TaGAT2-1B gene capable of resisting wheat stripe rust and application of TaGAT2-1B gene

Abstract

The invention belongs to the technical field of biology, and particularly relates to a TaGAT2-1B gene resistant to wheat stripe rust and application of the TaGAT2-1B gene, and the nucleotide sequence of the TaGAT2-1B gene is shown as SEQ ID NO.3. It is found that the TaGAT2-1B gene and the encoding protein thereof can effectively improve the wheat stripe rust resistance, the TaGAT2-1B gene is a new stripe rust resistance gene in the wheat adult-plant stage, infection of various stripe rust strains can be effectively resisted, the field phenotype of the TaGAT2-1B gene shows the characteristic of slow rust, and the TaGAT2-1B gene and the encoding protein thereof have good application prospects. The discovery of the gene provides a new genetic resource for the research of the wheat stripe rust resistance mechanism and the wheat stripe rust resistance breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a TaGAT2-1B gene resistant to wheat stripe rust and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.), one of the three most widely cultivated cereal crops in the world, is a major source of plant protein and energy in our daily diet. Wheat stripe rust is an airborne, obligate parasitic fungal disease caused by the fungus Puccinia striiformis Westend f.sp. tritic Eriks. It spreads rapidly, over long distances, and causes widespread damage. It has become one of the most damaging and widespread diseases. Generally, wheat stripe rust is prevalent in regions with cool, humid climates. While wheat stripe rust is found in wheat-producing areas worldwide, its prevalence in China is complex, varied, and unique.

[0003] Wheat stripe rust resistance is generally categorized as seedling-stage resistance and adult-stage resistance. Seedling-stage resistance is often also referred to as resistance throughout the entire growth period. Seedling-stage resistance is typically race-specific, generally controlled by a single resistance gene and classified as a quality trait. However, due to the variable physiological races of the pathogen, resistance effects are easily lost. Unlike seedling-stage resistance genes, adult-stage resistance genes are either non-specifi c or weakly specific to the pathogen's physiological races. Their resistance is not lost due to changes in the pathogen's physiological races, thus reducing the selection pressure on resistant varieties against the pathogen's physiological races, resulting in long-lasting and stable disease resistance. Long-term research has shown that the cultivation and use of disease-resistant varieties is the most economical, safe, and fundamental approach to controlling wheat stripe rust and a key strategy for green disease control. Therefore, discovering and cloning new adult-stage stripe rust resistance genes, determining their resistance roles, and clarifying their mechanisms of resistance are crucial for breeding for disease resistance. Summary of the Invention

[0004] The purpose of the present invention is to provide a wheat stripe rust-resistant TaGAT2-1B gene for improving the resistance of wheat to wheat stripe rust.

[0005] The technical solution adopted in the present invention is:

[0006] In a first aspect, the present invention provides a TaGAT2-1B gene resistant to wheat stripe rust. The nucleotide sequence of the TaGAT2-1B gene is shown in SEQ ID NO.3.

[0007] The second aspect of the present invention provides a nucleic acid molecule, the nucleotide sequence of which has 70% to 99% homology with the TaGAT2-1B gene.

[0008] The third aspect of the present invention provides a gene expression cassette, which comprises the TaGAT2-1B gene or the nucleic acid molecule.

[0009] The fourth aspect of the present invention provides a recombinant overexpression vector, which comprises the TaGAT2-1B gene or the nucleic acid molecule.

[0010] The fifth aspect of the present invention provides a method for preparing the recombinant overexpression vector, comprising the following steps:

[0011] extracting wheat DNA, and using the DNA as a template to amplify the TaGAT2-1B gene or nucleic acid molecule by PCR;

[0012] The TaGAT2-1B gene or nucleic acid molecule is cloned into the overexpression vector after enzyme digestion to obtain a recombinant overexpression vector.

[0013] Preferably, the overexpression vector is pCub.

[0014] Preferably, the restriction endonucleases used for enzymatic digestion of the overexpression vector are BamHI and SacI.

