GPC1 gene and application thereof

Abstract

The invention discloses a GPC1 gene and application thereof, and relates to the technical field of plant genetic engineering, and an amino acid sequence coded by the GPC1 gene is shown as SEQ ID NO.2. The increase of the single gene expression quantity of the CPC1 gene leads to the significant increase of the whole grain protein content of the rice, but the knockout of the gene leads to the significant reduction of the whole grain protein content of the rice, and the whole grain protein content of the rice in a near-isogenic line of the allele has significant difference. The gene is used for improving the characters of rice varieties, has huge application potential and prospect, and provides a new gene resource for providing rice nutritional quality and cooking quality breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically to a... GPC1 Gene and its application: This gene regulates the protein content of whole grain rice. Background Technology

[0002] Rice is a cereal crop. Whole grain rice refers to whole grain rice with the husk removed, also known as brown rice, which includes the starchy endosperm, germ, and bran. Analysis shows that adequate intake of whole grains can reduce the risk of various diseases, including cardiovascular and gastrointestinal diseases, by about 30%. Compared to refined rice, whole grains can increase the edible portion by more than 20%, and this increased portion is rich in various nutrients and functional components.

[0003] In whole rice, protein is the second most abundant nutrient after starch, making it a primary protein source for populations whose staple food is rice. Rice protein contains essential amino acids that the human body cannot synthesize, and the proportions of these amino acids are well-balanced. It is low in fat, cholesterol-free, and has low allergenicity, making it an ideal plant protein source. Researching rice functional genomics to identify and functionally analyze the genes controlling rice protein content can provide a crucial technological foundation for molecular design breeding to improve the nutritional value and cooking quality of whole-grain rice.

[0004] Given the importance of whole grains, the discovery and application of genes that control the protein content of rice, and the improvement of the nutritional quality and cooking taste of whole rice, are particularly urgent and are technical problems that need to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a GPC1 Genes and their applications.

[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution: A GPC1 gene, the GPC1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0007] Preferably, the GPC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] In a second aspect, a GPC1 protein is provided, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] Thirdly, it provides ways to improve the above. GPC1 Application of gene expression level or enhanced GPC1 protein activity in increasing the protein content of whole grain rice.

[0010] Fourthly, to provide improvements to the above. GPC1 The application of gene expression levels or enhanced GPC1 protein activity in rice genetic breeding, wherein the genetic breeding is for cultivating rice germplasm with high whole grain protein content.

[0011] Fifthly, providing improvements GPC1 Application of biomaterials with high gene expression levels in increasing the protein content of whole rice, wherein the biomaterial is one of the following: a. Nucleic acid molecules capable of encoding amino acid sequences such as SEQ ID NO.2; b. An expression cassette capable of overexpressing the nucleic acid molecules described in a; c. A recombinant vector containing the expression cassette described in a; d. Recombinant microorganisms containing the expression cassette described in a or the recombinant vector described in b; e. A non-renewable plant portion containing the expression cassette described in a, the recombinant vector described in b, or the recombinant microorganism described in c.

[0012] Sixthly, a method for increasing the whole grain protein content of rice is provided, wherein the method involves hybridization, backcrossing, self-pollination, or transgenic methods to increase the protein content of the rice. GPC1 Gene transfer to recipient varieties significantly increases the protein content of whole grain rice.

[0013] Beneficial effects: This invention provides CPC1 This gene and its applications show that increased expression of this single gene significantly increases the protein content of whole rice, while knocking out the gene significantly decreases the protein content. Furthermore, there are significant differences in the protein content of whole rice among near-isogenic lines of this allele. Utilizing this gene to improve rice varietal traits has enormous application potential and prospects, providing a new genetic resource for breeding rice to improve nutritional and cooking qualities. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 This is a comparison of the nutritional components of whole grains from near-isogenic lines; where AC represents the gluten and prolamin content, soluble sugar content, and fat content of whole grains from near-isogenic lines, respectively.

[0016] Figure 2 pU1301- GPC1Overexpression vector plasmid map.

[0017] Figure 3 For overexpression of plants and wild-type HHZ GPC1 Comparison of gene expression levels.

[0018] Figure 4 Overexpression GPC1 Comparison of whole grain nutritional components between genetically modified and wild-type HHZ; where AC represents the content of whole grain gluten and prolamins, soluble sugar content and fat content, respectively.

[0019] Figure 5 For the gene knockout vector pYLCRISPR / Cas9Pubi- GPC1 Schematic diagram.

[0020] Figure 6 for GPC1 The mutated sequence after gene knockout.

