the polynucleotides and/or nucleotides in the present invention can be under the control of the regulatory sequences of the target host.
As well known by one skilled in the art, DNA exists typically as double strands. In such an arrangement, one strand is complementary with the other, and vice versa. When DNA is replicated in plants, other complementary strands of DNA are also generated. Therefore, the polynucleotides exemplified in the sequence listing and complementary strands thereof are comprised in this invention. The “coding strand” generally used in the art refers to a strand binding with an antisense strand. To express a protein in vivo, one strand of the DNA is typically transcribed into a complementary strand of mRNA, which serves as the template of protein expression. In fact, a mRNA is transcribed from the “antisense” strand of DNA. “Sense strand” or “coding strand” contains a series of codons (codon is a triplet of nucleotides that codes for a specific amino acid), which might be read as open reading frames (ORF) to generate target proteins or peptides. RNA and PNA (peptide nucleic acid) which are functionally equivalent with the exemplified DNA were also contemplated in this invention.
Nucleic acid molecule or fragments thereof were hybridized with the herbicide-resistant gene under stringent condition in this invention. Any regular methods of nucleic acid hybridization or amplification can be used to identify the existence of the herbicide-resistant gene in present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing with other nucleic acid molecules under certain conditions. In present invention, if two nucleic acid molecules can form an antiparallel nucleic acid structure with double strands, it can be determined that these two molecules can hybridize with each other specifically. If two nucleic acid molecules are completely complementary, one of two molecules is called as the “complement” of the other one. In this invention, when every nucleotide of a nucleic acid molecule is complementary with the corresponding nucleotide of another nucleic acid molecule, it is identified the two molecules are “completely complementary”. If two nucleic acid molecules can hybridize with each other so that they can anneal to and bind to each other with enough stability under at least normal “low-stringency” conditions, these two nucleic acids are identified as “minimum complementary”. Similarly, if two nucleic acid molecules can hybridize with each other so that they can anneal to and bind to each other with enough stability under normal “high-stringency” conditions, it is identified that these two nucleic acids are “complementary”. Deviation from “completely complementary” can be allowed, as long as the deviation does not completely prevent the two molecules to form a double-strand structure. A nucleic acid molecule which can be taken as a primer or a probe must have sufficiently complementary sequences to form a stable double-strand structure in the specific solvent at a specific salt concentration.
In this invention, basically homologous sequence refers to a nucleic acid molecule, which can specifically hybridize with the complementary strand of another matched nucleic acid molecule under “high-stringency” condition. The stringency conditions for DNA hybridization are well-known to one skilled in the art, such as treatment with 6.0× sodium chloride/sodium citrate (SSC) solution at about 45° C. and washing with 2.0×SSC at 50° C. For example, the salt concentration in the washing step is selected from 2.0×SSC and 50° C. for the “low-stringency” conditions and 0.2×SSC and 50° C. for the “high-stringency” conditions. In addition, the temperature in the washing step ranges from 22° C. for the “low-stringency” conditions to 65° C. for the “high-stringency” conditions. Both temperature and the salt concentration can vary together or only one of these two variables varies. Preferably, the stringency condition used in this invention might be as below. SEQ ID NO:1 is specifically hybridized in 6.0×SSC and 0.5% SDS solution at 65° C. Then the membrane was washed one time in 2×SSC and 0.1% SDS solution and 1×SSC and 0.1% SDS solution, respectively.
Therefore, certain herbicide-resistant sequence which can hybridize with SEQ ID NO: 1 under stringent conditions was comprised in this invention. These sequences were at least about 40%-50% homologous or about 60%, 65% or 70% homologous, even at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homologous to the sequences of present invention.
The present invention provides functional proteins. “Functional activity” (or “activity”) as described herein means the activity of proteins/enzymes (alone or combined with other protein) in this invention to degrade herbicide or reduce the herbicide activity. The plants which produce the proteins of this invention preferably produce such an effective amount of proteins that, when treating plants with herbicides, the protein expression level is enough to provide the plants with complete or partial resistance or tolerance to herbicides (general dosage if there are no specific instructions). Herbicides are usually applied at the dosage capable of killing the target plants, normal dosage and concentration applied in the field. Preferably, plant cells and plants of this invention are protected from the growth inhibition or damage caused by herbicide treatment. The transformed plants and plant cells of the present invention preferably have resistance or tolerance to 2,4-D herbicides, which means that the transformed plants and plant cells can survive in the condition with effective amount of 2,4-D herbicides.
Genes and proteins described in the present invention include not only the specifically exemplified sequences, but also parts and/or fragments (including deletion(s) in and/or at the end of the full-length protein), variants, mutants, substitutes (proteins containing substituted amino acid(s)), chimeras and fusion proteins retaining the herbicide-resistant activity thereof. The said “variants” or “variation” refers to the nucleotide sequences encoding the same one protein or encoding an equivalent protein having herbicide-resistant activity. The said “equivalent protein” refers to the proteins that have the same or the substantially same bioactivity of herbicide-resistant activity as that of the claimed proteins.
The “fragment” or “truncation” of the DNA or protein sequences as described in this invention refers to a part or an artificially modified form thereof (e.g., sequences suitable for plant expression) of the original DNA or protein sequences (nucleotides or amino acids) involved in present invention. The sequence length of said sequence is variable, but it is long enough to ensure that the (encoded) protein is herbicide-resistant protein. In some cases (especially expression in plants), it is advantageous to use a truncated gene which encodes a truncated protein. The preferable, truncated gene usually encodes 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the whole protein.
Due to redundancy of the genetic codons, a variety of different DNA sequences can encode one same amino acid sequence. It is available for one skilled in the art to achieve substitutive DNA sequences encoding one same or substantially same protein. These different DNA sequences are comprised in this invention. The said “substantially same” sequence refers to a sequence in which certain amino acids are substituted, deleted, added or inserted, but herbicide-resistant activity thereof is not substantially affected, and also includes the fragments remaining the herbicide-resistant activity.
Substitution, deletion or addition of some amino acids in amino acid sequences in this invention is conventional technique in the art. Preferably, such an amino acid change includes: minor characteristics change, i.e. substitution of reserved amino acids which do not significantly influence the folding and/or activity of the protein; short deletion, usually a deletion of about 1-30 amino acids; short elongation of amino or carboxyl terminal, such as a methionine residue elongation at amino terminal; short connecting peptide, such as about 20-25 residues in length.
