Application of millet genes Seita.8G189900 and Seita.8G190200 in controlling prolamin quality

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a millet gene Seita.8G189900 and Seita.8G190200 application in controlling prolamine quality. The nucleotide sequence of the millet gene Seita.8G189900 is shown as SEQ ID NO. 1, and the nucleotide sequence of the millet gene Seita.8G190200 is shown as SEQ ID NO. 2. The application proves two genes that can down-regulate the content of prolamine in millet, and the prolamine content of millet can be reduced by reducing the expression level of the genes. Knocking out the two genes can not only down-regulate the content of prolamine, but also up-regulate the content of amino acid, increase the nutritional value of millet, and be more beneficial to gastrointestinal absorption and digestion, thereby providing a new gene resource for protein quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to millet genes. Seita.8G189900 and Seita.8G190200 Application in controlling the quality of prolysins. Background Technology

[0002] Millet is an important coarse grain crop and one of the earliest cultivated crops in my country, with a history of over 8,000 years. As a whole grain food high in dietary fiber, millet is rich in protein, starch, vitamins (especially folic acid, vitamin A, and carotenoids), and minerals. Mature millet grains consist of the seed coat, embryo, and endosperm. In the grain, the endosperm contains starch, protein, and lipids. Millet grain protein is mainly present in the form of prolamins (approximately 60%). Similar to corn zein, millet grain protein is classified into four categories based on its solubility in solvents: albumins, globulins, prolamins, and glutelins. Based on molecular weight and solubility, millet prolamins are further classified into α, β, or γ subunits. Millet prolamins are rich in essential amino acids and have been shown to have various beneficial effects, including anti-inflammatory, antioxidant, hypoglycemic, and lipid-lowering effects, making them highly nutritious. Overall, millet prolamins are a very nutritious food component, and their excellent bioactivity makes them a beneficial supplement to a comprehensive healthy diet.

[0003] Currently, there is considerable research on the characteristics of gliadin in different varieties of millet, but it remains unclear which specific genes regulate gliadin expression and how. Furthermore, millet gliadin is closely related to millet protein quality. Therefore, further research is needed on the relationship between different genes regulating gliadin expression and millet protein quality. Summary of the Invention

[0004] To facilitate research on the relationship between different genes regulating prolysin expression and the quality of millet protein, this invention provides millet genes. Seita.8G189900 and Seita.8G190200 Application in controlling the quality of prolysin. To achieve the above objectives, the present invention adopts the following technical solution.

[0005] This invention provides millet genes Seita.8G189900 and Seita.8G190200 Application in controlling the quality of prolysin, the millet gene Seita.8G189900 The nucleotide sequence is shown in SEQ ID NO.1, and the millet gene is described. Seita.8G190200 The nucleotide sequence is shown in SEQ ID NO.2. The millet gene... Seita.8G189900 and the millet gene Seita.8G190200The content of prolamin can be reduced by down-regulating the expression of the gene, and the effect of controlling the protein quality of millet can be achieved by reducing the content of prolamin. The application technology provided in the application not only reveals the relationship between the gene for regulating the expression of prolamin in millet and the protein quality of millet, but also reveals how the millet gene Seita.8G189900 and the millet gene Seita.8G190200 regulate the expression of prolamin, and provides a new gene resource for improving the protein quality of millet.

[0006] Preferably, the amino acid sequence of the protein expressed by the millet gene Seita.8G189900 is shown in SEQ ID NO. 3, and the amino acid sequence of the protein expressed by the millet gene Seita.8G190200 is shown in SEQ ID NO. 4.

[0007] Preferably, the protein quality of millet is improved by reducing the expression of the millet gene Seita.8G189900 and the millet gene Seita.8G190200 .

[0008] Preferably, the protein quality of millet is improved by reducing the expression of the millet gene Seita.8G189900 and the millet gene Seita.8G190200 , and then controlling the content of prolamin, essential and non-essential amino acids in millet.

[0009] The plurality of essential and non-essential amino acids include at least one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

[0010] Preferably, the method for reducing the expression of the millet gene Seita.8G189900 and the millet gene Seita.8G190200 includes knocking out, inhibiting or silencing the millet gene Seita.8G189900 and the millet gene Seita.8G190200 .