[0021] Figure 7 The nutritional composition of whole grains was compared between gene knockout mutant plants and wild-type HHZ; where AC represents the content of whole grain gluten and prolamins, soluble sugars, and oils, respectively. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1: Carrying JZ type GPC1 Near-isogenic lines of alleles significantly increase whole grain protein content. The NIL-HHZ and NIL-JZ(HHZ) near-isogenic lines were obtained by crossing the indica rice variety HHZ (Huang Huazhan) with the japonica rice variety JZ1560 (Ji Zi 1560) (JZ), using HHZ as the recurrent parent for multiple backcrosses and... GPC1 The background for functional gene marker-assisted selection was HHZ, and the target gene was... GPC1 The alleles were HHZ and JZ, respectively.

[0025] JZ1560 typeGPC1 Original CDS sequence of the gene: ATGACGGGCAGTAGTTGTCCGGCCGATTCTTCCCAGCTGCTGTACCCTCGCCGGGGTGCCCCCACCACCACCACCACCTCCCGTCCTCCTCTCCATCCGCTCATCGCTCATGCGCCCTACAACGTCGTCCTCCACCGATCTGTCGTCCTCTCCATCAGCTCAGCTCGTGATCAGGCTGAGCTCGCGAGCTTCTGGTGCTATATAAGGCTGGGTGGTGGTGCAAGTGCGAAGCGAGCGGCCGGTGAGGACGATCGATCGAGATCGAGCTTGACGGCGGCGAGAGGAGGAGGAGGGGAGACGATGAGCGGGCGGGGGAGGGGGGACCCGCTGGTGCTGGGGAGGGTGGTGGGGGACGTGGTGGACCCGTTCGTGAGGAGGGTGGCGCTGCGGGTGGCGTACGGAGCGCGGGAGGTGGCCAACGGCTGCGAGCTCCGCCCCTCCGCCGTCGGCGACCAGCCCCGCGTCGCCGTCGGCGGCCCCGACATGCGCACCTTCTACACCCTGGTGATGGTGGATCCGGACGCGCCGAGCCCGAGCGATCCAAACCTCAGGGAGTACCTGCACTGGCTGGTCACCGACATCCCGGCTACCACAGGAGTCTCTTTTGGGACAGAGGTGGTGTGCTACGAGAGCCCGCGGCCGGTGCTGGGGATCCACAGGCTGGTGTTCCTGCTGTTCGAGCAGCTGGGGCGGCACACGGTGTACGCACCGGGGTGGCGCCAGAACTTCAGCACCCGCGACTTCGCCGAGCTCTACAACCTCGGCCTCCCTGTCGCCGCCGTCTACTTCAACTGCCAGAGGGAGTCTGGAACCGGAGGAAGAAGAATGTGA, SEQ ID NO.1.

[0026] Amino acid sequence of GPC1 protein of JZ1560 type: MTGSSCPADSSQLLYPRRGAPTTTTTSRPPLHPLIAHAPYNVVLHRSVVLSISSARDQAELASFWCYIRLGGGASAKRAAGEDDRSRSSLTAARGGGGETMSGRGRGDPLVLGRVVGDVVDPFVRRVALRVAYGAREVAN GCELRPSAVGDQPRVAVGGPDMRTFYTLVMVDPDAPSPSDPNLREYLHWLVTDIPATTGVSFGTEVVCYESPRPVLGIHRLVFLLFEQLGRHTVYAPGWRQNFSTRDFAELYNLGLPVAAVYFNCQRESGTGGRRM, SEQ ID NO.2.

[0027] Functional gene marker primer sequences: Del5-F: CGTCGTCTCGTGTCGTCT, SEQ ID NO.3; Del5-R: GATCTCGTGGCTGCAAGT, SEQ ID NO.4.

[0028] Near-isogenic lines NIL-HHZ and NIL-JZ(HHZ) were planted in the field environment of the China National Rice Research Institute Experimental Farm in Fuyang District, Hangzhou City. Mature seeds were harvested, and after removing the husks, the nutritional components of the whole grain seeds were identified. The results showed that the NIL-JZ(HHZ) near-isogenic line had a significantly increased glutenin and prolamin content (17.6%) compared to NIL-HHZ, while there was no significant difference in soluble sugar and oil content (see Appendix). Figure 1 ).

[0029] Example 2 Overexpression GPC1 Genes significantly increased the whole grain protein content of transgenic plants. 1. Construction GPC1 Gene overexpression vector pU1301- GPC1 Based on the cDNA sequence of the GPC1 gene published in the Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ), a pair of PCR-specific primers with KpnI and BamHI linkers were designed (F: CGCGGATCCATGACGGGCAGTAGTTGT, SEQ ID NO.5; R: AAAACTGCAGCTCACATTCTTCTTCCTCCG, SEQ ID NO.6). Using cDNA from rice variety JZ1560 as a template, PCR amplification was performed. The reaction mixture consisted of 25 μL of 2×Taq Mix, 1 μL of cDNA, 1 μL of primer-F / R, and water to a final volume of 50 μL. The PCR program was as follows: 95℃ for 3 min, 95℃ for 30 sec, 55℃ for 30 sec, 72℃ for 60 sec, 72℃ for 5 min, 10℃ for 5 min, for 35 cycles. Finally, the full-length cDNA of the target gene, from the start codon to the stop codon, is obtained. The obtained gene... GPC1 The sequence was constructed into the pU1301 expression vector via homologous recombination (see attached plasmid map). Figure 2 ), to obtain the overexpression vector pU1301- GPC1 .