The examples of conservative substitution are the substitutions happening in the following amino acids groups: basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (e.g., glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (e.g., phenylalanine, tryptophan and tyrosine), and small molecular amino acids (such as glycine, alanine, serine and threonine and methionine). Amino acid substitutions generally not changing specific activity are well known in the art and have been already described in, for example, Protein edited by N. Neurath and R. L. Hill, published by Academic Press, New York in 1979. The most common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thu/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, and reverse substitutions thereof.
Obviously, for one skilled in the art, such a substitution may happen outside of the regions which are important to the molecular function and still cause the production of active polypeptides. For the polypeptide of the present invention, the amino acid residues which are required for their activity and chosen as the unsubstituted residues can be identified according to the known methods of the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g. Cunningham and Wells, 1989, Science 244:1081-1085). The latter technique is carried out by introducing mutations in every positively charged residue in the molecule and detecting the herbicide-resistant activity of the obtained mutation molecules, so as to identify the amino acid residues which are important to the activity of the molecules. Enzyme-substrates interaction sites can also be determined by analyzing its three-dimensional structure, which can be determined through some techniques such as nuclear magnetic resonance (NMR) analysis, crystallography, or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255:306-312; Smith, et al., 1992, J. Mol. Biol 224:899-904; Wlodaver, et al., 1992, FEBS Letters 309:59-64).
Therefore, amino acid sequences which have certain homology with the amino acid sequences set forth in SEQ ID No. 2 are also comprised in this invention. The sequence similarity/homology between these sequences and the sequences described in the present invention are typically more than 60%, preferably more than 75%, more preferably more than 80%, even more preferably more than 90% and more preferably more than 95%. The preferred polynucleotides and proteins in the present invention can also be defined according to more specific ranges of the homology and/or similarity. For example, they have a homology and/or similarity of 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequences described in this invention.
Regulatory sequences described in this invention include but are not limited to a promoter, transit peptide, terminator, enhancer, leading sequence, introns and other regulatory sequences that can be operably linked to the said 24DT22 gene.
The said promoter is a promoter expressible in plants, wherein said “a promoter expressible in plants” refers to a promoter which ensures that the coding sequences bound with the promoter can be expressed in plant cells. The promoter expressible in plants can be a constitutive promoter. The examples of promoters capable of directing the constitutive expression in plants include but are not limited to 35S promoter derived from Cauliflower mosaic virus, ubi promoter, promoter of GOS2 gene derived from rice and the like. Alternatively, the promoter expressible in plants can be a tissue-specific promoter, which means that the expression level directed by this promoter in some plant tissues such as in chlorenchyma, is higher than that in other tissues of the plant (can be measured through the conventional RNA test), such as the PEP carboxylase promoter. Alternatively, the promoter expressible in plants can be wound-inducible promoters as well. Wound-inducible promoters or promoters that direct wound-inducible expression manners refer to the promoters by which the expression level of the coding sequences can be increased remarkably compared with those under the normal growth conditions when the plants are subjected to mechanical wound or wound caused by the gnaw of insects. The examples of wound-inducible promoters include but are not limited to the promoters of genes of protease inhibitor of potatoes and tomatoes (pin I and pin II) and the promoters of corn protease inhibitor gene (MPI).
The said transit peptide (also called paracrine signal sequence or leader sequence) directs the transgenosis products into specific organelles or cellular compartments. For the receptor protein, the said transit peptide can be heterogeneous. For example, sequences encoding chloroplast transit peptide are used to lead to chloroplast; or ‘KDEL’ reserved sequence is used to lead to the endoplasmic reticulum or CTPP of the barley lectin gene is used to lead to the vacuole.
The said leader sequences include but are not limited to small RNA virus leader sequences, such as EMCV leader sequence (encephalomyocarditis virus 5′ non coding region); Potato virus Y leader sequences, such as MDMV (Corn dwarf mosaic virus) leader sequence; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of Alfalfa Mosaic virus (AMV RNA4); Tobacco Mosaic virus (TMV) leader sequence.
The said enhancer includes but is not limited to Cauliflower Mosaic virus (CaMV) enhancer, Figwort Mosaic virus (FMV) enhancer, Carnations Etched Ring virus (CERV) enhancer, Cassava Vein Mosaic virus (CsVMV) enhancer, Mirabilis Mosaic virus (MMV) enhancer, Cestrum yellow leaf curling virus (CmYLCV) enhancer, Cotton leaf curl Multan virus (CLCuMV), Commelina yellow mottle virus (CoYMV) and peanut chlorotic streak mosaic virus (PCLSV) enhancer.
For the application of monocotyledon, the said introns include but are limited to corn hsp70 introns, corn ubiquitin introns, Adh intron 1, sucrose synthase introns or rice Act1 introns. For the application of dicotyledonous plants, the said introns include but are not limited to CAT-1 introns, pKANNIBAL introns, PIV2 introns and “super ubiquitin” introns.
The said terminators can be the proper polyadenylation signal sequences playing a role in plants. They include but are not limited to polyadenylation signal sequence derived from Agrobacterium tumefaciens nopaline synthetase (NOS) gene, polyadenylation signal sequence derived from protease inhibitor II (pin II) gene, polyadenylation signal sequence derived from peas ssRUBISCO E9 gene and polyadenylation signal sequence derived from α-tubulin gene.
The term “operably linked” described in this invention refers to the linking of nucleic acid sequences, which provides the sequences the required function of the linked sequences. The term “operably linked” described in this invention can be the linkage of the promoter with the sequences of interest, which makes the transcription of these sequences under the control and regulation of the promoter. When the sequence of interest encodes a protein and the expression of this protein is required, the term “operably linked” indicates that the linking of the promoter and said sequence makes the obtained transcript to be effectively translated. If the linking of the promoter and the coding sequence results in transcription fusion and the expression of the encoding protein are required, such a linking is generated to make sure that the first translation initiation codon of the obtained transcript is the initiation codon of the coding sequence. Alternatively, if the linking of the promoter and the coding sequence results in translation fusion and the expression of the encoding protein is required, such a linking is generated to make sure that the first translation initiation codon of the 5′untranslated sequence is linked with the promoter, and such a linking way makes the relationship between the obtained translation products and the open reading frame encoding the protein of interest meet the reading frame. Nucleic acid sequences that can be “operably linked” include but are not limited to sequences providing the function of gene expression (i.e. gene expression elements, such as a promoter, 5′untranslated region, introns, protein-coding region, 3′untranslated region, polyadenylation sites and/or transcription terminators); sequences providing the function of DNA transfer and/or integration (i.e., T-DNA boundary sequences, recognition sites of site-specific recombinant enzyme, integrase recognition sites); sequences providing selectable function (i.e., antibiotic resistance markers, biosynthetic genes); sequences providing the function of scoring markers; sequences assistant with the operation of sequences in vitro or in vivo (polylinker sequences, site-specific recombinant sequences) and sequences providing replication function (i.e. origins of replication of bacteria, autonomously replicating sequences, centromeric sequences).
