Application of cotton 3-methyl-2-oxobutyric acid hydroxymethyltransferase gene in regulation and control of high temperature resistance of plants

Abstract

The invention discloses an application of a cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in regulation and control of high temperature resistance of plants, belongs to the technical field of molecular breeding of plants, and provides an application of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in regulation and control of high temperature resistance of plants. The nucleotide sequence of the cotton 3-methyl-2-oxobutyric acid hydroxymethyltransferase gene is as shown in SEQ ID NO. 6. The invention also discloses a cotton 3-methyl-2-oxobutyric acid The GhKPHMT gene for coding 3-methyl-2-oxobutyric acid hydroxymethyltransferase is cloned on the high-temperature-resistant gene locus qPV-D12 of the upland cotton D12 chromosome by analyzing the genetic characteristics of the high-temperature-resistant locus of the upland cotton, and participates in controlling the high-temperature resistance of cotton pollen; an overexpression transgenic line is created in upland cotton, rice and arabidopsis thaliana by utilizing an overexpression technology, and the effect and potential application capability of the upland cotton GhKPHMT gene in creating high-temperature-resistant materials in different species are proved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular breeding, and in particular relates to the application of cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in regulating plant high temperature resistance. Background Art

[0002] Upland cotton (Gossypium hirsutum.L) produces more than 90% of my country's natural textile fibers. As an important cotton producing area, Xinjiang cotton area frequently encounters extreme high temperature weather exceeding 35°C in summer, which leads to high temperature male sterility and causes a large number of buds and bolls to fall off during the concentrated boll-forming period. However, due to the complex high temperature resistance of cotton, the difficulty in material screening, and the inaccurate gene identification, there is a practical bottleneck in the current breeding of cotton for high temperature resistance. In order to solve the above-mentioned industrial problems, the patent with the prior art publication number CN118186135A has established a scheme for rapid identification of cotton pollen vitality under high temperature, and identified four high temperature resistance loci in the upland cotton breeding population, namely qPV-A01, qPV-D01, qPV-D05 and qPV-D12, and hopes to analyze the genetic variation and effects in the high temperature resistance loci and then determine the high temperature resistance genes. Through linkage analysis based on artificial populations and candidate gene association analysis, the effect block of the qPV-A01 high temperature resistance gene locus was determined. The patent with publication number CN118345099A announced that the high temperature resistance gene therein is serine / threonine kinase GhHRKs. The high temperature resistance genes in other loci still need to be studied and developed. Summary of the invention

[0003] In order to solve the above technical problems, the present invention proposes the application of cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in regulating plant high temperature resistance. By analyzing the genetic characteristics of the high temperature resistance site of upland cotton, a GhKPHMT gene encoding 3-methyl-2-oxobutyrate hydroxymethyltransferase located on the high temperature resistance gene locus qPV-D12 of upland cotton D12 chromosome was cloned. The gene is involved in controlling the high temperature resistance of cotton pollen. Overexpression transgenic lines were created in upland cotton, rice and Arabidopsis using overexpression technology, which proved the effect and potential application ability of the upland cotton GhKPHMT gene in creating high temperature resistant materials in different species.

[0004] To achieve the above object, the present invention provides the use of cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in regulating plant high temperature resistance, and the nucleotide sequence of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene is shown in SEQ ID NO.6.

[0005] Preferably, the high temperature resistance of the plant is improved by overexpressing the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene.

[0006] Preferably, the plant is one or more of cotton, rice or Arabidopsis thaliana.

[0007] The present invention also provides the use of an expression box, a vector or a recombinant bacterium containing the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in regulating plant high temperature resistance.

[0008] The present invention also provides the application of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in cultivating high temperature resistant transgenic plants.

[0009] The present invention also provides application of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in improving plant high temperature resistant germplasm resources.

[0010] The present invention also provides the use of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in improving pollen vitality of plants under high temperature environment.

[0011] Preferably, the plant is one or more of cotton, rice or Arabidopsis thaliana.