[0015] The sixth aspect of the present invention provides a recombinant bacterium, which comprises the recombinant overexpression vector.

[0016] Preferably, the starting strain of the recombinant bacteria is Escherichia coli or Agrobacterium

[0017] The seventh aspect of the present invention provides the use of the TaGAT2-1B gene, the nucleic acid molecule, the gene expression cassette, the recombinant overexpression vector or the recombinant bacteria, characterized in that the use is to improve the resistance of wheat to stripe rust.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a TaGAT2-1B gene for resistance to wheat stripe rust, the nucleotide sequence of which is shown in SEQ ID NO. 3. The present invention discovers that the TaGAT2-1B gene and its encoded protein can effectively improve wheat stripe rust resistance. The gene is a new gene for resistance to wheat stripe rust during the adult stage, effectively resisting infection by multiple stripe rust strains. Its field phenotype exhibits characteristics of a slow rust disease. The discovery of this gene provides a new genetic resource for research on the mechanism of wheat stripe rust resistance and for breeding wheat stripe rust resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Results of genome-wide association analysis, a: Manhattan plot of genome-wide association analysis; b: chromosome 1B interval where QYr.nwafu-1BL.3 is located; c: candidate genes within the QYr.nwafu-1BL.3 interval.

[0021] Figure 2 Figure 1. Localization of QYr.nwafu-1BL.3 and QYrXN3517-1BL on chromosome arm 1BL. a: Colocalization analysis of QYr.nwafu-1BL.3, QYrXN3517-1BL (red bars), and previously reported Pst resistance genes / QTLs (blue bars) on the integrated genetic map. The light blue shaded area indicates the resistance gene / QTL enrichment cluster on chromosome 1BL. b: Genetic linkage map of the 1BL region where QYr.nwafu-1BL.3 and QYrXN3517-1BL are located. Markers flanking QYrXN3517-1BL are indicated in blue, and QYr.nwafu-1BL.3 is indicated in red. c: Physical map of the 1BL region where QYr.nwafu-1BL.3 and QYrXN3517-1BL are located. Markers flanking QYrXN3517-1BL are indicated in blue. QYr.nwafu-1BL.3 is indicated in red; d: candidate genes in the YrFro interval

[0022] Figure 3 The expression of the YrFro candidate gene in adult plants of resistant and susceptible materials inoculated with and without inoculation is shown. Control: Uninoculated; Inoculated: Inoculated. Resistant materials: J411, Jing 411; JM44, Jimai 44; XN3517, Xinong 3517; QN142, Qinnong 142; susceptible materials: JM229, Jimai 229; MX169, Mingxian 169; AVS, Avocet S.

[0023] Figure 4 The results of wheat stripe rust resistance leaf phenotypic identification are shown in Figure 1. A: 10 parallel samples of the wild type; B: 15 parallel samples of the overexpression plant.

[0024] Figure 5 The phenotypes of wheat stripe rust-resistant plants in the field, A: wild type; B: overexpression plant. DETAILED DESCRIPTION

[0025] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0026] The inventive concept of the present invention is as follows:

[0027] γ-Aminobutyric acid, or GABA, is a ubiquitous four-carbon non-protein amino acid that exists in a free form in various organisms. Studies have shown that GABA in plants can play a dual role as a metabolic and signaling molecule, responding to a variety of biotic and abiotic stresses and regulating plant growth and development. Adversity stress can cause high levels of GABA to accumulate in plants and be transported within cells via transporters, thereby improving plant stress resistance. In plants, multiple amino acid transporters are involved in the GABA transport process, and GATs are one of them. The full name of GATs is γ-aminobutyric acid transporters.