This invention can confer new herbicide resistant trait(s) to the plants while adverse effects on phenotypes including yield are not observed. The plants of present invention can tolerate against 2×, 3×, 4× or 5× general application level of at least one subjected herbicide. The improvement of these resistance levels is in the scope of present invention. For example, it is possible to foreseeably optimize and further develop many kinds of known technologies in the art so as to increase the expression of a given gene.
In present invention, said herbicide-resistant protein is 24DT22 amino acid sequence as shown in SEQ ID NO: 2 of the sequence listing. Said herbicide-resistant gene is 24DT22 nucleotide sequence as shown in SEQ ID NO: 1 of the sequence listing. In order to be applied to plants, said herbicide-resistant gene also contains, besides coding region of the protein encoded by 24DT22 nucleotide sequence, other elements, such as encoding regions which encode transit peptides, the coding regions which encode selective marker proteins or the proteins which confer resistance to insect.
The herbicide-resistant protein 24DT22 as describe herein is tolerant to most phenoxy auxin herbicides. The genomes of the plants in present invention contain exogenous DNAs which contain 24DT22 nucleotide sequence. The plants are protected from the threat of herbicides by expressing effective amount of this protein. “Effective amount” refers to the amount which causes no damage or causes slight damage. At the same time, the plants should be morphologically normal, and could be cultivated under the common means for the consumption and/or generation of products.
The expression level of herbicide-resistance crystal proteins (ICP) in the plant materials can be determined using various methods described in this field, such as the method of quantifying mRNA encoding the herbicide-resistant protein in the tissue through using specific primers, or the method of quantifying the herbicide-resistant protein directly and specifically.
The present invention provides a herbicide-resistant protein, coding gene and use thereof with following advantages:
1. Strong herbicide-resistance activity. Herbicide-resistant protein 24DT22 of present invention is strongly resistant to herbicides, especially to phenoxy auxin herbicides, particularly 2,4-D.
2. Broad herbicide-resistance spectrum. The herbicide-resistant protein 24DT22 of present invention shows high resistance to a variety of plant phenoxy auxin herbicides, therefore it has broad application prospect on the plants.
The technical solutions of this invention will be further described through the appended figures and examples as following.
The technical solution of herbicide-resistant protein, coding gene and use thereof in present invention will be further illustrated through the following examples.
1. Obtaining of 24DT22 Gene Sequence
Amino acid sequence of the 24DT22 herbicide-resistant protein (292 amino acids) was shown as SEQ ID NO: 2 in the sequence listing; the nucleotide sequence (879 nucleotides) encoding the corresponding amino acid sequence of 24DT22 herbicide-resistant protein (292 amino acids) was shown as SEQ ID NO: 1 in the sequence listing.
2. Synthesis of the Nucleotide Sequence as Described Above
The 24DT22 nucleotide sequence (shown as SEQ ID NO: 1 in the sequence listing) was synthesized by GenScript Co., Ltd. Nanjing, P. R. China; The synthesized 24DT22 nucleotide sequence (SEQ ID NO: 1) was linked with a SpeI restriction site at the 5′ end, and a KasI restriction site at the 3′ end.
1. Construction of the Recombinant Cloning Vector DBN01-T Containing 24DT22 Nucleotide Sequence
The synthesized 24DT22 nucleotide sequence was cloned into cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), to get recombinant cloning vector DBN01-T following the instructions of Promega pGEM-T vector, and the construction process was shown in
The recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cell (Transgen, Beijing, China, the CAT: CD501) through heat shock method. The heat shock conditions were as follows: 50 μL of E. coli T1 competent cell and 10 μL of plasmid DNA (recombinant cloning vector DBN01-T) were incubated in water bath at 42° C. for 30 seconds. Then the E. coli cells were under shaking cultivation at 37° C. for 1 h (100 rpm in a shaking incubator) and then were grown on a LB plate (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Agar and pH was adjusted to 7.5 with NaOH) coated on the surface with IPTG (Isopropyl thio-beta-D-galactose glucoside), X-gal (5-bromine-4-chlorine-3-indole-beta-D-galactose glucoside) and ampicillin (100 mg/L) overnight. The white colonies were picked out and cultivated in LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 100 mg/L ampicillin and pH was adjusted to 7.5 with NaOH) at 37° C. overnight. The plasmids thereof were extracted using alkaline lysis method as follows: the bacterial liquid was centrifuged for 1 min at 12000 rpm, the supernatant was discarded and the pellet was resuspended in 100 μL in ice-chilled solution I (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid) and 50 mM glucose, pH=8.0); then 200 μL of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)) was added and the tube was reversed 4 times, mixed and then put on ice for 3-5 minutes; 150 μL of cold solution III (3 M potassium acetate and 5 M acetic acid) was added, thoroughly mixed immediately and incubated on ice for 5-10 minutes; the mixture was centrifuged at 12000 rpm at 4° C. for 5 minutes, two volumes of anhydrous ethanol were added into the supernatant, mixed and then placed at room temperature for 5 minutes; the mixture was centrifuged at 12000 rpm at 4° C. for 5 minutes, the supernatant was discarded and the pellet was dried after washed with 70% ethanol (V/V); 30 μL TE (10 mM Tris-HCl, 1 mM EDTA, pH=8.0) containing RNase (20 μg/mL) was added to dissolve the precipitate; the mixture was incubated at 37° C. in a water bath for 30 min to digest RNA and stored at −20° C. for the future use.
After the extracted plasmids were confirmed with restriction enzymes SpeI and KasI, the positive clones were verified through sequencing. The results showed that said 24DT22 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the sequence set forth in SEQ ID NO: 1 in the sequence listing, indicating that 24DT22 nucleotide sequence was correctly inserted.
