Wheat grain protein content gene tagpc-6a, protein encoded by the gene and application
By screening and confirming the application of the wheat grain protein content regulatory gene TaGPC-6A and its encoded protein, the technical problem of wheat quality improvement was solved, the technical effect of increasing wheat grain protein content was achieved, the problem of lack of grain protein content regulatory genes in the existing technology was solved, the molecular genetic basis of grain protein formation was revealed, and a new approach was provided for wheat quality breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HENAN AGRICULTURAL UNIVERSITY
- Filing Date
- 2024-08-09
- Publication Date
- 2026-05-15
AI Technical Summary
The lack of genes regulating wheat grain protein content in existing technologies has led to slow progress in wheat quality improvement breeding, and the unclear mechanism of grain protein formation has limited the improvement of wheat quality.
The wheat grain protein content regulatory gene TaGPC-6A and its encoded protein were screened and identified. By constructing a plant expression vector and overexpressing the gene in wheat, the grain protein content was increased using an Agrobacterium-mediated transformation system.
It significantly increased the protein content of wheat grains, revealed the molecular genetic basis of grain protein formation, and provided important genetic resources and new approaches for wheat quality breeding.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, specifically to a wheat grain protein content regulating gene TaGPC-6A, its encoded protein, and its applications. Background Technology
[0002] Wheat is one of the most widely cultivated food crops in the world, a staple food for humankind, providing approximately 20% of the calories and protein required for human life activities (FAO, http: / / www.fao.org / faostat / ). Statistics show that the protein content provided by wheat is almost equivalent to the combined protein content of meat, eggs, and milk. With economic and social development and the continuous improvement of people's living standards, the requirements for wheat quality are also gradually increasing. Therefore, while focusing on wheat yield, it is also important to improve wheat grain quality. Wheat grain protein content accounts for approximately 10% to 15% of the grain weight, containing a variety of amino acids required by the human body. Grain protein content is one of the important quality indicators of wheat, directly affecting its nutritional and processing quality. Improving the protein content of wheat grains will help improve the nutritional and processing quality of wheat.
[0003] Wheat grain protein content is influenced by both genetic factors and environmental conditions. Furthermore, wheat grain protein formation is the result of a complex process involving nitrogen absorption, accumulation, transport, and distribution. Researching the mechanisms of nitrogen accumulation and transport in wheat will help improve wheat grain protein content and thus wheat quality. Wheat grain protein content is a typical quantitative trait, controlled by multiple genes with minor effects. Currently, researchers have identified several QTLs associated with wheat grain protein content, covering almost all 21 wheat chromosomes. However, due to the complex genetic background of wheat and the instability of quality traits due to environmental influences, the discovery of relevant controlling genes has been very slow. Currently, only a few genes have been reported to participate in regulating wheat grain protein content. For example, the GPC-B1 gene, cloned from wild emmer wheat, is believed to participate in regulating grain protein content; the ortholog of rice OsAAP6, TaAAP6-3B, may play an important role in regulating wheat grain protein content; and TaGS2 may increase wheat grain protein content by regulating nitrogen metabolism.
[0004] Although much research has been conducted on the genetic mechanisms of wheat grain protein content, overall progress has been limited and the depth of understanding is insufficient, leaving a significant gap before elucidating gene function and practical applications. To date, few genes regulating wheat grain protein content with significant application value have been reported, and the mechanism of wheat grain protein formation remains unclear, greatly restricting the progress of wheat quality improvement breeding. Therefore, further exploration of genes regulating wheat grain protein content and their effective utilization is urgently needed. Discovering important genes regulating wheat grain protein content through genetic methods and then investigating their functions can deepen our understanding of the genetic mechanisms of wheat grain protein content and provide an important theoretical foundation and genetic resources for wheat quality improvement breeding. Summary of the Invention
[0005] This invention provides a wheat grain protein content regulating gene TaGPC-6A, its encoded protein, and its applications, aiming to solve the problem of lack of wheat grain protein content genes and provide more options for wheat quality improvement breeding.