[0030] The overexpression vector pU1301- GPC1 HHZ rice callus was genetically transformed using Agrobacterium-mediated transformation and identified as... GPC1 Plants with overexpressed genes.

[0031] RNA was extracted from transgenic plants during the seedling stage, and cDNA was obtained by reverse transcription and identified by qRT-PCR. GPC1 Gene expression levels. Specific operational procedures: Take wild-type HHZ and OE - GPC1 Total RNA was extracted from leaves of overexpressing plants using the Trizol method in triplicate. cDNA was synthesized using the Hifair® AdvanceFast 1st Strand cDNA Synthesis Kit. The cDNA was amplified using a 10 μl system with the SYBR Green kit. Amplification was performed using quantitative real-time PCR with primer sequences SEQ ID NO. 5 and SEQ ID NO. 6.

[0032] The amplification program consisted of three cycles: 95℃ for 3 minutes, 95℃ for 10 seconds, and 60℃ for 30 seconds, for a total of 38 cycles. The Ubiquitin gene was used as an internal control. -ΔΔCT The method calculates the relative expression level of genes. For example...Figure 3 As shown, it was successfully achieved. GPC1 The gene was overexpressed in OE-GPC1 plants.

[0033] 2. GPC1 Comparison of whole grain nutrients in plants with overexpressed genes Harvest wild-type HHZ and OE - GPC1 Mature seeds from overexpressing plants were dried to constant weight in a 42°C oven. After removing the seed coat (whole grain rice), the seeds were ground and sieved for identification of the whole grain nutritional components.

[0034] The whole grain protein content was identified using the Coomassie Brilliant Blue method. The specific procedure was as follows: Weigh 0.1 g of whole grain flour into a 2 ml centrifuge tube, add a steel ball and 1 mL of 0.1 mol / L sodium hydroxide solution, grind using a sampler (50 Hz 60 s), let stand for 30 minutes, centrifuge at 3500 rpm for 15 minutes, and then transfer the supernatant to another centrifuge tube to obtain the gluten solution. Replace the 0.1 mol / L sodium hydroxide solution with 70% ethanol solution and repeat the above procedure to obtain the alcohol-soluble protein solution. Take 3 μl from each of the gluten and alcohol-soluble protein solutions and mix with 297 μl of Coomassie Brilliant Blue G-250 staining solution, let stand for 2 minutes, and then measure the absorbance at 595 nm using a microplate reader. The concentrations of gluten and alcohol-soluble protein in the sample were then determined using a standard curve.

[0035] The main method for determining soluble sugars is as follows: Weigh 0.1g of whole grain rice flour and add it to a 15ml centrifuge tube containing 4ml of 80% alcohol. Place the tube in an 80℃ water bath and stir continuously for 50 min. Centrifuge at 2000 rpm for 3 min and collect the supernatant into a new 15ml centrifuge tube. Repeat the collection twice from the residue and combine the supernatants. Dry the residue in the centrifuge tube in an 80℃ oven and store it for starch determination. Add 10mg of activated carbon to the collected supernatant and stir continuously in an 80℃ water bath for 40 min. Filter the solution in a funnel and collect the filtrate. Make up to 15ml. Take 500μL of the alcohol extract and add it to a centrifuge tube containing 500μL of ddH2O and 3mL of 2% anthrone solution (prepared with concentrated sulfuric acid). Add 2M NaOH, boil in a 100℃ water bath for 5 min, boil in a 100℃ water bath for 15 min, cool to room temperature, and measure the absorbance at 620 nm. Plot a standard curve for soluble sugars and calculate the concentration of soluble sugars in the samples.

[0036] The extraction and determination of whole grain oil mainly followed the Folch method. The specific steps are as follows: Wash a 20 mL glass bottle, place it in an 80℃ oven to constant weight, and then allow it to cool to room temperature in a drying oven. Accurately measure and record the weight of the glass bottle, denoted as A. Weigh 1 g of whole grain rice flour (denoted as B) and place it in a 50 mL centrifuge tube. Add 20 mL of a chloroform and methanol (2:1) mixture to the 50 mL centrifuge tube, shake to mix the whole grain rice flour and the mixture thoroughly, and place in a shaker for 2 h. Centrifuge at 2000 rpm for 5 min, collect the supernatant, and transfer it to a 50 mL centrifuge tube. Add 0.2 times the volume of 0.9% NaCl solution to the sample supernatant, vortex for 5 sec, and centrifuge at 2000 rpm for 10 min to obtain a two-phase liquid separation state. Collect the lower liquid layer, which is the oil contained in the chloroform layer. Based on the volatility characteristics of chloroform, the glass bottle was dried on an 80°C stirred heater in a fume hood until its weight remained constant, denoted as C. The total fat content was calculated as follows: Fat content (%) = 100 * (CA) / B.