2. Constructing the Recombinant Expression Vectors DBN100301 of Arabidopsis thaliana and Soybean Containing 24DT22 Nucleotide Sequence
The recombinant cloning vector DBN01-T and expression vector DBNBC-01 (Vector backbone: pCAMBIA2301, available from CAMBIA institution) were digested with restriction enzymes SpeI and KasI. The cleaved 24DT22 nucleotide sequence fragment was ligated between the restriction sites SpeI and KasI of the expression vector DBNBC-01 to construct the recombinant expression vector DBN100301. It is a well-known conventional method for one skilled in the art to construct expression vector through restriction enzyme digestion. The construction scheme was shown in
The recombinant expression vector DBN100301 was transformed into E. coli T1 competent cells with heat shock method as follows: 50 μL of E. coli T1 competent cell and 10 μL of plasmid DNA (recombinant expression vector DBN100301) were incubated in water bath at 42° C. for 30 seconds. Then the E. coli cells were under shaking cultivation at 37° C. for 1 hour (100 rpm in a shaking incubator) and then were grown on a LB solid plate (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Agar and pH was adjusted to 7.5 with NaOH) containing 50 mg/L kanamycin at 37° C. for 12 hours. The white colonies were picked out and cultivated in LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 mg/L kanamycin and pH was adjusted to 7.5 with NaOH) at 37° C. overnight. The plasmids thereof were extracted using alkaline lysis method. After the extracted plasmids were confirmed with restriction enzymes SpeI and KasI, the positive clones were verified through sequencing. The results showed that the nucleotide sequence between restriction sites SpeI and KasI in the recombinant expression vector DBN100301 was the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing, i.e. 24DT22 nucleotide sequence.
3. Constructing the Recombinant Expression Vectors DBN100301N of Arabidopsis thaliana and Soybean Containing a Control Sequence
Following the process for constructing recombinant cloning vector DBN01-T comprising 24DT22 nucleotide sequence as described in part 1 of Example 2, recombinant cloning vector DBN01R-T containing control sequence was constructed by using control sequence (SEQ ID NO: 8). The positive clones were verified through sequencing. The results showed that the control nucleotide sequence inserted into the recombinant cloning vector DBN01R-T was the sequence set forth in SEQ ID NO: 8 in the sequence listing, indicating that control nucleotide sequence was correctly inserted.
Following the process for constructing recombinant expression vector DBN100301 containing 24DT22 nucleotide sequence as described in part 2 of Example 2, recombinant expression vector DBN100301N containing control sequence was constructed using the control sequence and the structure of the vector was shown in
1. Transformation of Agrobacterium tumefaciens with Recombinant Expression Vectors
The correctly constructed recombinant expression vectors DBN100301 and DBN100301N (control sequence) were transformed into Agrobacterium GV3101 following liquid nitrogen rapid-freezing method as follows: 100 μL Agrobacterium GV3101 and 3 μL plasmid DNA (recombinant expression vector) were put into liquid nitrogen for 10 minutes and then incubated in water bath at 37° C. for 10 minutes. Then the transformed Agrobacterium GV3101 cells were inoculated in LB broth and cultivated at 28° C., 200 rpm for 2 hours and spread on a LB plate containing 50 mg/L of rifampicin (Rifampicin) and 50 mg/L of kanamycin until positive mono colonies appeared. The positive mono colonies were picked up and cultivated and the plasmids thereof were extracted. Recombinant expression vector DBN100301 was verified with restriction enzymes SmaI and PstI and recombinant expression vector DBN100301N (control sequence) was verified with restriction enzymes SmaI and BglI. The results showed that the recombinant expression vectors DBN100301 and DBN100301N (control sequence) were correct in structure, respectively.
2. Obtaining Transgenic Arabidopsis thaliana Plants
The wild-type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution and kept at 4° C. for 2 days so as to meet the need for dormancy to ensure the synchronous germination of seeds. Vermiculite and horses dung were mixed together and irrigated wet with water underground. The soil mixture was dewatered for 24 hours. The pretreated seeds were cultivated in the soil mixture and covered with a moisturizing mask for 7 days. The seeds were germinated and the plants were cultivated in a greenhouse at a constant temperature of 22° C. with constant moisture of 40-50% and a long day condition with the light intensity of 120-150 μmol/m2s (16 hours of light/8 hours of darkness). The plants were irrigated with Hoagland nutrient solution at first and then with deionized water to keep the soil moist but not drenched.
Floral dip method was used to transform Arabidopsis. One or more YEP media containing 50 mg/L of kanamycin and 10 mg/L of rifampicin of 15-30 ml were inoculated with the selected Agrobacterium colonies as a pre-culture. The pre-culture was incubated at 28° C. and 220 rpm overnight. Each pre-culture was used to inoculate two cultures of 500 mL YEP media containing kanamycin (100 mg/L) and rifampicin (10 mg/L) and the cultures were incubated at 28° C. in a shaking incubator overnight. Cultures were centrifuged at 8700×g for 10 minutes at room temperature to precipate cells and the obtained supernatant was discarded. The cell pellets were gently re-suspended in 500 ml of permeable medium which contains ½×MS salts/vitamin B5, 10% (w/v) sucrose, 0.044 μM Benzylaminopurine (10 μL/L (1 mg/mL stock solution in DMSO)) and 300 μL/L Silvet L-77. About 1 month old plants were soaked in the medium for 15 seconds and the latest inflorescences were ensured to be submerged. Then plants were put down by side and covered (transparent or non-transparent) for 24 hours, then washed with water and placed vertically. The plants were cultivated at 22° C. in a light cycle of 16 hours of light/8 hours of darkness. Seeds were harvested after soaked for 4 weeks.
The newly harvested T1 seeds (24DT22 nucleotide sequence and control sequence) were dried at room temperature for 7 days. The seeds were cultivated in germination plates (26.5×51 cm), 200 mg T1 seeds (about 10000 seeds)/plate. The seeds have already been suspended in 40 mL of 0.1% (w/v) agarose solution and stored at 4° C. for 2 days to meet the need for dormancy to ensure the synchronous germination of seeds.
Vermiculite and horses dung were mixed together and irrigated wet with water underground and drained through gravity. The pretreated seeds (40 mL each one) were uniformly planted on the soil mixture by using pipette and covered with moisturizing mask for 4 to 5 days. The mask was removed 1 day before the initial transformant selection by spraying glufosinate-ammonium (selection of the co-transformed PAT gene) after germination.