[0006] This invention screened and obtained a wheat grain protein content regulating gene, TaGPC-6A, whose genomic nucleotide sequence is shown in SEQ ID NO:1, with a length of 1858 bp, containing one exon and no introns; the cDNA nucleotide sequence of the TaGPC-6A gene is shown in SEQ ID NO:2, with a length of 1858 bp; the CDS nucleotide sequence of the TaGPC-6A gene is shown in SEQ ID NO:3, which is the deoxyribonucleotide from position 90 to 1613 of the 5' end in sequence SEQ ID NO:2, and encodes the amino acid sequence shown in SEQ ID NO:4, with 507 amino acids.
[0007] This invention provides a primer pair for amplifying the above-mentioned gene, the nucleotide sequence of which is shown below:
[0008] TaGPC-6A-1F: 5'-CAGCCATTAGCACCTCCTAG-3';
[0009] TaGPC-6A-1R: 5'-TTACTTGAAGGAAGGTGTACG-3'; or
[0010] TaGPC-6A-2F: 5'-ATGGAGGTGGAGTCTAGC-3';
[0011] TaGPC-6A-2R: 5'-CACATGCGCACCTTCCAC-3'.
[0012] Using wheat genomic DNA as a template, the DNA fragment obtained by PCR amplification using primer pair composed of TaGPC-6A-1F and TaGPC-6A-1R is the genomic sequence of the TaGPC-6A gene.
[0013] Using wheat cDNA as a template, the DNA fragment obtained by PCR amplification with primer pair composed of TaGPC-6A-1F and TaGPC-6A-1R was the cDNA sequence of the TaGPC-6A gene.
[0014] Using wheat cDNA as a template, the DNA fragment obtained by PCR amplification with primers consisting of TaGPC-6A-2F and TaGPC-6A-2R was the CDS sequence of the TaGPC-6A gene.
[0015] This invention also provides a plant expression vector containing a wheat grain protein content regulating gene TaGPC-6A or a CDS of the TaGPC-6A gene, for example, by inserting the aforementioned wheat grain protein content regulating gene TaGPC-6A into the LGY-OE3 plant overexpression vector. When constructing a plant expression vector using a CDS of the TaGPC-6A gene or a TaGPC-6A gene, any enhancing promoter or inducible promoter can be added before its transcription initiation nucleotide. To facilitate the identification and screening of transgenic plant cells or plants, the vector can be processed, such as by adding plant-selective markers (GUS gene, luciferase gene, etc.) or antibiotic resistance markers (gentamicin, kanamycin, etc.).
[0016] The present invention also provides recombinant bacteria containing the above-mentioned plant expression vector.
[0017] The present invention also applies the wheat grain protein content regulating gene TaGPC-6A, the CDS of the TaGPC-6A gene, the protein encoded by the TaGPC-6A gene, the plant expression vector, the recombinant bacteria, or the primer pair to improve the wheat grain protein content or to cultivate wheat varieties / lines with high grain protein content.
[0018] This invention also provides a method for increasing the protein content of wheat grains, specifically, overexpressing the wheat grain protein content regulatory gene TaGPC-6A in wheat. The procedure involves transforming a plant expression vector containing the wheat grain protein content regulatory gene TaGPC-6A into immature wheat embryos, and then screening to obtain transgenic positive lines.
[0019] The present invention has the following beneficial effects:
[0020] This invention discloses and confirms for the first time a novel wheat grain protein content regulating gene, TaGPC-6A, located on wheat chromosome 6A. The protein expressed by this gene can regulate wheat grain protein content. In the embodiments of this invention, a plant expression vector carrying the TaGPC-6A gene was transformed into wheat embryos using an Agrobacterium-mediated transformation system. Compared with wild-type Fielder, the grain protein content of the overexpression positive plants was significantly increased, indicating that TaGPC-6A is a wheat grain protein content regulating gene.
