Patent Application
20110088121
The invention relates to a nucleotide coding sequence and another nucleotide coding sequence artificially synthesized according to biased codons of plant. The invention also relates to the application of the said nucleotides in improving salt tolerance and drought tolerance of plant.
Deinococcus radiodurans (D. radiodurans) is one of the organisms having highest radiation resistance known so far. The bacteria has extremely high resistance to lethal dose of radiation ionizing, UV radiation and DNA damaging agent and is capable of repairing hundreds of genome which DNA double strands are broken by radiation ionizing without mutation. There are great interests on its capability of radiation resistance and DNA damage repairing mechanism in the scientific world, these features are meaningful for basic subject exploring DNA repairing mechanism and have potential application prospects in environment protection, bioremediation, human health, biotechnology, or even exploiting and development of the outer space. The Institute for Genomic Research (TIGR) completed and brought out the whole genome sequence of D. radiodurans in 1999.
There is no any report on Deinococcus radiodurans for improving salt tolerance and drought tolerance of plant in the art.
A aim of the invention is to find and artificially synthesize the DNA sequences for improving salt tolerance and drought tolerance of plant, transport the sequences into plant and grow the transgenic plant with salt and drought tolerance.
The inventors found the DNA sequences set forth in SEQ ID NO:1 and SEQ ID NO:2 all have improved salt and drought tolerance. Among others, the sequence of SEQ ID NO:1 is derived from a region of D. radiodurans; SEQ ID NO:2 is derived from SEQ ID NO:1 and artificially synthesized according to biased codons of plant.
The invention also provides a recombinant vector, comprising DNA set forth in SEQ ID NO:1 or DNA set forth in SEQ ID NO:2. The host cells are transformed with the said recombinant vector and the host cells include prokaryotic cell and eukaryotic cell. The prokaryotic host cells commonly used include JM109 and the eukaryotic host cells commonly used include yeast cells and other plant cells. In an embodiment of the invention, the host cells are E. coli JM109 and tobacco.
In a aspect of the invention, it also provides a method for generating polypeptide with the protein activity of SEQ ID NO:1 or SEQ ID NO:2, comprising the steps of:
(1) SEQ ID NO:1 or SEQ ID NO:2 is operatively linked to expression regulation sequence to form SEQ ID NO:1 or SEQ ID NO:2 protein expression vector;
(2) The expression vector of the step (1) is transported into host cells to form recombinant cells;
(3) The recombinant cells from the step (2) are cultured under the condition suitable for the expression of SEQ ID NO:1 or SEQ ID NO:2 protein polypeptide;
(4) The substantially pure polypeptides are isolated and the sequence is SEQ ID NO:3.
The invention further provides a method to transform SEQ ID NO:1 or SEQ ID NO:2 into plant using transgenic technology in order to improve salt tolerance and drought tolerance of plant, comprising the steps of:
(1) The sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 is operatively linked to plant expression regulation sequence to form plant expression vector;
(2) The expression vector of the step (1) is transformed into plant cells;
(3) The transformed cells are obtained via screening and they are ultimately regenerated into transgenic plants and progenies thereof, including seeds and tissues of plant.
The term “operatively linked to” as described above means, i.e. the activity of certain region of linear DNA sequence to influence the remaining region of the same linear DNA sequence. For example, DNA of signal peptide (secretion leader sequence) is operatively linked to DNA of polypeptide if DNA of signal peptide is expressed as precursor and participates in the secretion of the polypeptides; the promoter control sequence is operatively linked to the coding sequence if it is transcribed; the ribosome bind site is operatively linked to the coding sequence if it is located in the position making it to be translated. In general, the term “operatively linked to” means to be adjacent and to be adjacent in reading frame for the secretion leader sequence.
In one embodiment of the invention, the expression vector of step (1) is transformed into Agrobacterium-Agrobacterium containing the expression vector is co-cultured with eukaryotic host cells at 22-28, the transformed cells containing SEQ ID NO:1 or SEQ ID NO:2 gene are obtained via screening such as antibiotics screening after dark cultivation for 1-2 days, and the cells are regenerated into transgenic plants and progenies thereof, including seeds and tissues of plant.