On 7 days after planting (DAP) and 11 DAP respectively, the T1 plants (cotyledon stage and 2-4 leaves stage, respectively) were sprayed with 0.2% of Liberty herbicide solution (200 g ai/L glufosinate-ammonium) using DeVilbiss compressed air nozzle at a spraying volume of 10 mL/disc (703 L/ha) so as to provide effective amount of glufosinate-ammonium (280 g ai/ha) for each application. The survival plants (actively growing plants) were verified 4 to 7 days after the last spraying and transferred into the square pot (7 cm×7 cm) made from vermiculite and horses dung (3-5 plants per pot). The transplanted plants were covered with moisturizing mask for 3-4 days and placed in culture room at 22° C. or directly into the greenhouse as described above. Then the mask was removed and the plants were planted in greenhouse (22±5° C., 50±30% RH, 14 hours of lighting: 10 hours of darkness, minimum 500 μE/m2s1 natural light+complement light) at least one day before testing the ability of 24DT22 to provide the resistance to phenoxy auxin herbicide.
24DT22 gene was used to transform Arabidopsis for the first time. At first, T1 transformants were selected from the background of un-transformed seeds, using glufosinate-ammonium selection scheme. About 20000 T1 seeds are screened among which 314 strains of T1 generation positive transformants (PAT gene) were identified, i.e. the transformation efficiency was about 1.6%. Herbicide resistance effect tests to 2,4-D dimethyl ammonium salt and agroxone of Arabidopsis T1 plants transformed with 24DT22 nucleotide sequence, control nucleotide sequence respectively and wild-type Arabidopsis plants were performed after 18 days of planting.
Arabidopsis T1 plants transformed with 24DT22 nucleotide sequence, control nucleotide sequence respectively and wild-type Arabidopsis plants were sprayed with 2,4-D dimethyl ammonium salt (560 g ae/ha, 1-fold concentration in field), agroxone (560 g ae/ha, 1-fold concentration in field) and blank solvent (water). Resistance conditions of the plants were counted 7 days and 14 days after spraying. Plants with growth conditions consistent with blank solvent (water) 7 days after spaying were classified as highly resistant plants; Plants with curly rosette leaves 7 days after spaying were classified as moderately resistant plants; Plants incapable of bolting 14 days after spaying were classified as low-resistant plants and the dead plants 14 days after spaying were classified as non-resistant plants. Because each Arabidopsis T1 plant is an independent transformation event, significant differences of individual T1 responses can be expected under a given dose. The results were shown in Table 1 and
Arabidopsis
For Arabidopsis, 560 g ae/ha of 2,4-D and agroxone is the effective dose to distinguish the sensitive plants from plants with average resistance. Results shown in Table 1 and
1. Transformation of Agrobacterium tumefaciens with Recombinant Expression Vectors
The correctly constructed recombinant expression vectors DBN100301 and DBN100301N (control sequence) were transformed into Agrobacterium LBA4404 (Invitrogen, Chicago, USA, CAT: 18313-015) following liquid nitrogen rapid-freezing method, the transformation conditions are: Agrobacterium LBA4404 and 3 μL plasmid DNA (recombinant expression vector) were put into liquid nitrogen for 10 minutes and then incubated in water bath at 37° C. for 10 minutes. Then the transformed Agrobacterium LBA4404 cells were inoculated in LB broth and cultivated at 28° C., 200 rpm for 2 hours and spread on a LB plate containing 50 mg/L of rifampicin (Rifampicin) and 50 mg/L of kanamycin until positive mono colonies appeared. The positive mono colonies were picked up and cultivated and the plasmids thereof were extracted. Recombinant expression vectors DBN100301 was verified with restriction enzymes SmaI and PstI and recombinant expression vector DBN100301N (control sequence) was verified with restriction enzymes SmaI and BglI. The results showed that the recombinant expression vectors DBN100301 and DBN100301N (control sequence) were correct in structure, respectively.
2. Obtaining Transgenic Soybean Plants
The cotyledonary node of wild-type soybean (Zhonghuang 13) was sterilely cultivated with the Agrobacterium tumefaciens described in Example 1 as to transfer the T-DNA of the recombinant expression vectors DBN100301 and DBN100301N described in Example 2 and 3 (containing promoter sequence of the Arabidopsis thaliana ubiquitin10 gene, 24DT22 nucleotide sequence, control sequence, Nos terminator, cauliflower mosaic virus 35S promoter, glufosinate acetyl transferase gene and cauliflower mosaic virus 35S terminator) into the soybean genome, soybean plants containing 24DT22 and control nucleotide sequences were obtained and at the same time wild-type soybean plant was taken as a control.