[0021] The TaGPC-6A gene of this invention helps to reveal the molecular genetic basis of wheat grain protein formation, can provide important gene resources for wheat quality breeding, and provide new ways to cultivate high-yield and high-quality new wheat varieties. Attached Figure Description
[0022] Figure 1 This is a comparison chart of the protein content of seeds from wild-type Kronos and TaGPC-6A mutants (K533 and K2650) in Example 2 of the present invention.
[0023] Figure 2 This is a plasmid map of the LGY-OE3 vector in Example 3 of the present invention.
[0024] Figure 3 This is a graph showing the relative expression levels of TaGPC-6A in its overexpression lines (OE-2 and OE-4) and the control Fielder in Example 3 of the present invention.
[0025] Figure 4 This is a comparison of the grain protein content of TaGPC-6A in its overexpression lines (OE-2 and OE-4) and the control Fielder in Example 3 of the present invention. Detailed Implementation
[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0027] Unless otherwise specified, all instruments and equipment used in the following embodiments are conventional instruments and equipment; all reagents, carriers, and other experimental materials used are commercially available conventional products; and all experimental methods and detection methods used are conventional methods. Quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged. Primer synthesis and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.
[0028] The wheat material involved in the examples:
[0029] A natural population of 243 representative wheat varieties / lines from the Huang-Huai wheat region (Zhao L, Pan Y, Dong Z, Zheng Y, Liu J, Geng J, Ren Y, Zhang N, Chen F. (2020) Investigation and genome-wide association study of grain copper content in Chinese common wheat. J Cereal Sci. 95: 102991.) was genotyped using a genome-wide 660K SNP array (Sun C, Dong Z, Zhao L, Ren Y, Zhang N, Chen F. (2020) The Wheat 660K SNP array demonstrates great potential for marker-assisted selection in polyploid wheat. Plant Biotechnol J. 18(6): 1354-1360.). This population was used for genome-wide association analysis (GWAS).
[0030] Kronos materials: wild-type tetraploid wheat Kronos, Kronos mutants: K533 and K2650 (both are premature termination types of the TaGPC-6A gene in the A genome) mutant materials, used to compare the TaGPC-6A gene effect; the mutant materials were purchased from the University of California, Davis (UC Davis), and the above materials were generated by Jorge Dubcovsky's group at the university using EMS mutagenesis.
[0031] Fielder material: Wild-type Fielder.
[0032] Example 1: Mining genes related to wheat grain protein content using genome-wide association analysis (GWAS), as detailed below:
[0033] 1. Group planting
[0034] The 243 wheat varieties used for genome-wide association analysis were planted at the Yuanyang Research Base of Henan Agricultural University during the 2019–2020 growing season. A completely randomized block design was used, with each planting plot being 2m long and 1.5m wide, containing 12 rows. Each variety was planted in two rows, with single-seed sowing. The spacing between plants in the rows and within the rows was 20cm and 10cm, respectively. The experimental fields were managed according to standards, and there were no droughts, lodging, or pests and diseases during the entire growing season. After harvest, the wheat was threshed, dried, and stored uniformly.
[0035] 2. Determination of grain protein content
[0036] The protein content of the grains was determined using a Perten DA7250 near-infrared analyzer (Perten, Sweden). To ensure the accuracy and reliability of the test results, each sample was tested in triplicate, with each replicate consisting of three measurements. A control variety, Yunong 804, was also measured every 20 samples.
