Application of cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in regulating high temperature resistance of plants

Abstract

The application discloses an application of a cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in regulation of high-temperature resistance of plants and belongs to the technical field of plant molecular breeding, and provides the application of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in regulation of high-temperature resistance of plants, wherein the nucleotide sequence of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene is shown as SEQ ID NO. 6. The application clones a GhKPHMT gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase on a high-temperature resistance locus qPV-D12 of Gossypium hirsutum by analyzing the genetic characteristics of the high-temperature resistance locus of the Gossypium hirsutum, the gene participates in controlling the high-temperature resistance of cotton pollen, overexpression technology is used to create overexpression transgenic lines in the Gossypium hirsutum, rice and Arabidopsis, and the effect and potential application capability of the Gossypium hirsutum GhKPHMT gene in creating high-temperature resistant materials in different species are proved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant molecular breeding, and particularly relates to application of a cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in regulation of high-temperature resistance of plants. BACKGROUND

[0002] Gossypium hirsutum L produces more than 90% of natural spinnable fibers in China. As an important cotton production area, Xinjiang cotton area frequently encounters extreme high temperature weather of more than 35℃ in summer, resulting in high-temperature male sterility and causing a large number of bud boll shedding in the concentrated bolling period. However, due to the complexity of cotton high-temperature resistance, the difficulty of material screening, and the inaccuracy of gene identification, there is a practical bottleneck in cotton high-temperature resistance breeding at the present stage. In order to solve the above industrial problems, the existing technology with the publication number CN118186135A establishes a scheme for rapidly identifying cotton pollen viability under high temperature, and identifies four high-temperature resistance loci in a cotton breeding population, namely qPV-A01, qPV-D01, qPV-D05 and qPV-D12, and expects to analyze the genetic variation and effect of the high-temperature resistance loci to determine the high-temperature resistance genes. Through linkage analysis based on an artificial population and candidate gene association analysis, the effect block of the qPV-A01 high-temperature resistance locus is determined, and the high-temperature resistance gene disclosed in the patent with the publication number CN118345099A is serine / threonine kinase GhHRKs, and the high-temperature resistance genes in other loci still need to be researched and developed. SUMMARY

[0003] In order to solve the above technical problems, the application provides application of a cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in regulation of high-temperature resistance of plants, a GhKPHMT gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase located at the qPV-D12 high-temperature resistance locus of Gossypium hirsutum D12 chromosome is cloned by analyzing the genetic characteristics of the high-temperature resistance loci of Gossypium hirsutum, which participates in controlling high-temperature resistance of cotton pollen, and overexpression technology is used to create overexpression transgenic lines in Gossypium hirsutum, rice and Arabidopsis thaliana, and it is proved that the Gossypium hirsutum GhKPHMT gene has the effect of creating high-temperature resistance materials and potential application ability in different species.

[0004] In order to achieve the above purpose, the application provides application of a cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in regulation of high-temperature resistance of plants, the nucleotide sequence of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene is shown in SEQ ID NO. 6.

[0005] Preferably, the high-temperature resistance of plants is improved by overexpression of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene.

[0006] Preferably, the plant is one or more of Gossypium hirsutum, Oryza sativa or Arabidopsis thaliana.

[0007] The application also provides application of the expression cassette, vector or recombinant bacteria containing the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in regulating high temperature resistance of plants.

[0008] The application also provides application of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in breeding high temperature resistant transgenic plants.

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

[0010] The application also provides application of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in improving pollen viability of plants under high temperature environment.

[0011] Preferably, the plant is one or more of Gossypium hirsutum, Oryza sativa or Arabidopsis thaliana.

[0012] The application also provides a method for improving high temperature resistance of plants by using the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene, which comprises the following steps: by means of transgenic, hybridization, backcross, selfing or vegetative reproduction, making the plant express or overexpress the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene, and the nucleotide sequence of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene is shown as SEQ ID NO. 6.

[0013] Preferably, the transgenic comprises the following steps: introducing the recombinant expression vector containing the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene into the plant by means of Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method to obtain the transgenic plant.