It is confirmed that the transgenic plant as described above has improved salt and drought tolerance through experiments.
The vectors as described above may be selected from various vectors known in the art, such as commercially available vectors, including plasmid and cosmid and the like.
In addition, the invention further provides nucleic acid which can be used as probe, the molecule generally has 8-100 consecutive nucleotides of SEQ ID NO:1 or SEQ ID NO:2 nucleotide coding sequence, preferably 5-15 consecutive nucleotides. The probe may be used to detect whether there is nucleic acid molecules coding SEQ ID NO:1 or SEQ ID NO:2 in the samples.
The invention also provides a method for detecting whether there is nucleic acid molecules coding SEQ ID NO:1 or SEQ ID NO:2 in the samples, comprising the samples are hybridized with the probe as described above and then determined whether the probe is combined. Preferably, the samples are the product of PCR amplification, wherein the primers of PCR amplification correspond to SEQ ID NO:1 or SEQ ID NO:2 nucleotide coding sequence and may be located in flank and middle of the coding sequence□The length of primer is generally 15-50 nucleotides.
In the invention, “SEQ ID NO:1 or SEQ ID NO:2” refers to nucleotide sequence coding polypeptide with protein activity of SEQ ID NO:1 or SEQ ID NO:2 and degenerate sequence thereof. The degenerate sequence refers to the sequence where one and more codons are substituted with degenerate codons coding the same amino acid. The degenerate sequence with as low as about 89% homology with SEQ ID NO:1 or SEQ ID NO:2 also can code the sequence of SEQ ID NO:2. The term also includes the nucleotide sequence hybridized with the nucleotide sequence of SEQ ID NO:1 under moderate stringent conditions, preferably high stringent conditions. The term further includes the nucleotide sequence with 89% homology with the nucleotide sequence of SEQ ID NO:1, preferably at least 80%, more preferably at least 90%, most preferably at least 95%.
The term also includes variant for coding protein with the same function with natural SEQ ID NO:1 or SEQ ID NO:2, which is in the open reading frame sequence of SEQ ID NO:1. These variants include, but are not limited to, several (generally 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) nucleotides deletion, insertion and/or substitution, as well as adding several (generally within 60, preferably within 30, more preferably within 10, most preferably within 5) nucleotides at 5′ and/or 3′ end.
In the invention, the term “substantially pure” proteins or peptides refer to it account for at least 20% of the total substance of the sample, preferably at least 50%, more preferably at least 80%, most preferably at least 90% (the percentage all are on the basis of dry weight or wet weight). The purity can be measured by any suitable method, such as column chromatography, PAGE or HPLC. The substantially pure polypeptides substantially contain no component accompanied with it in the nature state.
In the invention, SEQ ID NO:3 protein or peptide refers to polypeptide with the activity of the protein coded by SEQ ID NO:1, also includes variant with the same function as SEQ ID NO:3. The variants include, but are not limited to, several (generally 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) amino acids deletion, insertion and/or substitution, as well as adding several (generally within 60, preferably within 30, more preferably within 10, most preferably within 5) amino acids at 5′ and/or 3′ end. For example, in the said proteins, the function of the protein generally is not varied when substitution is carried out with the amino acids with the close or similar function. Another example is the function of the protein generally may not be varied by adding one or more amino acids at C-terminus and/or N-terminus. The term also includes active fragment or active derivative of SEQ ID NO:3 protein.
The variants of SEQ ID NO:2 polypeptide of the invention include: homologous sequences, conservative variants, allelic variants, natural variants, induction variants, proteins coded by DNA hybridized with SEQ ID NO:1 or SEQ ID NO:2 under high or low stringent conditions and polypeptides and proteins obtained by using antiserum of SEQ ID NO:3. The invention also provides other polypeptides, such as fusion protein containing SEQ ID NO:3 polypeptides and fragments thereof. The invention also include soluble fragments of SEQ ID NO:3 polypeptides in addition to nearly full length polypeptides. The fragments generally have at least about 10 consecutive amino acids, generally at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids.