[0011] Preferably, the knocking out is performed by using a CRISPR / Cas9 system. The protein translated by the gene after the knocking out by using the CRISPR / Cas9 system has no original function or cannot translate a functional protein.

[0012] Preferably, the nucleotide sequence of the target site of gRNA in the CRISPR / Cas9 system is shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0013] Preferably, improving the quality of millet protein refers to reducing the content of prolamine in millet, and increasing the content of various essential and non-essential amino acids in millet.

[0014] Compared with the prior art, the present application has the following beneficial effects:

[0015] 1. The present application provides a millet gene Seita.8G189900 and Seita.8G190200 application in controlling the quality of prolamine. The present application proves two genes that can improve the functional quality of millet, and can improve the eating quality of millet, thereby providing materials for breeding high-quality millet. The application technology provided by the present application not only reveals the relationship between the genes regulating the expression of prolamine in millet and the quality of millet protein, but also reveals how the millet gene Seita.8G189900 and the millet gene Seita.8G190200 regulate the expression of prolamine.

[0016] 2. The present application proves two genes in millet that can down-regulate the content of prolamine, and reducing the expression level of the two genes can reduce the content of prolamine in millet. Knocking out the two genes can not only down-regulate the content of prolamine, but also up-regulate the content of amino acids, thereby increasing the nutritional value of millet and providing new genetic resources for improving the quality of protein.

[0017] 3. The present application knocks out the genes Seita.8G189900 and Seita.8G190200 controlling the content of prolamine in millet, proves the relationship between prolamine and the two genes, and explores the influence of the two genes on the quality of prolamine.

[0018] 4. The present application knocks out and proves the genes Seita.8G189900 and Seita.8G190200 controlling the content of prolamine in millet, proves the relationship between prolamine and the two genes, and reduces the content of prolamine in millet by reducing the expression level. The difference in the content of prolamine between the transgenic material and the wild type material is caused by the change in the coding region of the gene, thereby reducing the level of protein encoded by the gene. By further determining the gelatinization parameters, it is found that the change in the content of prolamine in millet can improve the eating quality of millet. Knocking out the genes can not only improve the nutrition of millet, but also improve the eating quality of millet. Therefore, the present application provides new genetic resources for improving the quality of millet protein. BRIEF DESCRIPTION OF DRAWINGS

[0019] Seita.8G189900 are the homozygous mutant types of T0 lines in the present application: wherein the red color represents the position of the base editing mutation. Seita.8G190200 and Seita.8G189900 are the homozygous mutant types of T0 lines in the present application: wherein the red color represents the position of the base editing mutation.

[0020] Seita.8G190200WT and Seita.8G189900 , Seita.8G190200 grain phenotype: wherein, Seita.8G189900 A graph in is WT and Seita.8G190200 , Seita.8G189900 is the length-width ratio of unhusked grain, wherein A1 is WT and Seita.8G190200 , Seita.8G189900 is the length ratio of unhusked grain, A2 is WT and Seita.8G190200 , Seita.8G189900 is the width ratio of unhusked grain; Seita.8G190200 B graph in is WT and Seita.8G189900 , Seita.8G190200 unhusked grain width ratio, wherein B1 is WT and Seita.8G189900 unhusked grain width ratio, B2 is WT and Seita.8G190200 unhusked grain width ratio; wherein A1, A2, B1 and B2 are multiple samples of parallel experiments; the horizontal line is: scale: 1 cm; Seita.8G189900 C graph in is WT and Seita.8G190200 , Seita.8G189900 grain length statistics; Seita.8G190200 D graph in is WT and Seita.8G189900 , Seita.8G190200 grain width statistics; Seita.8G189900 E graph in is WT and Seita.8G190200 , Seita.8G189900 thousand-grain weight statistics.