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

[0015] The application provides application of a cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase (GhKPHMT) gene in regulation of high-temperature resistance of plants, discloses a high-temperature resistance gene in a qPV-D12 block, the gene ID of the gene is Ghir_D12G013040, and the gene encodes a 3-methyl-2-oxobutanoate hydroxymethyltransferase gene. Based on a GhKPHMT gene model and a reference sequence, an allelic genotype with a function loss of the gene is created by using a gene editing technology, it is proved that GhKPHMT positively regulates high-temperature resistance and is an important gene involved in organ development, and an advantageous genotype and material beneficial to industrial development cannot be generated by using a gene editing method. The application creates genetic materials in Gossypium hirsutum, rice and Arabidopsis thaliana by using overexpression means, and it is proved that overexpression of GhKPHMT can provide considerable high-temperature resistance for male gametes of the above species.

[0016] The application discloses a high-temperature resistance gene GhKPHMT on a high-temperature resistance qPV-D12 locus of Gossypium hirsutum, and enriches the genetic resources of high-temperature resistance of Gossypium hirsutum. Genetic materials with high-temperature resistance are created in cotton, rice and Arabidopsis thaliana by using a suitable genetic transformation vector based on the GhKPHMT gene, a candidate scheme for improving high-temperature resistance of a self-species by using an exogenous gene is provided, and the breeding process of effective high-temperature resistance germplasm is effectively promoted. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0018] Figure 1 The analysis results of the qPV-D12 gene locus and genetic effects for example 1 are shown in the following table, wherein A is a Manhattan plot based on whole genome association analysis of a natural population of Gossypium hirsutum, and B is a Q-Q plot corresponding to the whole genome association analysis;

[0019] Figure 2 The gene editing results of the causal gene GhKPHMT of the qPV-D12 locus in example 1 are shown in the following table, wherein A is the phenotype of anther organs of offspring, B is a gene editing genotype, and C is the phenotype of field plants;

[0020] Figure 3 The influence results of the expression amount of the GhKPHMT gene on high-temperature resistance of Gossypium hirsutum pollen in example 1 in the present application are shown in the following table, wherein A is pollen vitality of Gossypium hirsutum, B is the expression amount of the GhKPHMT gene, and C is an electrophoresis map;

[0021] Figure 4For the results of the influence of GhKPHMT gene overexpression on plant high temperature resistance in Example 1, A is GhKPHMT gene overexpression Gossypium hirsutum, B is GhKPHMT gene overexpression Arabidopsis thaliana, and C is GhKPHMT gene overexpression Oryza sativa. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description is not intended to limit the present application, but rather to explain certain aspects, features, and embodiments of the present application.

[0023] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for the purposes of the present application, the term "about" means plus or minus 10% of the specified value. Furthermore, the use of the term "or" in the

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0025] Many modifications and variations of this application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the

[0026] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0027] Example 1

[0028] I. Identification of qPV-D12 high temperature resistance locus in Gossypium hirsutum and discovery of GhKPHMT gene

[0029] Among more than 3000 published land cotton germplasms (referring to 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 Genet, 53, 916-924.), 517 core germplasms were selected as the material basis, and pollen viability after high temperature stress was used as the phenotype (referring to CN109752226B), and three high temperature resistance loci qPV-A01-1, qPV-D01-1 and qPV-D12-1 were identified in the genome of land cotton by whole genome association analysis (as shown in Figure 1 A and Figure 1 B of the specification), wherein the qPV-A01-1 locus has been disclosed, and the causal gene thereof is S-locus protein gene GhHRKs (referring to CN118345099A). The present application analyzes the undisclosed qPV-D12-1 locus, uses CRISPR-Cas9 gene editing technology, batch synthesizes sgRNA, and batch edits the candidate genes in the qPV-D12-1 locus. As shown in Figure 2 A of the specification, NT represents normal temperature, and HT represents high temperature. After gene editing, the transgenic offspring GhKPHMT-CR1, GhKPHMT-CR2 and GhKPHMT-CR3 targeting the Ghir_D12G13040 gene all showed severe developmental defects compared with the transgenic receptor Jin668 (wild type plant control), manifested as abnormal flower organ development, pollen sterility and the like.

