Patent Application
20240254506
The sequence listing provided in the file entitled C6351-123_PUS1232680_2.xml, which is an Extensible Markup Language (XML) file that was created on Apr. 17, 2024, and which comprises 24,850 bytes, is hereby incorporated by reference in its entirety.
The present invention relates to the technical field of genetic engineering, in particular to DMP protein, encoding gene and application thereof.
Haploid breeding has become one of the important methods for breeding new plant varieties, while improving the haploid induction rate and simplifying the haploid induction procedure are the critical procedures for haploid breeding technology. With the development and improvement of haploid induction technology, haploid breeding technology has been widely used in the breeding research of many important crops, showing the advantages of allowing rapid homozygosity of a gene, breeding time shorten, high breeding efficiency, etc. Legume plants are important economic crops, and the in vivo haploid induction system has not been developed yet. If haploid breeding can be achieved, it will have a wide application prospect in agricultural production.
Medicago truncatula, as a model plant of Leguminosae, has the common characteristics of Leguminosae. Therefore, it is of great application value to study haploid induction mechanism of Medicago truncatula and develop an in vivo haploid induction system suitable for Leguminosae.
The present invention aims to provide DMP proteins and encoding genes and applications thereof.
In a first aspect, the present invention claims a set of protein.
The set of proteins claimed by the present invention consists of protein A and protein B. The protein A and the protein B are both derived from Medicago truncatula, designated as DMP8 and DMP9, respectively.
The protein A (i.e., DMP8) may be any one of the followings:
The protein B is any one of the followings:
The above proteins may be synthesized artificially or obtained by synthesizing the encoding genes thereof and followed by biologically expression.
Of the above proteins, the tag refers to a polypeptide or protein that is expressed together in fusion with a target protein by using DNA in vitro recombination, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag and the like.
Of the above proteins, the identity refers to the identity to an amino acid sequence. The identity to the amino acid sequence may be determined using the homology search site on the international internet, such as BLAST webpage on the NCBI homepage website. For example, the value of identity (%) may be obtained by retrieving the identity of a pair of amino acid sequences for calculation in the advanced BLAST2.1, using blast program, setting the Expect value to 10, all Filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, Per residual gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively.
Of the above proteins, the identity of 99% or more may be an identity of at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%. The identity of 95% or more may be an identity of at least 96%, 97%, 98%. The identity of 90% or more may be an identity of at least 91%, 92%, 93%, 94%. The identity of 85% or more may be an identity of at least 86%, 87%, 88%, 89%. The identity of more than 80% may be an identity of at least 81%, 82%, 83%, 84%.
In a second aspect, the present invention claims a set of nucleic acid molecules.
The set of nucleic acid molecules claimed by the present invention consists of a nucleic acid molecule A and a nucleic acid molecule B.
The nucleic acid molecule A is a nucleic acid molecule capable of expressing the protein A described above; The nucleic acid molecule B is a nucleic acid molecule capable of express the protein B described above.
The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; The nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
Further, the nucleic acid molecule A (designated MtDMP8) may be a DNA molecule shown in any one of the followings:
Of the above nucleic acid molecules, the stringent conditions may be as follows: hybridizing in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by rinsing in 2×SSC and 0.1% SDS at 50° C.; alternatively, hybridizing in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by rinsing in 1×SSC and 0.1% SDS at 50° C.; alternatively, hybridizing in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by rinsing in 0.5×SSC and 0.1% SDS at 50° C.; alternatively, hybridizing in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by rinsing in 0.1×SSC and 0.1% SDS at 50° C.; alternatively, hybridizing in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA at 50° C., followed by rinsing in 0.1×SSC and 0.1% SDS at 65° C.; alternatively, hybridizing in a solution of 6×SSC and 0.5% SDS at 65° C., followed by washing the membrane once each in 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.
Of the above nucleic acid molecules, the homology refers to the identity to the nucleotide sequence. The identity to the nucleotide sequence may be determined using the homology search site on the international internet, such as BLAST webpage on the NCBI homepage website. For example, the value of identity (%) may be obtained by retrieving the identity of a pair of nucleotide sequences for calculation in the advanced BLAST2.1, using blast program, setting the Expect value to 10, all Filters to OFF, using BLOSUM62 as Matrix, and setting Gap existence cost, Per residual gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively.