[0021] Seita.8G190200 WT and Seita.8G189900 , Seita.8G190200 alcohol-soluble protein content, amino acid content schematic diagram: wherein: Seita.8G189900 A graph in is Seita.8G190200 , ko2 alcohol-soluble protein content; Seita.8G189900 B graph in is WT and Seita.8G190200 , Seita.8G189900 essential amino acid content; Seita.8G190200 C graph, D graph, E graph, F graph in are respectively WT and Seita.8G189900 , Seita.8G190200 non-essential amino acid content; Seita.8G189900 G graph in is WT and Seita.8G190200 , Seita.8G189900 serotonin and GABA content; Seita.8G190200 H graph in is WT and Seita.8G189900 , Seita.8G190200 sialic acid content; Seita.8G189900 I graph in is WT and Seita.8G190200 , Seita.8G189900 taurine content; Seita.8G190200 J graph in is WT and Seita.8G189900 , Seita.8G190200 cystathionine content; Seita.8G189900K graph in Figure 1 is the RVA analysis of WT and Seita.8G190200 , Seita.8G189900 the content of succinic acid.

[0022] Seita.8G190200 K graph in Figure 1 is the RVA analysis of WT and Seita.8G189900 , Seita.8G190200 K graph in Figure 1 is the RVA analysis of WT and Seita.8G189900 , Seita.8G190200 , Seita.8G189900 K graph in Figure 1 is the RVA analysis of WT and Seita.8G190200 , Seita.8G189900 K graph in Figure 1 is the RVA analysis of WT and Seita.8G190200 , Seita.8G189900 K graph in Figure 1 is the RVA analysis of WT and Seita.8G190200 , K graph in Figure 1 is the RVA analysis of WT and ,

[0023] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples, but should not be understood as a limitation of the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0024] Example 1: Genetic transformation of genes Seita.8G189900 and Seita.8G190200 in millet

[0025] 1. Two guide RNAs (Target: CGATGAACCCCACTGCCTTA, as shown in SEQ ID NO. 5; CAGGGCAGACTGTTGTTGTA, as shown in SEQ ID NO. 6) of genes Seita.8G189900 and gene Seita.8G190200 were designed on the knockout vector using the CRISPR-P website. The structure of genes Seita.8G189900 and gene Seita.8G190200 is shown in Seita.8G189900 .

[0026] Among them, gene Seita.8G190200 is millet gene Seita.8G189900 , and its nucleotide sequence is shown in SEQ ID NO. 1:

[0027] ATGGTGGCCAAGATATGTACCCTGCTTGTGCTCCTTGCTCTTTCAGCTAGCGCTGCAACTGCCGTCCTTATTCCGCAGTGCTCTGCCGCTATTCCCCAGTACCTCCCTCATGTGACAGCACTGGGATATGGAAACCCAATTGTGCAATCCTGCAGGCTACAACAGGCTCTCGCGGCAAGCATCTTATCATCACCGGCCGTGTTCTTACAACAACAGTCCGCCCTATTGCAGCAGCAATACTTGGCGCATTTGACATTACAGAGCATCATGGCACAACAACACCAGGTGCTTTCACCATTCAGCCGACTATCCTTGGCTAGCCCCGCCGCATACTGGCAACACCAACAGCTGCTTCCATTCAACCAGCTAGCCGTGGAGAATGCCTACTTGCAGCAACAGCAGCTGCTTCCACTCAACCCACTTGCTGTTGGCAACCCCGCCGCCTTCTGGCAACAACAACAGCTACTACGTGTCAACCCATTTTCTGCGATGAACCCCGCCGCATTCTGGCAACAACAGCAGCTATTGCGTGTCAACCCAGTTGCTGCGATGAACCCCGCCACTTTCTGGCAACAACAACAGCTACTGCGCATCAACCCACTTGCTGCGATGAACCCCACTGCCTTATGGCAACAACAGCAGCTACTGCGTATCAACCCACTTGCTACGATGAACCCTGCCGCTTTCTTGCAGCAACCAATCATTGGTTCTGGCCTTTTCTAG.

[0028] millet gene Seita.8G190200 The amino acid sequence of the expressed protein is shown as SEQ ID NO. 3:

[0029] MVAKICTLLVLLALSASAATAVLIPQCSAAIPQYLPHVTALGYGNPIVQSCRLQQALAASILSSPAVFLQQQSALLQQQYLAHLTLQSIMAQQHQVLSPFSRLSLASPAAYWQHQQLLPFN QLAVENAYLQQQQLLPLNPLAVGNPAAFWQQQQLLRVNPFSAMNPAAFWQQQQLLRVNPVAAMNPATFWQQQQLLRINPLAAMNPTALWQQQQLLRINPLATMNPAAFLQQPIIGSGLF*.