[0030] Table 1 PCR amplification system

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

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

[0033] Table 2 PCR amplification program

[0034]

[0035] Table 3 PCR product ligation system

[0036] Component Volume 5 x Ultra-Universal TOPO Cloning Mix 1 μL PCR product 1.5 μL Double distilled water Make up to 5 μL (reaction at 25 °C for 10-15 min)

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

[0038] By sgRNA typing, it is confirmed that the nucleotide sequence of the sgRNA targeting Ghir_D12G13040 gene is shown as SEQ ID NO. 1. The PCR amplification system shown in Table 1 and the PCR amplification program shown in Table 2 are used in this embodiment, and the nucleotide sequences of the upstream primer and the downstream primer used are shown as SEQ ID NO. 2 and SEQ ID NO. 3. The genotype of the gene editing site targeted by the sgRNA is analyzed. After the PCR is completed, the PCR product is connected using the connection system shown in Table 3, and after the E. coli is heat shocked, a single clone is picked for genotype sequencing. The cloning results are shown in Table 4. Figure 2 As shown in Table B, three independent gene editing lines all produce edits at the target position, and as shown in Table C, Ghir-D12G013040-CR1, Ghir-D12G013040-CR2 and Ghir-D12G013040-CR3 three independent gene editing lines also appear plant dwarf in the field, and cannot produce seed phenotype. Figure 2

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

[0040] The nucleotide sequences of the forward / reverse primers for amplifying the sgRNA targeting sequence are:

[0041] SEQ ID NO. 2: TTTTTCATTTTGTTACAGATCAGCA.

[0042] SEQ ID NO. 3: ATTGTTTGAATACTTGAACATTGGA.

[0043] II. Analysis of the Anti-high temperature effect of GhKPHMT gene and its application in creating anti-high temperature materials

[0044] ​Editing Ghir_D12G013040 gene in cotton produces a developmental defective phenotype, suggesting that the gene positively regulates upland cotton high temperature resistance, and is a developmental essential gene. To verify, the contribution of Ghir_D12G013040 gene to high temperature resistance effect is clear, based on the 217 upland cotton pollen high temperature resistance and transcriptome data already published (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, A. H. Khan, K. Lindsey, L. Zhu, X. Li & X. Zhang (2021) A combination of genome-wide and transcriptome-wide association studies reveals genetic elements leading to male sterility during high temperature stress in cotton. New Phytol, 231, 165-181.), the expression of Ghir_D12G013040 gene in different high temperature resistance materials is analyzed. The analysis results are shown in Figure 3 A and Figure 3 B, indicating that Ghir_D12G013040 gene presents high expression characteristics in upland cotton pollen activity high high temperature resistance materials, indicating that Ghir_D12G013040 gene may positively regulate upland cotton high temperature resistance at the population level. To further verify, the protein coding sequence of Ghir_D12G013040 gene is amplified, the upstream primer (SEQ ID NO. 4) and the downstream primer (SEQ ID NO. 5) of the reference sequence are used, based on the anther cDNA template (an experimental reagent dissolved with a large number of nucleotide molecules, used to amplify KPHMT gene) of upland cotton, the PCR system as shown in Table 1 is configured, the PCR program as shown in Table 2 is used, and the protein coding sequence of Ghir_D12G013040 gene is amplified and cloned. The PCR amplification results are shown in Figure 3As shown in C, three bands exist in GhKPHMT lane, under the indication of DNA Ladder, it can be concluded that the corresponding fragment size is 1000-1100 bp, 1100-1200 bp and 2000-2500 bp. After the PCR product connection and sequencing of Table 3, the product of 2000-2500 bp is the protein coding sequence of Ghir_D12G013040 gene (GhKPHMT), the nucleotide sequence is shown as SEQ ID NO. 6, the arrow in C indicates a non-specific amplification product, which cannot be aligned to the exact position on the genome of Gossypium hirsutum after cloning, and thus is excluded. Through protein sequence conversion and online database alignment (swiss-prot), Ghir_D12G013040 gene is annotated as a 3-methyl-2-oxobutanoic acid hydroxymethyltransferase (GhKPHMT) gene.

