A gene qPro7 that regulates the content of gliadin in rice grains and its application

Abstract

This invention discloses a gene that regulates the content of gliadin in rice grains. qPro7 This invention relates to the fields of genetic engineering and plant molecular breeding, and its applications. The amino acid sequence of the gene is shown in SEQ ID NO.1. Through genome-wide association analysis, this invention identified and cloned the gene on rice chromosome 7. qPro7 By constructing genes qPro7 Knockout vectors and self-promoted overexpression vectors were used to transform rice, and the results showed that knockout vectors... qPro7 The gene can significantly reduce the content of prolactin in rice grains, while overexpression qPro7 The gene significantly increases the content of prolamins in rice grains; this invention provides new genetic resources for the genetic improvement of rice quality, especially the regulation of protein content, and has important application value.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and plant molecular breeding technology, specifically relating to a gene that regulates the content of gliadin in rice grains. qPro7 And its applications. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops and the most consumed grain globally. Through high-yield breeding, super-high-yield breeding, super rice breeding, and green super rice breeding, rice yields have been significantly increased. However, with economic development and improved living standards, people are no longer just looking for enough to eat, but also for quality food, leading to increasingly higher demands for rice quality.

[0003] Rice quality is generally categorized into processing quality, appearance quality, cooking and eating quality, and nutritional quality, with consumers particularly concerned about cooking and eating quality and nutritional quality. The composition and proportions of rice largely determine its cooking and eating quality and nutritional value. Rice is mainly composed of starch and protein, with starch making up the vast majority. Although the protein content is low, only about 10%, both starch and protein content have a significant impact on rice quality. Recent progress in improving rice quality by focusing on starch content is encouraging, but it still cannot fully meet consumer demands.

[0004] In recent years, the excessive application of nitrogen fertilizer in production to ensure yield has led to an increase in rice protein content to some extent. Studies have shown that, with consistent starch content, moderately reducing the protein content of rice can improve its cooked and flavorful qualities. Rice protein content is regulated by multiple genes and is significantly influenced by the environment. While there are numerous studies on QTL mapping of rice protein content, currently only... OsAAP6 and OsGluA2 Cloning. Current progress in regulating rice protein content is slow, and there is an urgent need to identify the major genes controlling rice protein content to provide abundant genetic resources for rice quality improvement. Summary of the Invention

[0005] Objective of the Invention: To address the aforementioned technical problems, the objective of this invention is to provide a novel gene for regulating the content of gliadin in rice grains. qPro7 Another objective of this invention is to provide the application of this gene in regulating the content of gliadin in rice grains. This invention identifies and clones the gene on chromosome 7 through genome-wide association analysis of gliadin content. qPro7 The aim is to enrich the genetic resources for rice quality improvement and provide new technical means for improving rice quality, especially protein content.

[0006] Technical solution: A gene that regulates the content of prolysin in rice grains qPro7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the nucleotide sequence of the genomic region of the gene is shown in SEQ ID NO.2, the promoter nucleotide sequence is shown in SEQ ID NO.3, and the coding region CDS nucleotide sequence is shown in SEQ ID NO.4.

[0008] A gene comprising any of the above-described genes qPro7 The recombinant expression vector is a knockout vector or an overexpression vector.

[0009] Furthermore, the knockout vector is constructed based on the CRISPR / Cas9 system and is used to edit the gene. qPro7 The coding region; the overexpression vector is composed of the gene qPro7 Overexpression vectors driven by their own promoters.

[0010] A host cell containing any of the above-mentioned genes. qPro7 , or any of the above recombinant expression vectors.

[0011] Furthermore, the host cell is a rice cell.

[0012] One of the above-mentioned genes that regulates the content of gliadin in rice grains qPro7 The application of any of the above-mentioned recombinant expression vectors or any of the above-mentioned host cells in regulating the content of rice grain gliadin, by knocking out or inhibiting the gene. qPro7 The expression of [something] is used to reduce the content of prolactin in rice grains.

[0013] Furthermore, by overexpressing the gene qPro7 This is to increase the content of prolactin in rice grains.

[0014] This application also discloses a method for improving the content of alcohol-soluble protein in rice grains, including regulating any of the above-mentioned genes in rice. qPro7 The steps to express the level.