[0030] Gene Seita.8G189900 Millet genes Seita.8G190200 Its nucleotide sequence is shown in SEQ ID NO.2:

[0031] TGAGACCCAGTAGCAGAAATTGAAACCCACTAGCAGCCTCATAATAGCAACAATGGCAGCCAAGATATGTACCCTCCTTGTGCTCCTTGCTCTTTCAGCTAGCGCTGCCACTGCCATCCTTATTCCACAGTGCTCCACTATTCCCCAGTACCTCCCACATCTGACAGCCCTAGGATATGGAAACCCAATTGTGCAGCCCTACAGGCTGCAGCAGGCTCTTGCAGCAAGCATCTTATCATCACCGGCCATGTCCTTACAACAACAGTCTGCCCTGTGGCAGCATCAATCCTTGGCAAGCCCTGCCGCATACTGGCAACAACAACAGCTGCTTCCATTCAACCAGCTAGCTTTGGCGAATGCCTTCTCGCAGCAACAACAGCTGCTTCCATTCAACCAACTAGCTTTGGCGAATGCCTTCTCGCAGCAACAACAGCTTCTTCCATTCAACCAGCTAGCTTTGGCGAATGCCTTCTCGCAGCAACAACAGCTGCTTCCATTCAACCAGCTAGCTTCGGCGAATGTCTTCCGTCAACAACTGCTTCCATTCAACCAACTAGCTATCACCAACCCCGCCGCCTTCTGGCAACAACAGCAGCTACTGCGTGTCAACCCACTTGCTGCGATGAACGCCGCTGCCTTCTTGCAGCAACCAATCATTGGTTCTGGCCTTTGCTAGATCAATTATTTGTTGTACTTTTATAATAAAGTTTGCATGCCGACTCGTGTGATCCTAAGAAATAAGAAAACTGTTGGTTGAGATTCTATTGTCCTA.

[0032] millet gene Seita.8G189900 The amino acid sequence of the expressed protein is shown as SEQ ID NO. 4:

[0033] MAAKICTLLVLLALSASAATAILIPQCSTIPQYLPHLTALGYGNPIVQPYRLQQALAASILSSPAMSLQQQSALWQHQSLASPAAYWQQQQLLPFNQLALANAFSQQQQLLPFNQLALANAFSQQQQLLPFNQLALANAFSQQQQLLPFNQLASANVFRQQLLPFNQLAITNPAAFWQQQQLLRVNPLAAMNAAAFLQQPIIGSGLC*.

[0034] The specific operation is as follows:

[0035] 2, the website (https: / / home.firefoxchina.cn / ) is designed to knock out primers, and the pHUE411-cas9 knockout vector is constructed by the target vector pHUE411, which is transformed into E. coli, and after colony PCR identification, the sample is sequenced, then the bacteria are shaken, and the plasmid is extracted. Then the plasmid is sent to Wuhan Boyuan Biotechnology Co., Ltd., and is transformed into the mature seed-induced callus of millet variety Ci846 to perform genetic transformation.

[0036] Among them, the knockout primer and sequence designed are as follows:

[0037] The nucleotide sequence of MT1T2-F is shown in SEQ ID NO. 7:

[0038] 5'-AATAATGGTCTCAGGCGCGATGAACCCCACTGCCTTA-3'.

[0039] The nucleotide sequence of MT1T2-F0 is shown in SEQ ID NO. 8:

[0040] 5'-GCGATGAACCCCACTGCCTTAGTTTTAGAGCTAGAAATAGC-3'.

[0041] The nucleotide sequence of MT1T2-R0 is shown in SEQ ID NO. 9:

[0042] 5'-CAGGGCAGACTGTTGTTGTACGCTTCTTGGTGCC-3'.

[0043] The nucleotide sequence of MT1T2-R is shown in SEQ ID NO. 10:

[0044] 5'-ATTATTGGTCTCTAAACCAGGGCAGACTGTTGTTGTA-3'.

[0045] The construction of pHUE411-cas9 knockout vector is as follows:

[0046] (1) The saved pBUE411 E. coli was plasmid-extracted to obtain the target vector pHUE411.

[0047] (2) The four knockout primers were mixed in equal volumes at the normal primer concentration (10 micromoles) and F0 / R0 was diluted 20 times (2 micromoles). The target vector pBUE411 was used as a template, and the primers were mixed for PCR amplification.