[0012] The present invention also provides a method for improving the high temperature resistance of plants by utilizing the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene, wherein the plant expresses or overexpresses the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene by transgenic, hybridization, backcrossing, selfing or asexual reproduction. The nucleotide sequence of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene is shown in SEQ ID NO.6.

[0013] Preferably, the transgenic comprises introducing a recombinant expression vector containing cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene into the plant using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method to obtain transgenic plants.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects:

[0015] The present invention provides the application of cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase (GhKPHMT) gene in regulating plant high temperature resistance, and publishes the high temperature resistance gene in the qPV-D12 block, the gene ID is Ghir_D12G013040, and the gene encodes a 3-methyl-2-oxobutyrate hydroxymethyltransferase gene. Based on the GhKPHMT gene model and reference sequence, the gene editing technology was used to create an allele type with gene function loss, proving that GhKPHMT positively regulates high temperature resistance and is an important gene involved in organ development. The use of gene editing methods cannot produce favorable genotypes and materials that are conducive to industrial development. The present invention uses overexpression methods to create genetic materials in upland cotton, rice and Arabidopsis, proving that overexpression of GhKPHMT can provide considerable high temperature resistance for male gametes of the above species.

[0016] The present invention discloses the resistance gene GhKPHMT at the qPV-D12 locus of upland cotton, enriching the gene resources of upland cotton resistance to high temperatures. Targeting the GhKPHMT gene, using a suitable genetic transformation vector, genetic materials resistant to high temperatures were created in cotton, rice and Arabidopsis, providing a candidate solution for using exogenous genes to improve the high temperature resistance of own species, and effectively promoting the breeding process of high temperature resistant germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The analysis results of the qPV-D12 gene locus and genetic effect determined in Example 1, wherein A is a Manhattan plot based on the whole genome association analysis of a natural population of upland cotton, and B is a QQ plot corresponding to the whole genome association analysis;

[0019] Figure 2 is the result of gene editing of the causal gene GhKPHMT of the qPV-D12 locus in Example 1, wherein A is the anther organ phenotype of the offspring, B is the gene-edited genotype, and C is the phenotype of the plant in the field;

[0020] Figure 3 The results of the effect of the expression of the GhKPHMT gene on the high temperature resistance of upland cotton powder in Example 1, wherein A is the vigor of upland cotton powder, B is the expression of the GhKPHMT gene, and C is the electrophoresis diagram;

[0021] Figure 4These are the results of the effect of GhKPHMT gene overexpression on plant high temperature resistance in Example 1, wherein A is GhKPHMT gene overexpressed upland cotton, B is GhKPHMT gene overexpressed Arabidopsis, and C is GhKPHMT gene overexpressed rice. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1

[0028] I. Identification of the heat resistance locus of qPV-D12 in upland cotton and discovery of the GhKPHMT gene

[0029] Among the more than 3000 published upland cotton germplasms (reference He, S., G. Sun, X. Geng, W. Gong, P. Dai, Y. Jia, W. Shi, Z. Pan, J. Wang, L. Wang, S. Xiao, B. Chen, S. Cui, C. You, Z. Xie, F. Wang, J. Sun, G. Fu, Z. Peng, D. Hu, L. Wang, B. Pang & X. Du (2021) The genomic basis of geographic differentiation and fiber improvement in cultivated cotton. Nat 517 core germplasms were selected from the G. hirsutum Genet, 53, 916-924.) as the material basis, and pollen viability after high temperature stress was used as the phenotype (refer to CN109752226B). Three heat resistance gene loci, qPV-A01-1, qPV-D01-1 and qPV-D12-1, were identified in the G. hirsutum genome by genome-wide association analysis (refer to CN109752226B). Figure 1 Middle A and Figure 1 As shown in B), the qPV-A01-1 locus has been disclosed, and the causal gene is the S-locus protein gene GhHRKs (refer to CN118345099A). The present invention analyzes the undisclosed qPV-D12-1 locus, uses CRISPR-Cas9 gene editing technology, batch synthesizes sgRNA, and performs batch gene editing on candidate genes in the qPV-D12-1 locus. Figure 2 As shown in A, NT represents normal temperature and HT represents high temperature. Among the gene-edited offspring, the transgenic offspring GhKPHMT-CR1, GhKPHMT-CR2 and GhKPHMT-CR3 targeting the Ghir_D12G13040 gene all showed severe developmental defects compared to the transgenic receptor Jin668 (wild-type plant control), manifested as abnormal development of floral organs and pollen sterility.