[0015] Furthermore, by knocking out or suppressing the gene qPro7 The expression of the gene can be used to reduce the content of gliadin in rice grains, or by overexpressing the gene. qPro7 This is to increase the content of prolactin in rice grains.

[0016] Beneficial effects: 1. This invention achieves the effect of knocking out [something] in rice. qPro71. This invention can successfully reduce the content of gliadin in rice grains, thereby improving rice quality; 2. This invention is the first to clone and identify the gene that regulates the content of gliadin in rice grains. qPro7 This provides new key gene resources for improving rice quality; 3. This invention clarifies the gene through transgenic function verification. qPro7 Positive regulation of gliadin content in rice grains. By knocking out this gene, the gliadin content in rice grains can be successfully downregulated, thereby improving rice quality (especially cooking and eating quality); 4. This invention provides a method based on... qPro7 Gene knockout and overexpression techniques provide an operational method for rice molecular breeding and have significant industrial application value. Attached Figure Description

[0017] Figure 1 For the gene in this application qPro7 Screening and identification of prolysin content, Manhattan plot of genome-wide association analysis, where the top plot is from 2019 and the bottom plot is from 2020; Figure 2 For the gene in this application qPro7 The screening and identification of 18 genes in the candidate region under the significant peak repeatedly detected on chromosome 7 over two years; Figure 3 For the gene in this application qPro7 The filter chart; Figure 4 For the gene in this application qPro7 Identification diagram; Figure 5 For this application qPro7 The diagram shows the vectors of the transgenic materials and the content of gliadin in the knockout lines and the overexpression lines driven by their own promoters. A is a schematic diagram of the two vectors and the sequencing results of the knockout lines; B is a diagram showing the measurement results of the gliadin content of the transgenic lines and wild-type lines; and C is a diagram showing the measurement results of the overexpression driven by the own promoters of the transgenic lines and wild-type lines. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only for illustrating the invention and are intended to limit its scope. Unless otherwise specified, the experimental techniques and methods involved in the embodiments are common techniques in genetic engineering and plant molecular breeding, and are performed under conventional conditions.

[0019] Example 1: A gene regulating the content of gliadin in rice grains qPro7The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1: MKIIFVFALLAIVACNASARFDPLSQSYRQYQLQSHLLLQQQVLSPCSEFVRQQYSIVATPFWQPATFQLINNQVMQQQCCQQLRLVAQQSHYQAISIVQAIVQQLQLQQFSGVYFDQTQAQAQTLLTFNLPSICGIYPNYYSAPRSIATVGGVWY.

[0020]

[0021] The primer pairs used to amplify the gene promoter region and coding region include the forward primer SEQ ID NO.5 and the reverse primer SEQ ID NO.6, and the primer pairs used to amplify the gene coding region include the forward primer SEQ ID NO.7 and the reverse primer SEQ ID NO.8. Specifically, SEQ ID NO.5: aaccagcaccgcattacctg; SEQ ID NO.6: gcacaatataaaattatttg; SEQ ID NO.7: atgaagatcattttcgtatt; SEQ ID NO. 8: tcagtaccagacaccaccaa.

[0022] A gene comprising any of the above-described genes qPro7 The recombinant expression vector is a knockout vector or an overexpression vector.

[0023] Furthermore, the knockout vector is constructed based on the CRISPR / Cas9 system and is used to edit the gene. qPro7 The coding region; the overexpression vector is composed of the gene qPro7 Overexpression vectors driven by their own promoters.

[0024] A host cell containing any of the above-mentioned genes. qPro7 , or any of the above recombinant expression vectors.

[0025] Furthermore, the host cell is a rice cell.

[0026] One of the above-mentioned genes that regulates the content of gliadin in rice grains qPro7 The application of any of the above-mentioned recombinant expression vectors or any of the above-mentioned host cells in regulating the content of rice grain gliadin, by knocking out or inhibiting the gene. qPro7 The expression of [something] is used to reduce the content of prolactin in rice grains.

[0027] Furthermore, by overexpressing the gene qPro7 This is to increase the content of prolactin in rice grains.

[0028] Furthermore, the gene qPro7 The rice variety Nip was transformed with the overexpression vector driven by the knockout vector and its own promoter, and transgenic positive plants were screened.