[0048] (3) The gel was cut and the PCR product was recovered, and the DNA fragment was purified using a Simgen kit.

[0049] (4) The enzyme digestion and ligation system was prepared according to Table 1.

[0050] (5) The enzyme digestion and ligation were performed at 37°C overnight, and the ligation product was obtained by incubation at 80°C for 10 min, obtaining the pHUE411-cas9 knockout vector.

[0051] Table 1 Enzyme digestion and ligation system

[0052]

[0053] 10 μL of the ligation product was transformed into E. coli competent cells, and the transformed E. coli was cultured using kanamycin-resistant medium at 37°C for 12 hours. Bacterial colony PCR identification was performed.

[0054] Twenty colonies were picked and subjected to PCR identification to determine whether the ligation was successful. The identification primers were OsU3-F and TaU3-F. Three positive bands were selected for sequencing, and the correct sequencing results were used for bacterial preservation.

[0055] The nucleotide sequence of OsU3-F is shown in SEQ ID NO. 11:

[0056] 5'-GACAGGCGTCTTCTACTGGTGCTAC-3'.

[0057] The nucleotide sequence of TaU3-F is shown in SEQ ID NO. 12:

[0058] 5'-TTGACTAGCGTGCTGATAATTTGTG-3'.

[0059] The plasmid of the correct sequencing bacteria was extracted, and the transformation was performed by Wuhan Boyuan Biotechnology Co., Ltd.

[0060] 3. Then, the gene Seita.8G190200 and the gene Seita.8G189900The specific gene detection primer (the sequence of the specific gene detection primer is shown in Table 2) was identified by sanger sequencing to identify the mutant change type, and two homozygous millet transgenic seedlings with Ci846 as the background were selected Seita.8G190200 , Seita.8G189900 and Seita.8G190200 .

[0061] Among them, Seita.8G189900 The gene Seita.8G190200 Two T bases are replaced by two C bases, and the gene Seita.8G189900 An C base is inserted. Seita.8G190200 Among them, Seita.8G189900 Two T bases are replaced by two C bases, and the gene Seita.8G190200 An T base is inserted.

[0062] In order to verify the effect of double-target gene editing, we compared the grain length and width and thousand seed weight of the knockout line and wild type by WSeen SC-G automatic seed analyzer, and found that Seita.8G189900 The grain length of the grain length and wild type has no difference, and the grain width is significantly smaller than that of the wild type; Seita.8G190200 The grain length and grain width of the grain length and grain width are significantly smaller than that of the wild type (A figure in Seita.8G189900 and B figure in Seita.8G190200 ). And the data statistics also prove this point (C figure in Seita.8G189900 and D figure in Seita.8G190200 ), and Seita.8G189900 , Seita.8G190200 The thousand seed weight of the thousand seed weight is significantly reduced (E figure in Seita.8G189900 ).

[0063] Table 2 Specific gene detection primer and sequence

[0064]

[0065] Example 2: Knockout line Seita.8G190200 and Seita.8G189900 Alcohol-soluble protein and amino acid content analysis

[0066] The present application determines the alcohol-soluble protein content in WT and Seita.8G190200 and Seita.8G189900 Millet by plant wheat alcohol-soluble protein ELISA detection kit. The analysis of amino acids and their derivatives is completed by cooperating with MetWare (MetWare, http: / / www.metware.cn / ) and using AB Sciex QTRAP 6500 LC-MS / MS platform.

[0067] Firstly, the WT and Seita.8G190200 and Seita.8G189900Seed samples were mechanically homogenized. 0.05 g of the homogenized sample was extracted with 500 µL of 70% (w / w) methanol-water solution. The sample was vortexed at 2500 rpm for 3 minutes, followed by centrifugation at 12000 rpm and 4°C for 10 minutes. After centrifugation, 300 μL of the supernatant was transferred to a new centrifuge tube and incubated at -20°C for 30 minutes. This was then repeated under the same conditions (12000 rpm, 4°C, 10 minutes). Finally, after centrifugation, 200 μL of the supernatant was filtered through a protein precipitation plate for LC-MS analysis. The effects of [the study / analysis] were investigated. Seita.8G190200 and Seita.8G189900 Phenotypic values ​​of prolysin and amino acid content in millet. Among them, the knockout system... Seita.8G190200 and Seita.8G189900 Abbreviated as Seita.8G190200 and Seita.8G189900 .