As to the agrobacterium-mediated transformation of soybean, in brief, mature soybean seeds were germinated in a soybean germination medium (3.1 g/L B5 salt, B5 vitamin, 20 g/L sucrose, 8 g/L agar and pH5.6) and cultivated at following conditions: temperature, 25±1° C.; photoperiod (light/darkness), 16/8 h. Fresh green aseptic soybean with bulging cotyledon node was obtained after 4-6 days of germination, cut off the hypocotyl which is 3-4 mm below the cotyledon node, cut the cotyledon longitudinally, and remove the terminal bud, the lateral bud and the seminal roots from the cotyledon, make a damage in the cotyledonary node with the back of a scalpel and bring agrobacterium suspension into contact with the damaged cotyledonary node tissues, wherein the agrobacterium can transfer the 24DT22 nucleotide sequence to the damaged cotyledonary node tissues (Step 1, infection step: in this step, preferably, the cotyledonary node tissue were immersed in Agrobacterium suspension (OD660=0.5-0.8, infection medium (2.15 g/L MS salt, B5 vitamin, 20 g/L sucrose, 10 g/L glucose, 40 mg/L acetosyringone (AS), 4 g/L 2-(N-Morpholino) ethanesulfonic acid (MES), 2 mg/L zeatin (ZT), pH5.3)) to initiate the inoculation. Co-culture the cotyledonary node tissue with the agrobacterium for a period (3 days). (Step 2: co-cultivation step). Preferably, the colyledonary node tissues were cultured in a solid medium (4.3 g/L MS salt, B5 vitamin, 20 g/L sucrose, 10 g/L glucose, 4 g/L 2-(N-Morpholino) ethanesulfonic acid (MES), 2 mg/L zeatin, 8 g/L agar and pH5.6) after the infection. After this co-cultivation step, a selective “recovery” step can be preceded. In the “recovery” step, the recovery medium (3.1 g/L B5 salt, B5 vitamin, 1 g/L 2-(N-Morpholino) ethanesulfonic acid (MES), 30 g/L sucrose, 2 mg/L zeain (ZT), 8 g/L agar, 150 mg/L cephalosporin, 100 mg/L glutamic acid, 100 mg/L aspartic acid and pH5.6) contains at least one kind of known Agrobacterium-inhibiting antibiotics (cephalosporin) without the selective agent for plant transfectants (Step 3: recovery step). Preferably, the tissues were cultivated on solid medium culture containing antibiotics but without selective agent so as to eliminate Agrobacterium and to provide a recovery period for the infected cells. Then, the inoculated tissues were cultivated on a medium containing selective agent (glufosinate) and the transformed, growing callus was selected (Step 4: selection step). Preferably, the tissues were cultivated on a selective solid medium containing selective agent (3.1 g/L B5 salt, B5 vitamin, 1 g/L 2-(N-Morpholino) ethanesulfonic acid (MES), 30 g/L sucrose, 1 mg/L 6-benzyladenine (6-BAP), 8 g/L agar, 150 mg/L cephalosporin, 100 mg/L glutamic acid, 100 mg/L aspartic acid, 6 mg/L glufosinate, and pH5.6), resulting in the selective growth of the transformed cells. Then, callus regenerated into plants (Step 5: regeneration step), Preferably, the callus was cultivated on a solid medium containing selective agent (B5 differentiation medium and B5 rooting medium) to regenerate into plants.
The obtained resistant callus was transferred to said B5 differentiation medium (3.1 g/L B5 salt, B5 vitamin, 1 g/L 2-(N-Morpholino) ethanesulfonic acid (MES), 30 g/L sucrose, 1 mg/L zeatin (ZT), 8 g/L agar, 150 mg/L cephalosporin, 50 mg/L glutamic acid, 50 mg/L aspartic acid, 1 mg/L gibberellin, 1 mg/L auxin, 6 mg/L glufosinate and pH5.6) for cultivation and differentiation at 25° C. The differentiated seedlings were transferred to said B5 rooting medium (3.1 g/L B5 salt, B5 vitamin, 1 g/L 2-(N-Morpholino)ethanesulfonic acid (MES), 30 g/L sucrose, 8 g/L agar, 150 mg/L cephalosporin and 1 mg/L Indole-butyric acid (IBA)) and cultivated to about 10 cm in height at 25° C. Next, the seedlings were transferred to and cultivated in the greenhouse until fructification. In the greenhouse, the soybean plants were cultivated at 26° C. for 16 hours and at 20° C. for 8 hours every day.
3. Validating of Transgenic Soybean Plants with TaqMan Technique
100 mg of leaves from every transformed soybean plant (soybean plant transformed with 24DT22 nucleotide sequence or control nucleotide sequence) was taken as sample respectively. Genomic DNA thereof was extracted using DNeasy Plant Maxi Kit (Qiagen) and the copy number of 24DT22 gene was quantified through Taqman probe-based fluorescence quantitative PCR assay. Wild type soybean plant was taken as a control and analyzed according to the processes as described above. Experiments were carried out in triplicate and the results were the mean values.
The specific method for detecting the copy number of the PAT gene was described as follows:
The following primers and probes were used to detect the PAT nucleotide sequence:
PCR Reaction System:
Said 50× primer/probe mixture containing 45 μL of each primer (1 mM), 50 μL of the probe (100 μM) and 860 μL of 1×TE buffer and was stored in an amber tube at 4° C.
PCR Reaction Conditions:
Data were analyzed using software SDS 2.3 (Applied Biosystems).
The experimental results showed that all the nucleotide sequence of the 24DT22 nucleotide sequence has been integrated into said detected soybean plants. Furthermore, all soybean plants transformed with 24DT22 nucleotide sequence or control sequence contained single copy of gene, respectively.
Herbicide resistance effects tests to 2,4-D dimethyl ammonium salt and agroxone of soybean plants containing 24DT22 nucleotide sequence and control nucleotide sequence respectively and wild type soybean plants (stages V3-V4) were performed respectively.
Soybean plants containing 24DT22 nucleotide sequence, control nucleotide sequence and wild type soybean plants were taken and spayed with 2,4-D dimethyl ammonium salt (2240 g ae/ha, four-folds concentration in field), agroxone (2240 g ae/ha, four-folds concentration in field) and blank solvent (water). Take statistics of the damage degree of each plant caused by herbicides according to the curling degree of leaves and the damage degree of growing point six hours (GHAT), two days (2DAT), seven days (7DAT) and 14 days (14DAT) after spraying respectively: if leaves are flat like wild-type leaves and the growing point is intact, the damage degree is 0%; if leaves curl up and wilt and the growing point is died, the damage degree is 100%. There are three strains contained a transferred 24DT22 nucleotide sequence (S1, S2 and S3), two strains contained a transferred control sequence (S4 and S5) and one wild-type strain (CK1) in total; select 10-15 plants from each strain for testing. The results were shown in Table 2 and
For soybean, 2240 g ae/ha of 2,4-D and agroxone is the effective dose to distinguish the sensitive plants from plants with average resistance. Results shown in Table 2 and
1. Construction of the Corn Recombinant Expression Vector DBN100764 Containing 24DT22 Nucleotide Sequence
The recombinant cloning vector DBN01-T and expression vector DBNBC-02 (Vector backbone: pCAMBIA2301, available from CAMBIA institution) were digested with restriction enzymes SpeI and KasI. The cleaved 24DT22 nucleotide sequence fragment was ligated between the restriction sites SpeI and KasI of the expression vector DBNBC-02 to construct the recombinant expression vector DBN100764. It is a well-known conventional method to construct expression vector through restriction enzyme digestion. SpeI and KasI restriction sites in the expression vector DBNBC-02 were also introduced using conventional enzyme digestion method. The construction scheme was shown in
The recombinant expression vector DBN100764 was transformed into E. coli T1 competent cells with heat shock method as follows: 50 μL of E. coli T1 competent cell and 10 μL of plasmid DNA (recombinant expression vector DBN100764) were incubated in water bath at 42° C. for 30 seconds. Then the E. coli cells were incubated in shaking cultivation at 37° C. for 1 hour (100 rpm in a shaking incubator) and then were grown on a LB solid plate (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Agar and pH was adjusted to 7.5 with NaOH) containing 50 mg/L kanamycin (kanamycin) at 37° C. for 12 hours. The white colonies were picked out and cultivated in LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 mg/L kanamycin and pH was adjusted to 7.5 with NaOH) at 37° C. overnight. The plasmids thereof were extracted using alkaline lysis method. After the extracted plasmids were confirmed with restriction enzymes SpeI and KasI, the positive clones were verified through sequencing. The results showed that the nucleotide sequence between restriction sites SpeI and KasI in the recombinant expression vector DBN100764 was the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing, i.e. 24DT22 nucleotide sequence.