[0030] Table 1 PCR amplification system

[0031] Components Dosage cDNA template (diluted 100 times) 10μL 2×Buffer 20μL Forward primer F (10mM) 0.5μL Reverse primer R (10mM) 0.5μL dNTP(10mM) 0.6μL DNA polymerase (10U / μL) 0.4μL Double distilled water Make up to 40 μL

[0032] Note: Table 1 is Vazyme Phanta DNA Polymerase Kit #P505 (Nanjing Novozyme Biotechnology Co., Ltd., Vazyme).

[0033] Table 2 PCR amplification program

[0034]

[0035] Table 3 PCR product ligation system

[0036] Components volume 5×Ultra-Universal TOPO Cloning Mix 1μL PCR products 1.5μL Double distilled water Make up to 5 μL (react at 25℃ for 10-15 min)

[0037] Note: Table 3 is Ultra-Universal Topoisomerase Cloning Kit #C603 (Nanjing Novogene Biotech Co., Ltd., product name is Ultra-Universal TOPO Cloning Kit, product number is C603).

[0038] Through sgRNA typing, it was confirmed that the nucleotide sequence of the sgRNA targeting the Ghir_D12G13040 gene is shown in SEQ ID NO.1. This example uses the PCR amplification system shown in Table 1 and the PCR amplification program shown in Table 2. The nucleotide sequences of the upstream primers and downstream primers used are shown in SEQ ID NO.2 and SEQ ID NO.3, and the genotype analysis of the gene editing site targeted by the sgRNA is performed. After the PCR is completed, the PCR products are connected using the connection system shown in Table 3, and the single clones are picked for genotype sequencing after heat shocking Escherichia coli. The cloning results are shown in Figure 2 As shown in Figure 2B, three independent gene editing lines all produced edits at the target site, and Figure 2 As shown in middle C, three independent gene-edited lines, Ghir-D12G013040-CR1, Ghir-D12G013040-CR2 and Ghir-D12G013040-CR3, also showed a phenotype of short plants and inability to produce seeds in the field.

[0039] The nucleotide sequence of sgRNA targeting Ghir_D12G13040 gene is SEQ ID NO.1: CTTGGGTTACAAACAAACGT.

[0040] The nucleotide sequences of the forward / reverse primers used to amplify the sgRNA targeting sequence are:

[0041] SEQ ID NO. 2: TTTTTCATTTGTTACAGATCAGCA.

[0042] SEQ ID NO. 3: ATTGTTTGAATACTTGAACATTGGA.

[0043] 2. Analysis of the high temperature resistance effect of GhKPHMT gene and its application in the creation of high temperature resistant materials