In the invention, “SEQ ID NO:3 conservative variant polypeptides” refer to the polypeptides have at most 10, preferably at most 8, more preferably at most 5 amino acids substituted with amino acids with close or similar property compared to the amino acid sequence of SEQ ID NO:3. The conservation variant polypeptides are prepared with the substitutions according to table 1 as below.
The invention also embraces analogs of the proteins or polypeptides of SEQ ID NO:3. The differences between these analogs and the natural polypeptide of SEQ ID NO:3 may be the differences between the amino acid sequences, or the differences between the modified versions that are not influencing the sequences, or both. These polypeptides include natural or induced genetic variant. The induced variants can be obtained by various technologies, such as random mutagenesis via radiation or exposing to mutagenic agent, or via site-directed mutagenesis or any other molecular biologic technology known in the art. The analogs also include analogs which have residues differing from natural L-amino acids (such as D-amino acids) and analogs which have non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the invention aren't limited to the representative polypeptides as described above.
The versions of modification (the primary structure is generally not modified) include: chemical derivatization version of polypeptides in vivo or in vitro, such as acetylation or acylation. The modifications also include glycosylation, such as the polypeptides produced by glycosylation during synthesis and processing or further processing steps of polypeptides. The modification can be accomplished by exposing the polypeptides to the enzymes for glycosylation (such as the glycosidase or the deglycosidase of mammals). The versions of modification also include a sequence with phosphorylated amino acid residues (phosphotyrosine, phosphoserine and phosphothreonine). A polypeptide modified to improve the ability of resisting hydrolysis or optimizing the solubility property is also included.
The expression of the gene products of SEQ ID NO:1 or SEQ ID NO:2 is analyzed using the Northern bolt method, i.e. to analyze whether the RNA transcripts of SEQ ID NO:1 or SEQ ID NO:2 present in the cells and amount thereof.
The Northern blot analysis of RNA of SEQ ID NO:1 or SEQ ID NO:2 and the Western blot analysis of the specific antibody of SEQ ID NO:3 can be combined, to confirm the expression of SEQ ID NO:1 or SEQ ID NO:2 in a biological sample.
In addition, genes or proteins which are homologous with SEQ ID NO: 1 or SEQ ID NO:2 may be screened according to the nucleotide sequences and the amino acid sequences of the invention, on the basis of homology of nucleic acids and proteins expressed.
To obtain dot matrix of D. radiodurans cDNA that is related to the gene of SEQ ID NO:1 or SEQ ID NO:2, D. radiodurans cDNA may be screened using DNA probe, and these probes are produced by radioactively labeling the whole or partial SEQ ID NO:1 or SEQ ID NO:2 with 32P under the low stringent condition. The most suitable cDNA library to be screened is from D. radiodurans library. The methods for constructing cDNA library from interested cells or tissues are well known in the field of molecular biology. In addition, many of such libraries may be commercially available, for example, from Clontech, Stratagene, Palo Alto, Cal. The screening methods can recognize a nucleotide sequence of gene family which is related to SEQ ID NO:1 or SEQ ID NO:2.
Once the related sequences are obtained, the related sequences can be obtained in a large scale using recombinant method. It is generally cloned into a vector, transformed into a cell and then the related sequences are isolated from the host cells after proliferation by conventional method.
In addition, the related sequences are also synthesized by artificial chemical synthesis method. A plurality of small fragment of polynucleotide are firstly synthesized and then they are linked to give the nucleotide sequences coding for SEQ ID NO:2 protein of D. radiodurans of the invention according to the prior art prior to the invention. The nucleotide sequences can be subsequently introduced into various exiting DNA molecules (such as a vector) and cells in the art. Also, the mutation can be introduced into the protein sequences of the invention by chemical synthesis.