[0029] Furthermore, through CRISPR / Cas9 Systematic gene editing qPro7The coding region; amplified gene qPro7 The promoter (~2Kb) and the full-length genome sequence were linked together and constructed into the vector PC1300HB to form a self-promoted overexpression vector.

[0030] Furthermore, positive plant detection was performed on knockout lines using forward primer SEQ ID NO.9 and reverse primer SEQ ID NO.10, and positive detection was performed on overexpression lines driven by their own promoters using forward primer SEQ ID NO.11 and reverse primer SEQ ID NO.12. Specifically: SEQ ID NO.9: GCAACCGATGGGAAAGCATC; SEQ ID NO.10: GAGCAATACAATAGCAGGCC; SEQ ID NO.11: CTATAGCAGCGGTTGTAGCC; SEQ ID NO. 12: CTATTACGCCAGCTGGCGAA.

[0031] This application also discloses a method for improving the content of alcohol-soluble protein in rice grains, including regulating any of the above-mentioned genes in rice. qPro7 The steps to express the level.

[0032] Furthermore, by knocking out or suppressing the gene qPro7 The expression of the gene can be used to reduce the content of gliadin in rice grains, or by overexpressing the gene. qPro7 This is to increase the content of prolactin in rice grains.

[0033] The content of prolactin in 367 rice accessions after laboratory resequencing was determined, and genome-wide association analysis was performed. The results are as follows: Figure 1 As shown, significant loci (2019: Chr07: 5728364, 2020: Chr07: 5654806) were detected on chromosome 7 in both the 2019 and 2020 gliadin content results. Based on these two significant loci and the 150kb region before and after them, 18 potential candidate genes were screened, as shown in Table 1 below. Table 1. 18 candidate genes within the candidate region of chromosome 7 .

[0034] To further identify target genes among the 18 candidate genes, 8 varieties with high gliadin content and 8 varieties with low gliadin content were selected. The expression levels of the 18 candidate genes were measured and analyzed 15 days after flowering in these 16 varieties. The results are as follows: Figure 4As shown, only the gene designated XII exhibited a highly significant difference in expression levels between high- and low-glycan content varieties, while the expression levels of other genes did not differ significantly between the two groups. Based on this finding, it is speculated that the gene designated XII may be a key functional gene regulating grain alcohol solubility, and this gene is named... qPro7 .

[0035] To further verify qPro7 Gene function, utilizing CRISPR / Cas9 Technology knockout in qPro7 Two target sites were designed in the coding region to construct a knockout vector for knockout. qPro7 Simultaneously amplify genes qPro7 The promoter and full-length genome sequence were linked and constructed into the vector PC1300HB to form the overexpression vector P1300- driven by its own promoter. qPro7 The vectors were then transformed into NiP, respectively. Schematic diagrams of the two vectors and sequencing results of the knockout lines are shown below. Figure 5 As shown in Figure A, the content of gliadin in transgenic lines and wild-type lines was determined, and the results are as follows. Figure 5 China B and Figure 5 As shown in Figure C: the content of prolysin in the knockout lines was significantly lower than that in the wild type, while the content of overexpression lines driven by their own promoters was significantly higher than that in the wild type.

[0036] Example 2, Gene qPro7 Functional verification: 1) Determination of protein content in rice components: Weigh 100 ± 0.5 mg of rice flour into a 2.0 ml centrifuge tube, repeating the weighing process three times for each sample. Add 1 mL of ddH₂O to the centrifuge tube, mix well, and place in a shaker at 30℃ and 200 rpm for 2 hours. After shaking, centrifuge at 10000 rpm for 15 minutes at 4℃, then transfer the supernatant to a 10 mL centrifuge tube. Repeat the above steps three times, combining the supernatants. Add 1 mL of 1 mol / L NaCl to the residue obtained in the previous step, and extract under the same conditions as the previous step, combining the supernatants. Add 1 mL of ethylene glycol to the residue obtained in the second step, and extract under the same conditions as the previous step, combining the supernatants. Add 1 mL of 0.05 mol / L NaOH to the residue obtained in the third step, and extract under the same conditions as the previous step, combining the supernatants.