[0028] The GAT gene family belongs to a subfamily of the AAAP gene family. Its family members have been identified in Arabidopsis, rice, corn, potato and many other species. There are two GAT genes in Arabidopsis, AtGAT1 and AtGAT2. Studies have shown that AtGAT1 is a H-terminal gene located on the plasma membrane. + A transporter that transports GABA via proton coupling and has a very high affinity for GABA. Unlike AtGAT1, AtGAT2 is a low-affinity GABA transporter. While GABA is its primary substrate, AtGAT2 also transports glutamate, proline, and choline. AtGAT2 is located in the plasma membrane and endomembrane, and its physiological significance remains unclear. Studies in rice have shown that the expression of the rice OsGAT2 gene is upregulated in response to drought and salt stress.

[0029] Currently, research on GAT gene function is still in the exploratory stage and is mostly focused on a few model plants such as Arabidopsis thaliana. Although rice, wheat, and maize have 4, 14, and 2 GAT homologous genes, respectively, their transport activity and regulatory mechanisms have not been deeply explored. In addition, existing research has mostly focused on abiotic stress responses, while the potential role of GAT genes in plant resistance to biotic stresses such as pathogen infection has not been explored. Based on this, the present invention is the first to conduct a preliminary analysis of the stripe rust resistance function of the GAT gene in wheat, preliminarily verifying its stripe rust resistance function, providing available genetic resources for wheat stripe rust resistance breeding, and also laying the foundation for the analysis of GAT gene function.

[0030] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0031] The primer sequences of the present invention are shown in Table 1.

[0032] Serial number Primer sequences SEQ ID NO.5 CGACCGATCGAGAGTCTT SEQ ID NO.6 GCTTTGACCGGCCTACTTTA SEQ ID NO.7 ATGGATCCGAACATGGCC SEQ ID NO.8 TCAGCTGATGACATGGTCAC SEQ ID NO.9 AGGTCGACTCTAGAGGATCCATGGATCCGAACATGGCCGG SEQ ID NO.10 GATCGGGGAAATTCGAGCTCTCAGCTGATGACATGGTCAC

[0033] Example 1

[0034] A TaGAT2-1B gene resistant to wheat stripe rust, specifically as follows:

[0035] Using the genomic data of 2191 natural population materials collected by the Wheat Disease Resistance Genetics and Molecular Breeding Laboratory of Northwest Agriculture and Forestry University, combined with the wheat stripe rust resistance phenotypes of 12 natural populations in 4 years and 5 points, the unbiased linear mixed model MLM of GEMMA software was used for genome-wide association analysis. A significant and stable QTL locus associated with stripe rust resistance in the adult stage was identified in the 681.75Mb to 683.17Mb interval of chromosome 1BL. Taking Jiangyou, Sichuan in 2021 as a typical representative, the results are as follows Figure 1 As shown, this QTL was named QYr.nwafu-1BL.3. The wheat reference genome in the present invention is IWGSCRefSeq v.2.1.

[0036] By referring to the physical location of the QTL in the Chinese Spring reference genome and closely linked markers flanking the QTL, the relationship between QYr.nwafu-1BL.3 and previously reported Yr genes or QTLs was determined. The results showed that the QYr.nwafu-1BL.3 locus overlaps with the QTL site QYrXN3517-1BL identified on chromosome 1BL during genetic analysis of stripe rust resistance in the Xinong 3517 population, explaining 8.5% to 22.3% of the phenotypic variation. This result validates the accuracy of the GWAS QTL identified in this study.

[0037] Further comparative analysis revealed that QYr.nwafu-1BL.3 does not overlap with the known gene Yr29 on chromosome 1BL, and the two differ in physical location by approximately 3.5 Mb, suggesting that QYr.nwafu-1BL.3 may be a novel adult-stage stripe rust resistance locus. Based on the source of resistance and pedigree analysis, this locus likely originated from Xinong 88, the parent of Xinong 3517. Xinong 88 may contain Frontana ancestry, and Frontana also contains this locus. Therefore, to facilitate subsequent research, QYr.nwafu-1BL.3 was renamed YrFro.