2. Construction of the Corn Recombinant Expression Vector DBN100764N Containing Control Nucleotide Sequence
Following the process for constructing recombinant cloning vector DBN01-T containing 24DT22 nucleotide sequences described in part 1 of Example 2, recombinant cloning vector DBN02R-T containing control sequence was constructed by using control sequence (SEQ ID NO: 8). The positive clones were verified through sequencing. The results showed that the control nucleotide sequence inserted into the recombinant cloning vector DBN02R-T was the sequence set forth in SEQ ID NO: 8 in the sequence listing, indicating that control nucleotide sequence was correctly inserted.
Following the process for constructing recombinant expression vector DBN100764 containing 24DT22 nucleotide sequence as described in part 1 of example 7, recombinant expression vector DBN100764N containing natural sequence was constructed by using the control sequence and the construction process was shown in
3. Transformation of Agrobacterium tumefaciens with Corn Recombinant Expression Vectors
The correctly constructed recombinant expression vectors DBN100764 and DBN100764N (control sequence) were transformed into Agrobacterium LBA4404 (Invitrogen, Chicago, USA, CAT: 18313-015) following liquid nitrogen rapid-freezing method as follows: 100 μL Agrobacterium LBA4404 and 3 μL plasmid DNA (recombinant expression vector) were put into liquid nitrogen and kept for 10 minutes and then incubated in water bath at 37° C. for 10 minutes. Then the transformed Agrobacterium LBA4404 cells were inoculated in LB tube and cultivated at 28° C., 200 rpm for 2 hours and spread on a LB plate containing 50 mg/L of rifampicin (Rifampicin) and 50 mg/L of kanamycin until positive mono colonies appeared. The positive mono colonies were picked up and cultivated and the plasmids thereof were extracted. Recombinant expression vector DBN100764 was verified with restriction enzymes SmaI and EcoRV and DBN100764N (control sequence) was verified with restriction enzymes StyI and BglI. The results showed that the recombinant expression vectors DBN100764 and DBN100764N (control sequence) were correct in structures, respectively.
According to the conventional Agrobacterium transformation method, the corn cultivar Zong 31 (Z31) was cultivated in sterilized conditions and the young embryo was co-cultivated with the Agrobacterium strains constructed in part 3 of Example 7 so as to introduce T-DNAs in the recombinant expression vectors DBN100764 and DBN100764N (control sequence) constructed in part 1 and 2 of Example 7 (including corn Ubiquitin 1 gene promoter sequence, 24DT22 nucleotide sequence, control nucleotide sequence, PMI gene and Nos terminator sequence) into the corn genome. Corn plants containing 24DT22 nucleotide sequence and control nucleotide sequence respectively were obtained and at the same time wild type corn plant was taken as a control.
As to the Agrobacterium-mediated transformation of corn, in brief, immature corn young embryo was isolated from corns and contacted with Agrobacterium suspension, in which the Agrobacterium can deliver the 24DT22 nucleotide sequence into at least one cell of one young embryo. (Step 1: infection step). In this step, preferably, young embryo was immersed in Agrobacterium suspension (OD660=0.4˜0.6, infection medium (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 68.5 g/L of sucrose, 36 g/L of glucose, 40 mg/L of Acetosyringone (AS), 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), pH=5.3)) to initiate the inoculation. Young embryo and Agrobacterium were cocultivated for a period (3 days) (Step 2: co-cultivation step). Preferably, the Young embryo was cultivated on a solid medium (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 20 g/L of sucrose, 10 g/L of glucose, 100 mg/L of Acetosyringone (AS), 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 8 g/L of Agar, pH=5.8) after the infection step. After this cocultivation step, a selective “recovery” step can be preceded. In the “recovery” step, the recovery medium (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3 g/L of phytagel, pH=5.8) contains at least one kind of known Agrobacterium-inhibiting antibiotics (cephalosporin) without the selective agent for plant transfectants (Step 3: recovery step). Preferably, the young embryo was cultivated on a solid medium culture containing antibiotics but without selective agent so as to eliminate Agrobacterium and to provide a recovery period for the infected cells. Then, the inoculated young embryo was cultivated on a medium containing selective agent (mannose) and the transformed, growing callus was selected (Step 4: selection step). Preferably, the young embryo was cultivated on a selective solid medium containing selective agent (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 12.5 g/L of mannose, 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3 g/L of phytagel, pH=5.8), resulting the selective growth of the transformed cells. Then, callus regenerated into plants (Step 5: regeneration step). Preferably, the callus was cultivated on a solid medium containing selective agent (MS differentiation medium and MS rooting medium) to regenerate into plants.
The obtained resistant callus was transferred to said MS differentiation medium (4.3 g/L MS salt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 2 mg/L of 6-benzyladenine, 5 g/L of mannose and 3 g/L phytagel, pH=5.8) and cultivated and differentiated at 25° C. The differentiated seedlings were transferred to said MS rooting medium (2.15 g/L of MS salt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 1 mg/L indole-3-acetic acid and 3 g/L phytagel, pH=5.8) and cultivated to about 10 cm in height at 25° C. Next, the seedlings were transferred to and cultivated in the greenhouse until fructification. In the greenhouse, the corn plants were cultivated at 28° C. for 16 hours and at 20° C. for 8 hours every day.