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

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

[0047]

[0048] Table 4 Restriction enzyme system

[0049] Component Amount Plasmid Total amount 1.5 μg Endonuclease corresponding 10 x Buffer 2 μL Restriction endonuclease (10 U / μL) 1 μL Double distilled water Make up to 20 μL

[0050] Table 5 Homologous recombination reaction system

[0051] Component Volume 5 x CE II Buffer 1 μL Exnase 0.5 μL Endonuclease-digested plasmid 1 μL (concentration of 75 ng / μL) PCR product 1 μL Double distilled water Make up to 5 μL (reaction at 50 °C for 30 min)

[0052] Note: In Table 5 is ClonExpress Ultra One Step Cloning Kit V2#C116 (Nanjing Novozyme Bio-tech Co., Ltd., commodity name ClonExpress Ultra One Step Cloning Kit V2, product number C116).

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

[0054] The genetically transformed, using the forward primer (SEQ ID NO. 13) and reverse primer (SEQ ID NO. 14) for the expression of GhKPHMT for the detection of transgenic positive and overexpression effect, genotype purification and field high temperature identification, overexpression of GhKPHMT gene of upland cotton, rice and Arabidopsis materials, all showed the phenotype of high temperature resistance. Overexpression of GhKPHMT gene of cotton, rice and Arabidopsis materials after suffering from high temperature stress, pollen viability compared with transgenic receptor, all increased by 20-35%, 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, OverExpression, null represents the transgenic negative line separated from the corresponding transgenic line, and all transformations use parallel overexpression of green fluorescent protein gene OE-GFP as a transgenic control. In the experiment of overexpression of GhKPHMT gene, the transgenic receptor Jin668 was used as the wild type control of upland cotton, the transgenic receptor Zhonghua 11 (ZH11) was used as the wild type control of rice, and the transgenic receptor Columbia type Col-0 was used as the wild type control of Arabidopsis.

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

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

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

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

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

[0060] Reverse primer SEQ ID NO. 12 for constructing Arabidopsis overexpression vector pMDC84-GhKPHMT: ttctcctttgcccattctagaACCAAGCAAGCTCTCCCAAC.

[0061] Forward primer SEQ ID NO. 13 for detecting GhKPHMT expression: TACCAGCGATGTTGTTCCGAG.

[0062] Reverse primer SEQ ID NO. 14 for detecting GhKPHMT expression: GAATCCAGGCACGACGACAG.

[0063] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. The use of cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene in enhancing pollen viability under high temperature environment of Gossypium hirsutum, Oryza sativa or Arabidopsis thaliana, characterized in that, The nucleotide sequence of the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene is shown as SEQ ID NO.

6.

2. Use according to claim 1, characterized in that, By overexpressing the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene, the pollen viability of Gossypium hirsutum, rice or Arabidopsis under high temperature environment is improved.

3. Application of an expression cassette, a vector or a recombinant bacterium containing a cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene with a nucleotide sequence shown as SEQ ID NO. 6 in enhancing the pollen viability of Gossypium hirsutum, rice or Arabidopsis under high temperature environment.

4. A method for improving pollen viability under high temperature environment of Gossypium hirsutum, Oryza sativa or Arabidopsis thaliana by using a 3-methyl-2-oxobutanoate hydroxymethyltransferase gene with a nucleotide sequence as shown in SEQ ID NO. 6, characterized in that, By a transgenic method, Gossypium hirsutum, rice or Arabidopsis is made to overexpress the cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene with a nucleotide sequence shown as SEQ ID NO.

6.

5. The method of claim 4, wherein the method is for improving pollen viability in Gossypium hirsutum, Oryza sativa or Arabidopsis thaliana under high temperature environment. The transgene includes introducing a recombinant expression vector containing a cotton 3-methyl-2-oxobutanoate hydroxymethyltransferase gene into Gossypium hirsutum, rice or Arabidopsis by a method of Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium mediation, to obtain transgenic Gossypium hirsutum, rice or Arabidopsis.

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