[0038] To further clarify the candidate interval of YrFro, the GWAS signal peak interval of QYr.nwafu-1BL.3 and the fine positioning results of QYrXN3517-1BL were combined, and the target interval was finally locked in the overlapping region between s1B_681080223 and s1B_682409491. The physical position of this interval is 681.08Mb to 682.41Mb, which contains 24 candidate genes, of which 17 are high-confidence genes. Figure 2 .

[0039] The 17 high-confidence genes in the YrFro candidate interval were further analyzed in terms of gene annotation and gene expression.

[0040] (1) Functional annotation: Among the 17 high-confidence genes, five genes, including TraesCS1B03G1234700, TraesCS1B03G1234800, TraesCS1B03G1236000, TraesCS1B03G1236100, and TraesCS1B03G1236400, contained two transcripts. According to gene annotation, the candidate genes encode twelve protein classes, including indoleacetic acid-amide synthase GH3.3, very large G-like proteins, γ-aminobutyric acid GABA transporters GATs, transducin / WD40 repeat-like superfamily proteins, lipid transfer proteins, disease resistance proteins NBS-LRR-like family, TFIIIC B-block binding subunits, polyol transporters, Agenet and bromo-neighbor homology BAH domain proteins, pfkB-like carbohydrate kinase family proteins, cardiolipin synthase B, and GDSL-like lipase / acyl hydrolase superfamily proteins, as shown in Table 2.

[0041] (2) Expression analysis: Transcriptome analysis was performed on the flag leaves of the disease-resistant wheat varieties Jimai 44, Jing 411, Xinong 3517, Qinnong 142 and the susceptible varieties AVS, Mingxian 169, and Jimai 229 at the adult stage with and without inoculation. The expression of 17 highly credible candidate genes in the YrFro segment was retrieved (see Table 3). The results showed that among the 17 high-confidence genes, the expression levels of eight genes were 0 or close to 0 in the resistant and susceptible materials regardless of whether they were inoculated or not, namely TraesCS1B03G1234900, TraesCS1B03G1235200, TraesCS1B03G1235500, TraesCS1B03G1235600, TraesCS1B03G1235900, TraesCS1B03G1236600, TraesCS1B03G1236700 and TraesCS1B03G1236900; the expression levels of seven genes were lower than 2, respectively. The expression levels of the two genes, TraesCS1B03G1235000 and TraesCS1B03G1235100, were greater than 5 in both inoculated and uninoculated conditions of the resistant and susceptible materials, which were significantly higher than those of other genes. Figure 3 .

[0042] Combined with gene function annotation and transcriptome gene expression analysis, TraesCS1B03G1235000 and TraesCS1B03G1235100 were preliminarily identified as key candidate genes for YrFro, encoding GABA transporters GATs and transducin / WD40 repeat superfamily proteins, respectively. TraesCS1B03G1235000 was temporarily named TaGAT2, and TraesCS1B03G1235100 was temporarily named TaWD40.

[0043] Prior art indicates that GABA participates in plant immunity through metabolic regulation, signal transduction, and direct involvement in disease resistance. Furthermore, resequencing results revealed sequence differences in the TaGAT2 gene between the susceptible and resistant materials, Xinong 3517 and AvS, while no sequence variation was detected in TaWD40. Based on these results, the present invention identified TaGAT2 as the first key candidate gene for YrFro and conducted subsequent analyses.

[0044] Table 2 Functional annotation of candidate genes in the YrFro interval

[0045]

[0046] Table 3 Expression levels of candidate genes in different resistant and susceptible materials

[0047]

[0048]

[0049] Example 2

[0050] An application of the wheat stripe rust resistance TaGAT2-1B gene, specifically as follows:

[0051] 1. Cloning of the full-length TaGAT2-1B gene.

[0052] (1) Take seeds of Xinong 3517 and germinate them at 25°C for 3 days. Then transfer the germinated seeds to nutrient soil and culture them at 25°C for two weeks to obtain Xinong 3517 wheat seedlings.