[0037] 3. Gene mining of wheat grain protein content
[0038] The phenotypic data of grain protein content obtained from the survey were combined with the population 660K SNP chip genotype (Beijing Compson Biotechnology Co., Ltd.) to conduct genome-wide association analysis (GWAS) to identify SNP loci associated with wheat grain protein content. The analysis revealed that the detected significant SNPs were mainly enriched in a 40Kb segment between 15.74 and 15.78 Mb on wheat chromosome 6A; referring to Ensembl... Analysis of annotated genome data of *Traes. chinensis* from the *Plants* database revealed four annotated genes within this region, all encoding high-affinity nitrate transporters. Analysis of the expression levels of these four genes in wheat roots, stems, leaves, ears, and grains showed that *TraesCS6A02G030900* had the highest expression level. Further analysis of the physical location of significant SNPs on chromosomes revealed that one SNP located within the *TraesCS6A02G030900* gene had the highest significance level, and two other SNPs located in the promoter region of this gene also had high significance levels. No significant SNPs were found within or near the other three genes. Therefore, *TraesCS6A02G030900* was identified as a target candidate gene and named TaGPC-6A.
[0039] Based on the published Chinese spring reference sequence, TaGPC-6A in wheat variety Yu Nong 804 was cloned, ultimately yielding a complete open reading frame.
[0040] The specific steps are as follows:
[0041] ① DNA extraction: DNA was extracted from the seedlings of Yunong 804 using the SLS method.
[0042] The specific implementation steps are as follows:
[0043] (1) Take an appropriate amount of wheat leaves and put them into a 2mL centrifuge tube containing steel balls. Quickly freeze the tube in liquid nitrogen and then grind it with a grinder. Add 800μL SLS and shake for 20min to mix it thoroughly. The SLS contains 288mM NaCl, 200mM Tris-HCl, 25mM EDTA, and 0.5% SDS.
[0044] (2) Then add an equal volume of the three-in-one solution (phenol:chloroform:isoamyl alcohol = 25:24:1);
[0045] (3) Shake for 10 minutes to mix thoroughly;
[0046] (4) Centrifuge at 12,000 rpm for 15 min, transfer the supernatant to another new 2 mL centrifuge tube, add an equal volume of pre-cooled isopropanol, and let stand for 10 min to precipitate;
[0047] (5) Centrifuge at 12,000 rpm for 15 min, discard the supernatant, add 0.5 mL of 75% (volume percentage) ethanol aqueous solution, and let stand for 5 min.
[0048] (6) Centrifuge at 12,000 for 5 min, discard the supernatant, vacuum dry, add 100 μL TE to dissolve the precipitate, and obtain wheat leaf genomic DNA.
[0049] ② Total RNA extraction and cDNA synthesis: Total RNA was extracted from the wheat variety Yunong 804 using the TRIzol method. 2 μL of RNA was electrophoresed on 2% agarose gel, and RNA integrity was assessed. The extracted total RNA showed a generally clear 28S and 18S main band. OD was measured using a UV spectrophotometer. 260 / OD 280 The ratio is between 1.80 and 2.00, OD 260 / OD 230 >2.0. Collect total RNA that meets the required quality standards, referring to PrimeScript. TM The cDNA was obtained by reverse transcription using the RT Reagent Kit (perfect real time) instructions.
[0050] ③ Cloning and sequence analysis of the TaGPC-6A gene
[0051] Nested PCR primers were designed, with TaGPC-6A-1F and TaGPC-6A-1R positioned at opposite ends of the start codon ATG and stop codon TGA, respectively; TaGPC-6A-2F and TaGPC-6A-2R were positioned at the positions of the start codon ATG and stop codon TAG, respectively. Specific details are as follows:
[0052] TaGPC-6A-1F: 5'-CAGCCATTAGCACCTCCTAG-3';
[0053] TaGPC-6A-1R: 5'-TTACTTGAAGGAAGGTGTACG-3';
[0054] TaGPC-6A-2F: 5'-ATGGAGGTGGAGTCTAGC-3';
[0055] TaGPC-6A-2R: 5'-CACATGCGCACCTTCCAC-3'.
[0056] Using DNA from Yu Nong 804 as a template, PCR amplification was performed using a primer pair consisting of TaGPC-6A-1F and TaGPC-6A-1R. The PCR amplification system is shown in Table 1, and the PCR amplification program is shown in Table 2.