[0044] Editing the Ghir_D12G013040 gene in cotton produced a developmental defect phenotype, suggesting that this gene positively regulates the high temperature resistance of upland cotton and is a developmentally essential gene. To verify and clarify the contribution of the Ghir_D12G013040 gene to the high temperature resistance effect, based on the published high temperature resistance and transcriptome data of 217 upland cotton powders (reference Ma, Y., L. Min, J. Wang, Y. Li, Y. Wu, Q. Hu, Y. Ding, M. Wang, Y. Liang, Z. Gong, S. Xie, X. Su, C. Wang, Y. Zhao, Q. Fang, Y. Li, H. Chi, M. Chen, AHKhan, K. Lindsey, L. Zhu, X. Li & X. Zhang (2021) A combination of genome-wide and transcriptome-wide association studies reveals genetic elements Leading tomalesterility during high temperature stress in cotton. New Phytol, 231, 165-181.) analyzed the expression of Ghir_D12G013040 gene in different high temperature resistant materials. The analysis results are as follows Figure 3 Middle A and Figure 3 As shown in B, it is shown that the Ghir_D12G013040 gene is highly expressed in the high temperature resistant materials with high powder activity of upland cotton, indicating that the Ghir_D12G013040 gene may positively regulate the high temperature resistance of upland cotton at the population level. For further verification, the protein coding sequence of the Ghir_D12G013040 gene was amplified using the upstream primer (SEQ ID NO.4) and downstream primer (SEQ ID NO.5) of the reference sequence, based on the upland cotton drug cDNA template (experimental reagents dissolved with a large number of nucleotide molecules, used to amplify the KPHMT gene), the PCR system shown in Table 1 was configured, and the PCR program shown in Table 2 was used to amplify and clone the protein coding sequence of the Ghir_D12G013040 gene. The PCR amplification results are shown in Table 2. Figure 3As shown in C, there are three band types in the GhKPHMT swimming lane. Under the instruction of DNALadder, the corresponding fragment sizes of 1000-1100bp, 1100-1200bp and 2000-2500bp can be obtained. After the PCR products in Table 3 are connected and sequenced, the product of 2000-2500bp is the protein coding sequence (GhKPHMT) of the Ghir_D12G013040 gene, and its nucleotide sequence is shown in SEQIDNO.6. The arrow in C indicates a non-specific amplification product, which cannot be aligned to the precise position on the upland cotton genome after cloning, so it is excluded. After protein sequence conversion and online database comparison (swiss-prot), the Ghir_D12G013040 gene is annotated as a 3-methyl-2-oxobutyrate hydroxymethyltransferase (GhKPHMT) gene.

[0045] The upstream primer for amplifying the protein of the Ghir_D12G013040 gene is SEQ ID NO. 4: ATGTTATCTTTGCAAACTCAAACC.

[0046] The downstream primer for amplifying the protein of the Ghir_D12G013040 gene is SEQ ID NO. 5: TTAACCAAGCAAGCTCTCCCA.

[0047]

[0048] Table 4 Restriction endonuclease system

[0049] Components Dosage Plasmids Total 1.5 μg Endonuclease corresponding to 10×Buffer 2μL Restriction endonuclease (10U / μL) 1μL Double distilled water Make up to 20 μL

[0050] Table 5 Homologous recombination reaction system

[0051] Components volume 5×CE II Buffer 1μL Exnase 0.5μL Endonuclease digestion of plasmid 1μL (75ng / μL concentration) PCR products 1μL Double distilled water Make up to 5 μL (50℃ reaction for 30 min)

[0052] Note: Table 5 shows the ClonExpress Ultra One Step Cloning Kit V2#C116 (Nanjing Novogene Biotech Co., Ltd., product name is ClonExpress Ultra One Step Cloning Kit V2, product number is C116).

[0053] Further, the forward primer (SEQ ID NO.7) and reverse primer (SEQ ID NO.8) for constructing the cotton overexpression vector pGWB402-GhKPHMT, the forward primer (SEQ ID NO.9) and reverse primer (SEQ ID NO.10) for constructing the rice overexpression vector pCAMBIA1300S-GhKPHMT, and the forward primer (SEQ ID NO.11) and reverse primer (SEQ ID NO.12) for constructing the Arabidopsis overexpression vector pMDC84-GhKPHMT, a total of 3 primer pairs, were used, and the PCR system shown in Table 1 and the PCR program shown in Table 2 were used to amplify the GhKPHMT fragment with a recombination linker. Further, according to the restriction digestion system shown in Table 4, pGWB402 (for cotton genetic transformation, endonuclease is XbaI), pCAMBIA1300S (for rice genetic transformation, endonuclease is EcoRI), and pMDC84 (for Arabidopsis genetic transformation, double restriction digestion using XbaI and KpnI) vectors were digested, and the recombination system shown in Table 5 was used to recombinant the GhKPHMT sequence into the corresponding restriction digested vector to form a gene overexpression binary vector that can be used for genetic transformation.