[0068] The results showed that, compared to WT, genes Seita.8G190200 and genes Seita.8G189900 After the function is lost, Seita.8G190200 and Seita.8G189900 The content of prolysin decreased by 29.90% and 28.47%, respectively. Seita.8G190200 (Figure A in the text); except Seita.8G189900 The levels of tryptophan and serotonin in the samples were not significantly different from those in the WT group. Seita.8G190200 and Seita.8G189900 The content of all other amino acids increased significantly. Seita.8G190200 Figure B in the middle - Seita.8G189900 (K-chart in the diagram).

[0069] Based on the above results, it is proven that reducing gene... Seita.8G190200 and genes Seita.8G189900 The expression of this substance can increase the amino acid content of millet grains. Here, the amino acids include essential amino acids, non-essential amino acids, and some functional amino acids.

[0070] Example 3: Knockout System Seita.8G190200 and Seita.8G189900 Gelatinization degree analysis

[0071] This invention weighed 3g of ground millet and used a rapid viscosity analyzer (RVA) to detect changes in starch quality in the knockout lines, analyzing their starch gelatinization characteristics. The results are shown below. Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 Seita.8G189900 Seita.8G190200 .

[0072] turn out, ko1 and ko2The peak viscosity level of the millet is significantly increased compared with the wild type, and the lowest viscosity and final viscosity are also significantly increased. However, the attenuation degree and the rebound value are not different. Therefore, the knockout gene can improve the nutritional quality of the millet to a certain extent and improve the taste quality.

[0073] With the continuous improvement of the quality of life requirements of contemporary young people, the nutritional components of food are more and more concerned by the public, and the exploration of the nutritional quality of millet will become an important direction of millet quality breeding in the future. The grain is an important organ for nutrient storage. Among them, the protein in the grain of millet mainly exists in the form of alcohol-soluble protein (about 60%). Studies have shown that the higher the protein content, the worse the cooking and taste quality of rice. Therefore, the present application finds that the gene Seita.8G189900 and the gene Seita.8G190200 not only can determine the content of alcohol-soluble protein in millet, but also can change the taste quality of millet, and may be an ideal gene for improving the nutritional quality of millet.

[0074] It should be noted that when numerical ranges are involved in the present application, it is understood that each intervening value, to the utmost limits of the ranges specified, is also contemplated. For the purpose of this application, the conventional "parts by weight" and "percentage by weight" units used to express the relationships between the individual components or amounts have the weight relationship indicated. All numerical values are "approximate", meaning close to, but not necessarily the precise value specified. In this case, the numerical values are derived from instrument readouts and are reported as obtained.

[0075] Although preferred embodiments of the application have been described, a person of ordinary skill in the art, once aware of the basic inventive concept, can make additional changes and modifications to these embodiments, and these changes and modifications all fall within the scope of all changes and modifications of the present application.

Claims

1. Millet genes Seita.8G189900 and Seita.8G190200 Its application in improving the quality of gluten protein is characterized by... By reducing the millet gene Seita.8G189900 and the millet gene Seita.8G190200 The expression of the millet gene is used to improve the quality of millet protein; Seita.8G189900 The amino acid sequence of the expressed protein is shown in SEQ ID NO.3, which is the millet gene. Seita.8G190200 The amino acid sequence of the expressed protein is shown in SEQ ID NO.4; Improving the quality of millet protein means reducing the content of prolamins in millet while increasing the content of essential and non-essential amino acids in millet. Reduce the millet gene Seita.8G189900 and the millet gene Seita.8G190200 The expression method includes knocking out the millet gene. Seita.8G189900 and the millet gene Seita.8G190200 .

2. The application according to claim 1, characterized in that, The knockout was performed using the CRISPR / Cas9 system.

3. The application according to claim 2, characterized in that, The nucleotide sequences of the target sites of gRNA in the CRISPR / Cas9 system are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. The application according to claim 1, characterized in that, The millet gene Seita.8G189900 The nucleotide sequence is shown in SEQ ID NO.1, and the millet gene is described. Seita.8G190200 The nucleotide sequence is shown in SEQ ID NO.2.

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