The fragments of the protein of the invention can be produced through solid phase method and direct peptide synthesis (Stewart et al. (1969), Solid-Phase Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am. Chem. Soc 85:2149-2154) in addition to recombinant method. The synthesis of protein in vitro may be performed by hand or automatically. For example, the peptides can be synthesized using 431A Model Peptide Synthesizer from Applied Biosytems (Foster City, Calif.). The respective fragments of the protein of the invention can be chemically synthesized and then they are linked into full length molecular by chemical methods.
The invention will be further described by the following examples. It should be understood that the examples are intended to illustrate the methods of the invention and aren't intended to limit the scope of the invention. All experiment conditions not described are according to the conventional conditions well known in the art, for example the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufactures.
The published genome sequences of Deinococcus radiodurans relate to a pair of PCR specific primers and the whole nucleotide sequences are amplified from the genomic DNA of Deinococcus radiodurans.
2. Construction of E. coli Expression Vector and Molecular Verification
The cloned fragments as described above are digested with two enzymes, NdeI and SacII, and linked to vector pTtSacB containing E. coli universal promoter groE to replace SacB gene, in order to produce E. coli expression vector for the genes. E. coli JM109 is transformed, plated onto LB solid medium containing Amp, the white clones are selected, the plasmids are extracted using alkaline lysis to screen the different recombinants, digested with Bgl II and verified by sequencing, one strain of E. coli JM109 containing the nucleotide sequence expression vector is obtained. The analysis of enzyme cutting shows the gene fragment containing groE promoter is 1.2 kb.
3. Verification of Salt and Drought Tolerance of E. coli Expression Vector
E. coli JM109 strain containing SEQ ID NO:1 nucleotide sequence expression vector, pMD18T and host strain E. coli JM109 as controls, the OD value of which are all same, are inoculated into MM medium with 0.75M NaCl in 1% inoculum size respectively, shaking cultured at 37° C. for 15 hours, and OD value is detected at 550 nm. From
According to the known SEQ ID NO:1 nucleotide sequence, it is firstly divided into 7 regions, and then single strand oligonucleotide fragments in 150-200 bp length and with cohesive terminus are synthesized according to the positive strand and the negative strand, respectively. The 7 complementary single strand oligonucleotide fragments corresponding to the positive strand and the negative strand are annealed to form 7 double strands oligonucleotide fragments with cohesive terminus. The double strands oligonucleotide fragments are combined, and assembled catalytically into a whole gene via T4 ligase. The two ends of the synthesized DNA fragments contain XbaI and Sad sites.
SEQ ID NO:2 which is artificially synthesized as described above and 5′ and 3′ end enzyme cutting sites of which are XbaI and Sad is used to construct the plant expression vector with high salt and drought tolerance as described below.
Primers for amplifying the whole coding reading frame are designed according to the full length coding sequence (SEQ ID NO:2), and the cutting sites of restriction enzyme are introduced into the positive and negative primers (depending to the vectors selected), in order to construct the expression vector. The amplified product from example 1 is used as template, the sequence of cDNA is cloned into intermediate vector (such as pBluescript) after PCR amplification, and then it is further cloned into binary expression vector, such as pBI121 and pCAMBIA2200, the good expression vector is identified under the premise of ensuring the reading frame, it is transformed into Agrobacterium, and the model plant tobacco is transformed using leaf disc cocultivation.
1. Pick the positive clone on the selective plate using sterile toothpick, seed into 2 ml YEB liquid (Sm+, Kan+), shaking culture for 24-36 hours at 28° C., 200 rpm;
2. Centrifuge at 4,000 g at room temperature for 10 min;
3. Discard the supernatant, suspend the bacteria with ½ MS medium, and dilute to 5-20 times as the initial volume to make the OD600 of liquid to be about 0.5;
4. Take a sterile leaf of tobacco grown for about two weeks, remove the main vein, cut it into small pieces of about 1 cm2;
5. Place the leaf pieces into the liquid containing the bacteria prepared, immerse for 2-5 min, and suck off the liquid on the sterile filter paper;
6. Place the infected leaf pieces onto the MS medium and culture for 48 hours at 28° C. in the dark;
7. Transfer the leaf pieces onto the Callus medium (MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+Kan 50 mg/L+cb 250 mg/L), culture at 25-28° C. in the light and observe the formation of the callus tissues after 7-15 days;
8. Observe the differentiated buds coming out after about 20 days, cut the buds after it grown up, put them into the root medium (½ MS+NAA 0.5 mg/L+Kan 25 mg/L) to perform rooting culture and observe the roots after about 2-7 days;
9. Remove the plant after the root system is large, wash off the attached solid medium with sterile water, transfer it into the soil, cover it with a glass cover for several days at first, remove the cover after the plant is robust and plant it in the greenhouse.