[0037] The results showed that overexpression OE - GPC1 Compared to the wild-type HHZ, the transgenic plants showed a 27.5% increase in the content of whole grain gluten and prolamins, while there was no significant difference in the content of soluble sugars and oils in whole grains (see appendix). Figure 4 This is consistent with the results of the near-isogenic line experiment in Example 1.

[0038] Example 3 Knockout GPC1 Genes significantly reduce the whole grain protein content of transgenic plants. 1. Construction GPC1 Gene knockout vector pYLCRISPR / Cas9Pubi- GPC1 .

[0039] According to GPC1 Gene sequence (e.g., SEQ ID No. 1), and primers gRNA-GPC1-F (GGCACCAGCTGCTGTACCCTCGC, SEQ ID No. 7) and gRNA-GPC1-R (AAACGCGAGGGTACAGCAGCTGG, SEQ ID No. 8) were designed based on appropriate target sequences. The primer pair gRNA-GPC1-F / R was denatured and annealed to obtain the gRNA-GPC1-F / R dimer. The gRNA-GPC1-F / R dimer was ligated into the pYLgRNA-U3 vector to obtain an intermediate vector. The intermediate vector was then ligated into the pYLCRISPR / Cas9Pubi-H final vector (see Appendix) using a cut-and-ligate method. Figure 5 ), to obtain a single target point GPC1CRISPR / Cas9 gene editing vector pYLCRISPR / Cas9Pubi- GPC1 After successful vector construction, HHZ callus was transformed using Agrobacterium-mediated transformation to obtain T0 generation transgenic plants.

[0040] DNA was extracted from transgenic plants, primers were designed to amplify the target sequence for gene knockout, and identification was performed. GPC1 Sequence variation after gene knockout, amplification primers: Crgpc1-F: AGAGACCATTTCCCTCCT, SEQ ID NO.9; Crgpc1-R: AGGACGACGTCGTAGG, SEQ ID NO. 10.

[0041] Sequencing results of the amplified products showed that GPC1 Gene knockout mutant plants gpc1 - 1 and gpc1 - 2 Compared to wild-type HHZ, GPC1 The genes were missing 7 and 2 bases respectively (see appendix). Figure 6 The results confirmed that the constructed knockout vector pYLCRISPR / Cas9Pubi was successfully mutated. GPC1 Gene.

[0042] 2. GPC1 Comparison of whole grain nutrients in gene knockout mutant plants Harvest wild-type HHZ and knockout mutants gpc1 - 1 and gpc1 - 2 Mature seeds were used to determine the concentrations of gluten and prolamins, soluble sugar content, and oil content in the samples using the method described in Example 2.

[0043] The results showed that, compared to wild-type HHZ, the knockout mutant gpc1 - 1 and gpc1 - 2 The content of whole grain gluten and prolamins decreased significantly by 11.2%, while the content of soluble sugars and fats showed no significant difference (see appendix). Figure 7 ).

[0044] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0045] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kind GPC1 Genes, characterized by, The GPC1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. A GPC1 protein, characterized in that, The amino acid sequence of the GPC1 protein is shown in SEQ ID NO.

2.

3. Improvement of claim 1 GPC1 Application of gene expression level or enhanced GPC1 protein activity as described in claim 2 in increasing the protein content of whole grain rice.

4. Improvement of claim 1 GPC1 The application of gene expression level or enhanced GPC1 protein activity as described in claim 2 in rice genetic breeding is characterized by, The genetic breeding program aims to cultivate rice germplasm with high whole grain protein content.

5. Improve GPC1 The application of biomaterials with high gene expression levels in increasing the protein content of whole rice is characterized by: The biomaterial is one of the following: a. Nucleic acid molecules capable of encoding amino acid sequences such as SEQ ID NO.2; b. An expression cassette capable of overexpressing the nucleic acid molecules described in a; c. A recombinant vector containing the expression cassette described in a; d. Recombinant microorganisms containing the expression cassette described in a or the recombinant vector described in b; e. A non-renewable plant portion containing the expression cassette described in a, the recombinant vector described in b, or the recombinant microorganism described in c.

6. A method for increasing the protein content of whole rice, characterized in that, The method described in claim 1 is to use hybridization, backcrossing, self-crossing, or transgenic methods to... GPC1 Gene transfer to recipient varieties significantly increases the protein content of whole grain rice.

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