[0053] (2) Total DNA from seedlings of Xinong 3517 was extracted and used as a template for PCR amplification using primers F1 and R1 to obtain a PCR amplification product of approximately 2382 bp. The sequences of primers F1 and R1 are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0054] (3) The obtained PCR amplification product was sequenced. The sequencing results showed that the nucleotide sequence of the PCR amplification product was shown in SEQ ID NO. 4, which contained the full-length gDNA sequence of the TaGAT2-1B gene (2258 bp), as well as 37 bp upstream and 87 bp downstream. The full-length gDNA sequence of the TaGAT2-1B gene was shown in SEQ ID NO. 3.

[0055] 2. Cloning of the coding region of TaGAT2-1B gene.

[0056] (1) Take seeds of Xinong 3517 and germinate them at 25°C for 3 days. Then transfer the germinated seeds to nutrient soil and culture them at 25°C for two weeks to obtain Xinong 3517 wheat seedlings.

[0057] (2) Total RNA was extracted from the seedlings of Xinong 3517, and cDNA of Xinong 3517 was obtained by reverse transcription.

[0058] (3) Using the cDNA of Xinong 3517 as a template, primers F2 and R2 were used to perform PCR amplification, and a PCR amplification product of approximately 1425 bp was obtained. The sequences of primers F2 and R2 are shown in SEQ ID NO. 7 and SEQ ID NO. 8.

[0059] (4) The obtained PCR amplification product was sequenced. The sequencing results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID NO.2.

[0060] The gene shown in SEQ ID NO. 2 or SEQ ID NO. 3 is named TaGAT2-1B gene. The TaGAT2-1B gene encodes TaGAT2-1B protein, and the amino acid sequence of the TaGAT-1B protein is shown in SEQ ID NO. 1.

[0061] 3. Construction of recombinant overexpression vector.

[0062] The overexpression vector pCub was digested with BamHI and SacI restriction endonucleases; using wheat gDNA as a template, the DNA fragment of TaGAT2-1B shown in SEQ ID NO.3 was amplified using primers F3 and R3. The digested products of the overexpression vector pCub and the amplified product of the TaGAT2-1B gene were detected by agarose gel electrophoresis, then excised and recovered, and homologous recombination was performed. The full-length sequence of TaGAT2-1B was then ligated between the BamHI and SacI restriction sites of the pCub vector, which are located downstream of the Ubi promoter. After transformation with E. coli and sequencing, the recombinant overexpression vector pCub-TaGAT2-1B was obtained, which expressed the TaGAT2-1B protein shown in SEQ ID NO.1. The sequences of primers F3 and R3 are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0063] 4. Obtaining recombinant Agrobacterium tumefaciens.

[0064] The recombinant overexpression vector pCub-TaGAT2-1B was transformed into Agrobacterium tumefaciens GV3101 (pSoup-p19) to obtain recombinant Agrobacterium X containing the recombinant overexpression vector pCub-TaGAT2-1B.

[0065] 5. Obtaining overexpression plants.

[0066] The recombinant Agrobacterium X was sent to the Shandong Academy of Agricultural Sciences for transgenic plant cultivation, and wheat plants overexpressing TaGAT2-1B were obtained.

[0067] The stripe rust resistance phenotype of the overexpressing plants was analyzed as follows:

[0068] The seeds of the overexpressing plants were planted in the transgenic field of Caoxinzhuang Experimental Farm of Northwest Agriculture and Forestry University in Yangling District, Shaanxi Province, and were inoculated with stripe rust in April of the following year.

[0069] First, spray Tween water prepared at a volume ratio of 1:500 evenly on the induced wheat leaves. Then, mix CYR32, CYR34, and Pst-V26 wheat stripe rust species with talcum powder at a ratio of 1:20. Use the "shaking powder method" to inoculate the induced plant leaves with the wheat stripe rust strain spore powder. Then use black plastic bags to block light and uncover the bags the next day. In June, the wheat stripe rust phenotypic identification was carried out at the adult stage. The results are as follows: Figure 4 and Figure 5 As shown, the overexpressing plants were more resistant to stripe rust than the wild-type line.