[0057] Table 1. PCR amplification system of TaGPC-6A gene using primer pairs composed of TaGPC-6A-1F and TaGPC-6A-1R.
[0058] Components volume 2×Phanta buffer 25μL dNTP 1μL Phanta Enzyme 1μL Primer F 2μL Primer R 2μL DNA 2μL <![CDATA[ddH2O]]> To 50μL
[0059] Table 2. PCR amplification program for the TaGPC-6A gene using primer pairs composed of TaGPC-6A-1F and TaGPC-6A-1R.
[0060]
[0061]
[0062] The PCR products were subjected to 1% agarose gel electrophoresis, recovered, and ligated into the pMD18-T vector (Takara), and sent to Shanghai Sangon Biotech for sequencing.
[0063] Using a similar method as described above, cDNA from YuNong 804 was used as a template for PCR amplification with a primer pair consisting of TaGPC-6A-1F and TaGPC-6A-1R. After electrophoresis, the DNA was recovered and ligated into the pMD18-T vector (Takara), and then sent to Shanghai Sangon Biotech for sequencing. The PCR reaction system and amplification procedure were the same as described above.
[0064] Sequencing results showed that the DNA fragment obtained by PCR amplification using Yunong 804 DNA as a template was the genomic-level sequence of the TaGPC-6A gene, as shown in SEQ ID NO:1. The total length of the gene sequence was 1858 bp, containing only one exon and no introns. The DNA fragment obtained by PCR amplification using Yunong 804 cDNA as a template was the cDNA sequence of the TaGPC-6A gene, as shown in SEQ ID NO:2 in the sequence listing, also 1858 bp in length. The DNA fragment obtained by nested PCR amplification using the TaGPC-6A cDNA sequence as a template and primers consisting of TaGPC-6A-2F and TaGPC-6A-2R was the CDS sequence of the TaGPC-6A gene, as shown in SEQ ID NO:3 in the sequence listing. Its length was 1524 bp, representing the deoxyribonucleotide sequence from position 90 to 1613 from the 5' end of SEQ ID NO:2 in the sequence listing, and encoding the sequence shown in SEQ ID NO:2. The protein shown in NO:4 has 507 amino acids.
[0065] Example 2: Identification of protein content in TaGPC-6A mutant grains
[0066] Screening of the tetraploid wheat Kronos EMS mutant library yielded two premature termination type TaGPC-6A mutants, named K533 (G-603A) and K2650 (C-452T), respectively. K533 and K2650 were then backcrossed with wild-type Kronos and self-crossed to obtain homozygous BC2F3 mutant lines. Protein content was determined in grains of the BC2F3 homozygous mutant lines and wild-type Kronos harvested in the field. Specific determination methods are described in Example 1. Results are as follows: Figure 1 As shown, the protein content of the mutant grains was significantly lower compared to the wild-type plants.
[0067] Example 3: Obtaining TaGPC-6A transgenic wheat
[0068] 1. Construction of TaGPC-6A plant overexpression vector
[0069] Based on the full-length cDNA sequence of TaGPC-6A cloned in Example 1, protective primers were designed, incorporating recognition sites for restriction endonucleases BamHI and SacI, as well as protective bases. The primer sequences are as follows:
[0070] TaGPC-6A-3F: 5'-GGATTCATGGAGGTGGAGTCTAGC-3';
[0071] TaGPC-6A-3R: 5'-GAGCTCCACATGCGCACCTTCCAC-3'.
[0072] The expression vector chosen was LGY-OE3, which carries the maize ubiquitin promoter. The vector map is shown below. Figure 2 As shown.
[0073] The 1524bp DNA fragment (the CDS sequence of the TaGPC-6A gene) from Example 1 was cloned into the restriction sites between BamHI and SacI of the plant expression vector LGY-OE3 to obtain a recombinant expression vector containing the wheat TaGPC-6A gene, named LGY-OE3-TaGPC-6A.