Of the above nucleic acid molecules, the homology of 95% or more may be an identity of at least 96%, 97%, and 98%. The homology of 90% or more may be an identity of at least 91%, 92%, 93%, and 94%. The homology of 85% may be an identity of at least 86%, 87%, 88%, and 89%. The homology of 80% or more may be an identity of at least 81%, 82%, 83%, and 84%.
In a third aspect, the present invention claims any of the following biomaterials:
In a specific embodiment of the present invention, P5-P7, the target sequences of the sgRNA molecule A are SEQ ID No. 5 and SEQ ID No. 6. The target sequences of the sgRNA molecule B are SEQ ID No. 7 and SEQ ID No. 8. The CRISPR-Cas9 knockout vectors are MtCRISPR/CaS9::MtDMP8, MtCRISPR/CaS9::MtDMP9 and/or MtCRISPR/CaS9::MtDMP8MtDMP9.
In a fourth aspect, the present invention claims the application of a set of proteins according to the first aspect described above or a set of nucleic acid molecules according to the second aspect described above or a set of biomaterials according to the third aspect described above in any of the followings:
In the applications, the expression and/or activity of the set of proteins (i.e. the protein A and the protein B as described above) in the plant is reduced (e.g., by causing the translation of the corresponding protein to terminate prematurely) and the haploid may be obtained from offsprings of the resulting positive plants by self-crossing or hybridization.
In a fifth aspect, the present invention claims a method of constructing a plant haploid induction line.
The method of constructing a plant haploid induction line as claimed in the present invention may include the steps of: reducing the expression and/or activity of both the aforementioned protein A and the protein B in the recipient plant (e.g., by causing the translation of the corresponding protein to terminate prematurely) and the haploid may be obtained from offsprings of the resulting positive plants by self-crossing or hybridization.
Further, the method may include the steps of: depressing expression of both the nucleic acid molecule A and the nucleic acid molecule B as described above in the recipient plant to obtain a transgenic plant; and obtaining a haploid induction line from self-crossed or hybridized offsprings of the transgenic plant.
Wherein depressing expression of both the nucleic acid molecule A and the nucleic acid molecule B in the recipient plant as described hereinabove may be achieved by any technical means include, but not limited to, knockout of the nucleic acid molecule A and the nucleic acid molecule B in the recipient plant using the CRISPR-Cas9.
For example, it may be achieved by introducing into the recipient plant the set of CRISPR-Cas9 system in P6 or the CRISPR-Cas9 knockout vector in P7 (due to the insertion or deletion of nucleotides, the reading frame is shifted and the translated protein is terminated prematurely) of the previous third aspect.
The hybrid offspring is obtained by crossing the transgenic plant with other varieties of the plant.
Specifically, the method may include the steps of:
Wherein the functional deletion mutation refers to the occurrence of a terminator or reading frame shift at the target site in the natural encoding sequences for MtDMP8 and MtDMP9.
The Cas9 nuclease expression frame is located in the same vector containing the sgRNA expression frame.
In step (3), the binary expression vectors MtCRISPR/Cas9::MtDMP8, MtCRISPR/Cas9::MtDMP9 and MtCRISPR/Cas9::MtDMP8 MtDMP9 are introduced into the target graft cells so that the cells contain both the sgRNA and the Cas9 nuclease, of the target fragment of step. Under the joint action of the sgRNA and the Cas9 nuclease, the double-stranded target fragments of the MtDMP8 and MtDMP9 genes are cut, and then through the DNA repair function of the target plant cell itself, the random insertion and/or random deletion of the target fragments of MtDMP8 and MtDMP9 genes are finally realized in the cells.
The method of introducing the recombinant vector into the target plant cell is Agrobacterium-mediated stable transformation of callus. It is because the Agrobacterium-mediated method is utilized in the process of introducing the obtained recombinant vector into the cell of the target plant, the recombinant vector is introduced into the genetic DNA of the target plant, the fragment of the genetic DNA of the target plant is cut when cutting is performed.
In the present invention, the method for regenerating a plant is to obtain a plant by tissue culture of cells or tissues.
In step (5), the DNA fragments of the MtDMP8 and MtDMP9 genes containing the target fragments in the regenerated plant may be cloned by genomic PCR, and the targets of the amplified products are subjected to next generation sequencing. The method genomic PCR comprises the following steps of: designing site-specific primers for the genomic region containing the target fragment, and amplifying the genomic region containing the target fragment by taking genomic DNA of a regenerated plant as a template.