[0070] CYR32 and CYR34 are disclosed in the reference: Wang N, Tang C, Fan X, He M, Gan P, Zhang S, HuZ, Wang X, Yan T, ShuW, Yu L, Zhao J, He J, Li L, Wang J, Huang X, Huang L, Zhou JM, KangZ, Wang X. Inactivation of a wheat protein kinase gene confers broad-spectrum resistance to rust fungi. Cell. 2022 Aug 4; 185(16): 2961-2974.e19. doi: 10.1016 / j.cell.2022.06.027. Epub 2022 Jul 14. PMID: 35839760.

[0071] Pst-V26 is disclosed in the reference: Zhou Xinli, Zhan Gangming, Huang Lili, Han Dejun, Kang Zhensheng. Evaluation of stripe rust resistance of 80 foreign spring wheat germplasm resources[J]. Chinese Agricultural Science, 2015, 48(8): 1518-1526.

[0072] SEQ ID NO.1:

[0073] MDPNMAGHTGDSPFLLSPSTGRAMTPSASYDGKPGPAAGDAGAAFVLESKGTWWHAGFHLTAMVGPTVLTLPYALRGMGWALGLSALTAVAAVTFYTYYLMSRVLDRCEAAGRRHIRFRELAADVLGSG WVFYAVVTVQTAINAGITIGSILIAGNCLQIMYESLAPNGTLKLYHFIIIVAVVLSLFSQMPSFHSLRYINLGSLVLAFGYTILVSGACIRAGMMSNAPVKDYSLSPSKSGKMYDAFLSISILATVFGNG ILPEIQATLAPPAAGKMVKALVLCYTVVFFTFYLSAISGYWAFGNKVQSNALQSLMPDSGPSLAPTWLLGLAVVLVLLQLLAIALVYSQVAYEIMEKGSADAGHGRF SWRNLAPRVALRTLYVAACAFVAAALPFFGDIVGVVGALGFIPLDFVLPVVMYNIALAPPKRSAVYVINVAIMALFTGVGIIGAIASVRKLVLDAGQFQLFSDHVIS.

[0074] SEQ ID NO.2:

[0075]

[0076] SEQ ID NO.3:

[0077]

[0078] SEQ ID NO.4:

[0079]

[0080] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0081] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A TaGAT2-1B gene resistant to wheat stripe rust, characterized in that: The nucleotide sequence of the TaGAT2-1B gene is shown in SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule has 70% to 99% homology with the TaGAT2-1B gene according to claim 1.

3. A gene expression cassette, characterized in that The gene expression cassette comprises the TaGAT2-1B gene of claim 1 or the nucleic acid molecule of claim 2 .

4. A recombinant overexpression vector, characterized in that: The recombinant overexpression vector comprises the TaGAT2-1B gene according to claim 1 or the nucleic acid molecule according to claim 2 .

5. The method for preparing a recombinant overexpression vector according to claim 4, wherein: The following steps are involved: extracting wheat DNA, and using the DNA as a template to amplify the TaGAT2-1B gene or nucleic acid molecule by PCR; The TaGAT2-1B gene or nucleic acid molecule is cloned into the overexpression vector after enzyme digestion to obtain a recombinant overexpression vector.

6. The preparation method according to claim 5, wherein The overexpression vector is pCub.

7. The preparation method according to claim 5, wherein The restriction endonucleases used for digesting the overexpression vector are BamHI and SacI.

8. A recombinant bacterium, characterized in that The recombinant bacteria comprises the recombinant overexpression vector according to claim 4.

9. The recombinant bacterium according to claim 8, characterized in that The starting strain of the recombinant bacteria is Escherichia coli or Agrobacterium.

10. Use of the TaGAT2-1B gene according to claim 1, the nucleic acid molecule according to claim 2, the gene expression cassette according to claim 3, the recombinant overexpression vector according to claim 4, or the recombinant bacterium according to claim 8, characterized in that: The application is to improve the resistance of wheat to stripe rust.

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