[0054] After genetic transformation, the forward primer (SEQ ID NO.13) and the reverse primer (SEQ ID NO.14) for detecting the expression of GhKPHMT were used to detect the positive and overexpression effects of transgenes, genotype purification and field high temperature identification. The upland cotton, rice and Arabidopsis materials overexpressing the GhKPHMT gene all showed a phenotype of high temperature resistance. After being subjected to high temperature stress, the pollen vitality of the cotton, rice and Arabidopsis materials overexpressing the GhKPHMT gene increased by 20-35% compared with the transgenic receptors, indicating that GhKPHMT can positively regulate high temperature resistance in plants and has the potential to create high temperature resistant genetic materials in different species. In the figure, all NT abbreviations represent normal temperature, HT abbreviations represent high temperature, OE represents overexpression, and null represents the transgenic negative line separated from the corresponding transgenic line. All transformations used parallel overexpression of the green fluorescent protein gene OE-GFP as a transgenic control. In the experiment of overexpressing the GhKPHMT gene, the transgenic receptor Jin668 was used as the wild-type control in upland cotton, the transgenic receptor Zhonghua 11 (ZH11) was used as the wild-type control in rice, and the transgenic receptor Columbia Col-0 was used as the wild-type control in Arabidopsis.

[0055] The forward primer SEQ ID NO.7 for constructing the cotton overexpression vector pGWB402-GhKPHMT is ttactatttacaatttctagaATGTTATCTTTGCAAACTCAAACC.

[0056] The reverse primer SEQ ID NO.8 for constructing the cotton overexpression vector pGWB402-GhKPHMT is ttctcctttgcccattctagaACCAAGCAAGCTCTCCCAA.

[0057] The forward primer SEQ ID NO.9 for constructing the rice overexpression vector pCAMBIA1300S-GhKPHMT is gagctcggtacccggggatccATGTTATCTTTGCAAACTCAAACC.

[0058] The reverse primer SEQ ID NO.10 for constructing the rice overexpression vector pCAMBIA1300S-GhKPHMT is: ttctcctttgcccatgaattcACCAAGCAAGCTCTCCCAA.

[0059] The forward primer SEQ ID NO.11 for constructing the Arabidopsis overexpression vector pMDC84-GhKPHMT is ttactatttacaatttctagaATGTTATCTTTGCAAACTCAAACC.

[0060] The reverse primer SEQ ID NO.12 for constructing the Arabidopsis overexpression vector pMDC84-GhKPHMT is ttctcctttgcccattctagaACCAAGCAAGCTCTCCCAAC.

[0061] The forward primer for detecting the expression level of GhKPHMT is SEQ ID NO.13: TACCAGCGATGTTGTTCCGAG.

[0062] The reverse primer for detecting the expression level of GhKPHMT is SEQ ID NO.14: GAATCCAGGCACGACGACAG.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene in regulating plant high temperature resistance, characterized in that: The nucleotide sequence of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene is shown in SEQ ID NO.

6.

2. The application according to claim 1, characterized in that: Improving plant high temperature resistance by overexpressing cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene.

3. The application according to claim 1, characterized in that: The plant is one or more of upland cotton, rice or Arabidopsis thaliana.

4. Use of an expression cassette, vector or recombinant bacterium containing the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene as claimed in claim 1 in regulating plant high temperature resistance.

5. Use of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene as claimed in claim 1 in cultivating high temperature resistant transgenic plants.

6. Use of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene as claimed in claim 1 in improving plant high temperature resistant germplasm resources.

7. Use of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene as claimed in claim 1 in improving pollen vigor of plants under high temperature environment.

8. The use according to claim 7, characterized in that: The plant is one or more of upland cotton, rice or Arabidopsis thaliana.

9. A method for improving plant high temperature resistance using the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene as claimed in claim 1, characterized in that: The cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene is expressed or overexpressed in plants by transgenic, hybridization, backcrossing, selfing or asexual reproduction. The nucleotide sequence of the cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene is shown in SEQ ID NO.

6.

10. The method for improving the high temperature resistance of plants according to claim 9, characterized in that: The transgenic method comprises introducing a recombinant expression vector containing cotton 3-methyl-2-oxobutyrate hydroxymethyltransferase gene into plants by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method to obtain transgenic plants.

Images