[0037] Before quantification, the supernatant should be frozen at -20°C. After extraction, the protein was measured using the BCA protein quantification kit from Beijing Tiangen Biotech Co., Ltd.

[0038] Results: First, after measuring the gliadin content of 367 resequencing rice germplasms, genome-wide association analysis was performed, successfully identifying a significant locus on chromosome 7. Second, by measuring the gliadin content of transgenic lines, a gene was successfully identified. qPro7 .

[0039] 2) Genome-wide association analysis: Genome-wide association analysis of gliadin content was performed using a mixed linear model (mlm) in EMMAX software. A 1.0 × 10⁻⁶ m² model was used. -5 Significant sites were screened using a threshold, and candidate genes were screened within a 75 kb interval upstream and downstream of the significant site.

[0040] Results: In the association analysis of prolysin content between 2019 and 2020, a significant site that was repeatedly detected was identified on chromosome 7. Genes within the candidate region of this site were screened, and a total of 18 candidate genes were identified.

[0041] 3) Expression level measurement: Fifteen days after flowering, the seeds were ground into powder, and RNA was extracted using the Tiangen reagent kit (DP419). Quantitative PCR was then performed using TransGen reverse transcription (AE341) and SYBR (AQ621).

[0042] Results: The expression levels of 18 candidate genes were determined and analyzed 15 days after flowering in 8 varieties with high and 8 varieties with low gliadin content. Only gene XII showed a highly significant difference in expression level between the high and low gliadin varieties, successfully identifying [the gene]. qPro7 .

[0043] 4) qPro7 Gene cloning: use qPro7 The amplification primers (forward primer SEQ ID NO.7: ATGAAGATCATTTTCGTATT, reverse primer SEQ ID NO.8: TCAGTACCAGACACCACCAA) were cloned from endosperm cDNA 15 days after flowering. qPro7 The CDS sequence is shown in SEQ ID NO.4.

[0044] 5) CRISPR / Cas9 Carrier construction: First, design the target points on the website (http: / / skl.scau.edu.cn / targetdesign / ). qPro7 The primers for knocking out the target site are shown in Table 2 below: Table 2 qPro7 Knockout target primer information .

[0045] according to CRISPR / Cas9 The vector construction steps involve connecting the designed specific target sites to promoters U3 and U6A respectively, and then connecting them to pYL using a cut-and-connect method. CRISPR / Cas9 Sequencing on -MH (B) yielded the knockout plasmid KO- qPro7, The plasmid was sent to Boyuan Company to obtain transformed seedlings.

[0046] Identification of positive plants: For the knockout transgenic lines, PCR detection was performed using the forward primer GCAACCGATGGGAAAGCATC (SEQ ID NO.9) and the reverse primer GAGCAATACAATAGCAGGCC (SEQ ID NO.10) to screen for homozygous mutant lines.

[0047] Results: Homozygous mutant lines were screened using SEQ ID NO.9 and SEQ ID NO.10. The content of gliadin in the homozygous knockout lines was measured, and it was found that the content of gliadin in the knockout lines was significantly lower than that in the wild type.

[0048] 6) Construction of overexpression vectors driven by their own promoters: Amplified genes qPro7 The promoter and full-length genome sequence were linked and constructed into the vector PC1300HB to form the overexpression vector P1300- driven by its own promoter. qPro7 The plasmid was sent to Boyuan Company to obtain transformed seedlings.

[0049] For the overexpressing transgenic lines, PCR detection was performed using the forward primer CTATAGCAGCGGTTGTAGCC (SEQ ID NO.11) and the reverse primer CTATTACGCCAGCTGGCGAA (SEQ ID NO.12) to screen for positive lines. These lines were then self-crossed until segregation ceased, resulting in stably inherited overexpressing lines.

[0050] Results: Mutants were screened from T0 using SEQ ID NO.11 and SEQ ID NO.12, and self-crossing was continued until no further segregation occurred. These mutants were named P1300- qPro7 Through analysis of P1300- qPro7 After measuring the content of prolactin, it was found that the prolactin content in the overexpression lines driven by the self-promoter was significantly increased compared with the wild type. Following validation with transgenic materials, the gene was successfully identified. qPro7 .

[0051] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

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