In a sixth aspect, the present invention claims application of the method described in the fifth aspect described above in plant haploid breeding.
In a seventh aspect, the present invention claims a plant haploid induction line constructed using the method described in the preceding fifth aspect.
In all of the above aspects, the plant may be a leguminous plant.
Further, the plant may be an alfalfa plant.
In a specific embodiment of the present invention, the plant is specifically Medicago truncatula, and more specifically, Medicago truncatula R108. Correspondingly, the hybrid offspring mentioned above is the hybrid offspring of the positive plant obtained by knocking out the DMP8 and DMP9 genes in Medicago truncatula R108 and Medicago truncatula A17.
The following examples facilitate a better understanding of the present invention but do not limit it. Methods of the experiments in the following examples are conventional methods unless otherwise specified. The test materials used in the examples below, unless otherwise specified, were all commercially available from the conventional biochemical reagent store. The quantitative tests in the following examples were repeated three times and the results were averaged.
Medicago truncatula R108 was provided by The Nobel Foundation (Available at: https://www.nobelprize.org/the-nobel-prize-organisation/the-nobel-foundation/).
Agrobacterium tumefaciens AGL1 was provided by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences (i.e., the Applicant's workplace) and is available to the public from the Applicant.
YEP liquid medium: 10 g of peptone, 10 g of yeast extract and 5 g of sodium chloride were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water, and autoclaved at 121° C. for 15 min.
Liquid medium for callus induction: 100 mL of mother liquor of macroelements, 1 mL of mother liquor of trace elements, 1 mL of mother liquor of organic elements, 20 mL of mother liquor of iron salts, 100 mg of inositol, 30 g of sucrose, 4 mg of auxin, and 0.5 mg of cytokinin were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water, adjusted to pH 5.8, and autoclaved at 121° C. for 15 min.
Solid medium for callus induction: 100 mL of mother liquor of macroelements, 1 mL of mother liquor of trace elements, 1 mL of mother liquor of organic elements, 20 mL of mother liquor of iron salts, 100 mg of inositol, 30 g of sucrose, 4 mg of auxin, 0.5 mg of cytokinin, 200 mg of cephalosporin, 250 mg of Timentin, 2 mg of kanamycin and 3.2 g of Phytagel were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water, adjusted to pH 5.8, and autoclaved at 121° C. for 15 min.
Medium for cell differentiation: 100 mL of mother liquor of macroelements, 1 mL of mother liquor of trace elements, 1 mL of mother liquor of organic elements, 20 mL of mother liquor of iron salts, 100 mg of inositol, 20 g of sucrose, 200 mg of cephalosporin, 250 mg of Timentin, 2 mg of kanamycin and 3.2 g of Phytagel were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water, adjusted to pH 5.8, and autoclaved at 121° C. for 15 min.
Medium for rooting: 2.215 g of Murashige&Skoog Basal Medium with Vitamins (a product available from PhytoTechnology Laboratories, catalog number of 16B0519138A) was dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water, adjusted to pH 5.8, and autoclaved at 121° C. for 15 min.
Mother liquor of iron salts: 37.3 mg of disodium ethylenediamine tetraacetic acid and 27.8 mg of ferrous sulfate heptahydrate were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water.
Mother liquor of macroelements: 1.85 g of magnesium sulfate heptahydrate, 28.3 g of potassium nitrate, 4.63 g of ammonium sulfate, 1.66 g of calcium chloride dihydrate and 4 g of potassium dihydrogen phosphate were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water.
Mother liquor of trace elements: 1 g of manganese sulfate monohydrate, 500 mg of boric acid, 100 mg of zinc sulfate heptahydrate, 100 mg of potassium iodide, 10 mg of sodium molybdate dihydrate, 20 mg of copper sulfate pentahydrate and 10 mg of cobalt chloride hexahydrate were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water.
Mother liquor of organic elements: 500 mg of nicotinic acid, 500 mg of thiamine hydrochloride and 500 mg of pyridoxine hydrochloride were dissolved in an appropriate amount of distilled water, followed by volume adjustment to 1 L with distilled water.