1. Extract RNA: refer to “molecular cloning” (Sambrook et al., 1989).
2. Quantify RNA: refer to “molecular cloning” (Sambrook et al., 1989), measure the OD260 using spectrophotometer, calculate the amount of RNA: 1 OD260=40 μg/ml.
3. Isolate the total RNA though agarose gel electrophoresis: 1) take 6 ml 25*electrophoretic buffer, add 117 ml sterile water into it and mix. 2) weight 1.5 g agarose into the solution as above, heat it to melt in the micro oven, and transfer it to the water bath at 55° C. 3) take 26.8 ml formaldehyde in a fume cupboard, add it into the gel solution at 55° C. and mix. 4) pour it rapidly into the plate for making gel, stand horizontally at the room temperature for 30 mins to make the gel set. 5) allow 30 μg extracted RNA to dissolve into 15 ml RNA dilute solution, heat it at 55-65° C. for 10 min and then immediately put it onto the ice. 6) add 2 μl 10*loading buffer into the sample and mix. 9) dot the sample under the condition that the electrophoresis buffer doesn't cover the gel, allow it to electrophorese at 80 v for 10 min, add the electrophoresis buffer to exceed the surface of the gel about half-centimeter after the sample comes into the gel totally. Allow it to electrophorese at 80-100V for 5 hours.
4. Transfer RNA onto the nylon membrane: 1) immerse the nylon membrane into 10*SSC before the transfer. 2) Put the wetted membrane correctly onto the membrane, immerse two pieces of filter paper as big as the membrane into 2*SCC solution to wet, put them onto the membrane and exclude air bubbles. 3) Place a pile of cleaning paper as big as membrane onto the filter paper, put a piece of glass and a heavy object onto the cleaning papers, stand horizontally to transfer for 12-20 hours. 4) Dry the membrane at 80° C. for 1-2 hours after transfer.
5. Detect RNA on the membrane: 1) immerse the membrane in 4*SSC for 10 min, remove the membrane to put it onto the filter paper to suck the excess liquid, put the membrane into prehybridization solution (50% formamide, 5*SSC, 50 mmol/L sodium phosphate (Ph 6.4), 5*Dendart 0.1% SDS, 0.1 mg/ml salmon sperm DNA), hybridize at 42° C. overnight. 2) pour out the prehybridization solution, replace it with the same volume of hybridization solution, put DNA probe labeled with 32P into the boiling water to denature for 5 min, add hybridization solution (50% formamide, 5*SSC, 50 mmol/L sodium phosphate (Ph 6.4), 10% dextran sulfate, 5*Dendart 0.1% SDS, 0.1 mg/ml salmon sperm DNA), hybridize at 42° C. for 24-48 hours. 3) remove the membrane, put it into wash buffer I (1*SSC, 1% SDS), wash three times at 42° C., 5 min every time. Transfer it into wash buffer II (0.1*SSC, 1% SDS), wash one to three times at 55-65° C. Press the X-ray film onto the membrane for 1-7 days, develop the film and fix.
The salt and drought tolerance of transgenic plant is further identified in view the fact that the sequence is confirmed to have salt tolerance in E. coli.
The transgenic tobacco and non-transgenic tobacco are cultured in the medium with 0 mmol or 250 mmol NaCl to investigate the viability and development of the plant by 5d, 10d and 15d observation. From
| Number | Date | Country | Kind |
|---|---|---|---|
| 200710176153.1 | Oct 2007 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2007/071043 | 11/9/2007 | WO | 00 | 12/30/2010 |