2. Verification of Transgenic Corn Plants with Inserted 24DT22 Gene Using TaqMan Technique
100 mg of leaves from every transformed corn plant (corn plant transformed with 24DT22 nucleotide sequence or control nucleotide sequence, respectively) was taken as sample respectively. Genomic DNA thereof was extracted using DNeasy Plant Maxi Kit (Qiagen) and the copy number of PMI gene was quantified through Taqman probe-based fluorescence quantitative PCR assay in order to determine the copy number of 24DT22. Wild type corn plant was taken as a control and analyzed according to the processes as described above. Experiments were carried out in triplicate and the results were the mean values.
The specific method for detecting the copy number of PMI gene was described as follows:
The following primers and probes are used to detect the PMI nucleotide sequence:
PCR Reaction System:
The 50× primer/probe mixture containing 45 μL of each primer (1 mM), 50 μL of the probe (100 μM) and 860 μL of 1×TE buffer and was stored in an amber tube at 4° C.
PCR Reaction Conditions:
Use the SDS2.3 software (Applied Biosystems) to analyze data.
The experimental results showed that all the nucleotide sequences of 24DT22 nucleotide sequence and the control nucleotide sequence have been integrated into the genomes of the detected corn plants, respectively. Furthermore, all corn plants transformed 24DT22 nucleotide sequence and the control nucleotide sequence respectively contained single copy of 24DT22 gene.
Herbicide resistance effects tests to 2,4-D dimethyl ammonium salt and agroxone of corn plants containing 24DT22 nucleotide sequence, control nucleotide sequence respectively and wild type corn plants (stages V3-V4) were performed respectively.
Corn plants containing 24DT22 nucleotide sequence, control nucleotide sequence respectively and wild type corn plants were taken and spayed with 2,4-D dimethyl ammonium salt (8960 g ae/ha, 16-folds concentration in field), agroxone (8960 g ae/ha, 16-folds concentration in field) and blank solvent (water) respectively. Prop root development was counted 21 days after spaying. Three strains (S6, S7 and S8) of corn plants transformed with 24DT22 nucleotide sequence, two strains (S9 and S10) of corn plants transformed with control nucleotide sequence and 1 strain of wild type (CK2) corn were selected and 10-15 plants from each stain were tested. The results were shown in Table 3.
Results in Table 3 indicated that the 24DT22 gene conferred high resistance against herbicides to the transgenic corn plants, especially the phenoxy auxin herbicides (since the monocotyledon plants inherently have certain resistance to phenoxy auxin herbicides, high levels of resistance appeared); while none of the wild type of corn plants and the corn plants transformed with control sequences showed high levels of resistance against herbicides.
Above all, corn, soybean and Arabidopsis thaliana plants transformed with 24DT22 nucleotide sequence had high herbicide-resistance ability. Preferred codons of plant were employed in the herbicide-resistant gene 24DT22 in present invention, resulting that the herbicide-resistant gene of present invention is suitable to be expressed in plants. 24DT22 herbicide-resistant protein of present invention has a broad herbicide-resistance spectrum, especially phenoxy auxin herbicides.
Finally what should be explained is that all the above examples are merely intentioned to illustrate the technical solutions of present invention rather than to restrict present invention. Although detailed description of this invention has been provided by referring to the preferable examples, one skilled in the art should understand that the technical solutions of the invention can be modified or equivalently substituted while still fall within the spirit and scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2015 1 0078810 | Feb 2015 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2016/073182 | 2/2/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/127867 | 8/18/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 8283522 | Wright | Oct 2012 | B2 |
| 20110203017 | Wright et al. | Aug 2011 | A1 |
| 20130040815 | Lira et al. | Feb 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 103361316 | Oct 2013 | CN |
| 103826444 | May 2014 | CN |
| 104611308 | May 2015 | CN |
| 2009-513139 | Apr 2009 | JP |
| 2011107239 | Sep 2012 | RU |
| 2007053482 | May 2007 | WO |
| 2012075429 | Jun 2012 | WO |
| Entry |
|---|
| International Search Report from PCT/CN2016/073182; dated Apr. 21, 2016. |
| Australian Patent Office, Examination Report, Application No. 2016218739, dated Sep. 3, 2018. |
| Australian Patent Office, Examination Report, Application No. 2016218739, dated Nov. 19, 2018. |
| Canadian Patent Office, Examination Report, Application No. 2975773, dated Jul. 11, 2018. |
| Canadian Patent Office, Examination Report, Application No. 2975773, dated Apr. 11, 2019. |
| European Patent Office, Supplementary European Search Report, Application No. 167486687, dated Jun. 8, 2018. |
| European Patent Office, Examination Report, Application No. 167486687, dated Apr. 30, 2019. |
| Japan Patent Office, Examination Report, Application No. 2017-560856, dated Aug. 28, 2018. |
| Japan Patent Office, Examination Report, Application No. 2017-560856, dated Dec. 11, 2018. |
| New Zealand Intellectual Property Office, Examination Report, Application No. 734705, dated Jan. 26, 2018. |
| Russian Patent Office, Patent Search Report, Application No. 201712/10(049745), dated Jun. 28, 2018. |
| Russian Patent Office, Office Action, Application No. 201712/10(049745), dated Jun. 28, 2018. |
| Russian Patent Office, Office Action, Application No. 201712/10(049745), dated Oct. 17, 2018. |
| GenBank Accession AAP88277.1 “S-2,4-dichlorophenoxypropionate/alpha-ketoglutarate dioxygenase (plasmid) [Delftia acidovorans]”, Sep. 7, 2010. |
| GenBank Accession AY327575.2 “Delftia acidovorans plasmid pMC1, partial sequence”, Sep. 7, 2010. |
| Hogan et al., “Site-directed Mutagenesis of 2,4 Dichlorophenoxyacetic Acid/a-Ketoglutarate Dioxygenase”. vol. 275, No. 17, p. 12400-12409. |
| Hogan et al., Distribution of the tfdA Gene in Soil Bacteria That Do Not Degrade 2,4-Dichlorophenoxyacetic Acid (2,4-D), Microbial Ecology 34:90-96, 1997, 1 page. |
| Wright et al., “Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes” vol. 107, No. 47, Nov. 8, 2010, p. 20240-20245. |
| Schleinitz KM et al, “Localization and characterization of two novel genes encoding stereospecific dioxygenases catalyzing 2(2,4-dichlorophenoxy)propionate cleavage in Delftia acidovorans MC1”, Applied and Environmental Microbiology, 2004, 70(9):5357-5365. |
| Number | Date | Country | |
|---|---|---|---|
| 20180030470 A1 | Feb 2018 | US |