Formula of the Alexander staining solution: 5 mL of 95% ethanol, 500 μL of 1% malachite green, 2.5 mL of 1% acid fuchsin, 250 μL of 1% orange G, 12.5 mL of glycerol, and 2 mL of glacial acetic acid. The volume was then fixed to 50 mL with distilled water.
Formula of the LB01 buffer: 1M tris(hydroxymethyl) aminomethane (pH 7.5) 1.5 mL, 0.5M ethylened diamine tetra-acetic acid (pH 8.0), 8 mL of 1M potassium chloride, 400 μL of 5M sodium chloride, 5 mM spermine tetrahydrochloride, 200 μL of β-mercaptoethanol, and 100 μL of polyethylene glycol octyl phenyl ether.
Sequencing results showed that the intermediate vector for the DMP8 gene contained the DNA molecule shown in SEQ ID No. 3. The DNA molecule shown in SEQ ID No. 3, i.e., the DMP8 gene, encodes DMP8 protein shown in SEQ ID No. 1. The intermediate vector for the DMP9 gene contains the DNA molecule shown in SEQ ID No. 4. The DNA molecule shown in SEQ ID No. 3, i.e., the DMP9 gene, encodes the DMP9 protein shown in SEQ ID No. 2.
Among them, the two targets to the MtDMP8 gene were:
Two targets to the MtDMP9 gene were:
The detailed information on each primer is as follows:
The vectors that have been verified to be correct by sequencing were named MtCRISPR/Cas9::MtDMP8, MtCRISPR/Cas9::MtDMP9 and MtCRISPR/Cas9::MtDMP8MtDMP9.
The MtCRISPR/Cas9::MtDMP8MtDMP9 binary vector was introduced into Agrobacterium tumefaciens AGL1 to obtain a recombinant Agrobacterium, named AGL1/MtCRISPR/Cas9::MtDMP8MtDMP9.
The MtCRISPR/Cas9::MtDMP8 binary vector was introduced into Agrobacterium tumefaciens AGL1 to obtain a recombinant Agrobacterium, named AGL1/MtCRISPR/Cas9::MtDMP.
The MtCRISPR/Cas9::MtDMP9 binary vector was introduced into Agrobacterium tumefaciens AGL1 to obtain a recombinant Agrobacterium, named AGL1/MtCRISPR/Cas9::MtDMP9.
Sequencing confirmed the availability of the single mutant dmp8, the single mutant dmp9, and the double mutant dmp8dmp9 (due to the insertion or deletion of nucleotides, the reading frame is shifted and the translated protein is terminated prematurely).
Alexander staining of pollen was performed as follows:
The results are shown in
The self-crossed offsprings of the dmp8, dmp9, and dmp8 dmp9 mutants were analyzed by flow cytometry. The procedure for flow cytometry analysis of DNA content in the nucleus was as follows:
The results showed that no haploid was observed in the self-crossed offsprings of the single mutant dmp8, the single mutant dmp9, and the wild-type R108. In contrast, haploid plants were obtained from the self-crossing offsprings of the double mutant dmp8 dmp9, which were then subjected to phenotypic analysis, and the results were shown in
VI. Phenotype Analysis of the Haploid from Hybridized Offsprings
The dmp8 dmp9 double mutant was hybridized with Medicago truncatula A17, and the hybridized offsprings were analyzed by flow cytometry. The results showed that the it may induce the haploid material of maternal origin by crossing the dmp8 dmp9 double mutant with their parents of different ecotypes of Medicago truncatula, and the haploid plant growth was consistent with the leaf phenotype of A17 plant. The results were shown in
By the method of designing the sgRNAs specifically targeting the genes encoding DMP8 and DMP9 of the Medicago truncatula, followed by utilizing the CRISPR-Cas9 system for knocking out the genes of DMP8 and DMP9 of the Medicago truncatula, a haploid induction system of the Medicago truncatula was created in the present invention. The present invention has great significance for haploid breeding of leguminous plants, and can effectively shorten the breeding period.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111225771.7 | Oct 2021 | CN | national |
The present application is a Continuation of International Application Number PCT/CN2022/140182 filed Dec. 20, 2022, which claims priority to Chinese Application Number 202111225771.7, filed Oct. 21, 2021, the disclosures of which are hereby incorporated by reference herein in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/140182 | Dec 2022 | WO |
| Child | 18640590 | US |