[0074] 2. Obtaining TaGPC-6A transgenic wheat
[0075] LGY-OE3-TaGPC-6A was transformed into wheat Fielder embryo callus tissue using Agrobacterium tumefaciens infection method. After screening, pre-differentiation, and differentiation, regenerated transgenic wheat plants were obtained.
[0076] 3. Positive identification of genetically modified wheat
[0077] First, positive identification of T1 generation transgenic plants was performed using hygroscopic enzyme tag primers Hyg-F and Hyg-R. The primer sequences are as follows:
[0078] Hyg-F: 5'-TCTGCACCATCGTCAACCAC-3';
[0079] Hyg-R: 5'-AAACCCACGTCATGCCAGTT-3'.
[0080] A total of 11 positive transgenic plants were obtained. After multiple generations, T2 generation homozygous transgenic lines OE-2 and OE-4 were obtained.
[0081] 4. Identification of TaGPC-6A expression level in transgenic wheat
[0082] The expression level of TaGPC-6A in transgenic lines was detected using TaGPC-6A-3F and TaGPC-6A-3R primers. The primer sequences are as follows:
[0083] TaGPC-6A-4F: 5'-CGCCGCGCCGTTGGTG-3';
[0084] TaGPC-6A-4R: 5'-GAAGACCGTTGGCGCTGAGAG-3'.
[0085] Real-time quantitative PCR was used to detect the expression level of TaGPC-6A in the T2 generation positive transgenic lines. The results are as follows: Figure 3 As shown, the expression level of TaGPC-6A in the leaves of the T2 generation positive transgenic lines OE-2 and OE-4 was significantly higher than that of the wild-type Fielder receptor, at 94.2 times and 73.3 times that of the control, respectively.
[0086] Example 4: Determination of protein content in TaGPC-6A transgenic wheat grains
[0087] Protein content was determined in the seeds of the T2 generation overexpression transgenic lines (OE-2 and OE-4) obtained in Example 3 and the control Fielder. The specific determination method is as described in Example 1. Results are as follows: Figure 4 As shown, the grain protein content of the T2 generation overexpressing transgenic lines (OE-2 and OE-4) was significantly higher than that of the recipient control Fielder. This confirms that TaGPC-6A plays an important role in wheat grain protein formation and accumulation.
[0088] In summary, the wheat grain protein content regulating gene TaGPC-6A described in this application can promote the formation and accumulation of wheat grain proteins. It can be used for further research on the molecular mechanisms of wheat grain protein formation and has important application value in wheat quality breeding.
[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. Genes regulating protein content in wheat grains TaGPC-6A The aforementioned gene TaGPC-6A The encoded protein, containing the gene TaGPC-6A The application of plant expression vectors or recombinant bacteria containing said plant expression vectors in the cultivation of wheat varieties / lines with high grain protein content, characterized in that... Gene TaGPC-6A The CDS nucleotide sequence is shown in SEQ ID NO:
3.
2. Genes regulating protein content in wheat grains TaGPC-6A The aforementioned gene TaGPC-6A The encoded protein, containing the gene TaGPC-6A The application of plant expression vectors or recombinant bacteria containing plant expression vectors in increasing the protein content of wheat grains, characterized in that... Gene TaGPC-6A The CDS nucleotide sequence is shown in SEQ ID NO:
3.
3. A method for increasing the protein content of wheat grains, characterized in that, Overexpression of wheat grain protein content regulatory genes in wheat TaGPC-6A ,Gene TaGPC-6A The CDS nucleotide sequence is shown in SEQ ID NO:
3.
4. The method according to claim 3, characterized in that, The gene containing the wheat grain protein content regulation gene TaGPC-6A The plant expression vector was transformed into immature wheat embryos, and transgenic positive lines were obtained by screening.