Gene Glb1 for regulating and controlling chalkiness and globulin of rice and application of gene Glb1

Abstract

The invention belongs to the technical field of biotechnology and plant genetic engineering, and particularly relates to a gene Glb1 for regulating and controlling chalkiness and globulin of rice and application of the gene Glb1. The nucleotide sequence of the Glb1 gene is as shown in SEQ ID NO. 1, and the CDS sequence of the Glb1 gene is as shown in SEQ ID NO. 2. A gene Glb1 for controlling chalkiness and globulin of rice is cloned through bioinformatics and reverse genetics methods, the gene is highly specifically expressed in rice development endosperm, and a mutant glb1-3 gene and a mutant glb1-6 gene are obtained through gene knockout. Compared with a wild type, the chalky grain rate and chalkiness degree of the mutant are remarkably increased, composite starch grains are abnormally ellipsoidal or spherical, the grain thickness and thousand grain weight are remarkably reduced, the total protein content is remarkably increased, the globulin content is remarkably reduced, the amylose content is remarkably reduced, and the taste value is remarkably increased; the content of secondary metabolites is increased.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology and plant genetic engineering, and particularly relates to a gene for regulating chalkiness and globulin of rice Glb1 and application thereof. BACKGROUND

[0002] Rice chalkiness affects the appearance quality, processing quality, milled rice yield and market value of rice. Rice globulin is an important storage protein trace component, but it is increasingly concerned by researchers. Therefore, mining more genes for simultaneously regulating chalkiness and globulin traits and elucidating the molecular mechanism thereof have important scientific value and production guiding significance for genetic improvement of rice quality.

[0003] Globulin is an important salt-soluble storage protein in rice endosperm, and the content accounts for about 10% of the total amount of rice endosperm protein (Wu et al., 1998). As a special protein in the rice gene family, although the proportion of globulin in the total protein of rice seeds is low, due to the small number of family members, the relative expression amount of rice globulin gene is high, and it is specifically highly expressed in seed endosperm. The 26kDa globulin gene promoter cloning and sequence analysis has important significance and value in the research of improving rice quality (Lü Yinghai et al., 2005). Recently, Korean and Chinese scientists have found that globulin has an important role in the cooking taste quality of rice (Nawade et al., 2025; Xu et al., 2025). Therefore, it is necessary to further analyze the biological function of globulin synthesis gene Glb1 , provide new important gene reserves and germplasm resources for genetic improvement of rice quality and yield, lay a scientific theoretical foundation for breeding more excellent rice varieties, and also provide a reference for similar research of other crops. SUMMARY

[0004] In view of the problems in the prior art, the purpose of the present application is to design a gene for regulating chalkiness and globulin of rice Glb1 and application thereof.

[0005] The present application specifically adopts the following technical solutions to achieve the above purposes. The first aspect of the present application provides a gene for regulating chalkiness and globulin of rice Glb1 , the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the CDS sequence is shown as SEQ ID NO. 2.

[0006] The second aspect of the present application provides a mutant Glb1 of the gene glb1-3 , the mutant glb1-3The nucleotide sequence is shown in SEQ ID NO.3, and the CDS sequence is shown in SEQ ID NO.4.

[0007] The third aspect of the present invention provides genes Glb1 mutant glb1-6 The mutant glb1-6 The nucleotide sequence is shown in SEQ ID NO.5, and the CDS sequence is shown in SEQ ID NO.6.

[0008] The fourth aspect of the present invention provides a gene Glb1 Gene mutants glb1-3 or gene mutant glb1-6 In the application of regulating rice quality and yield, the rice quality and yield are selected from any one or more of the following: 1) the chalky grain rate and chalkiness of mutants; 2) the shape of complex starch granules; 3) grain thickness and thousand-grain weight; 4) total protein content; 5) globulin content; 6) amylose content; 7) taste value; 8) content of secondary metabolites.

[0009] Furthermore, the rice quality and yield are selected from any one or more of the following: 1) increased chalky grain rate and chalkiness of mutants; 2) abnormally ellipsoidal or spherical shape of complex starch granules; 3) decreased grain thickness and thousand-grain weight; 4) increased total protein content; 5) decreased globulin content; 6) decreased amylose content; 7) increased taste value; 8) increased content of secondary metabolites.

[0010] Furthermore, the secondary metabolites include alkaloids, amino acids and their derivatives, terpenoids, phenolic acids, organic acids, nucleotides and their derivatives, lignans and coumarins, quinones, steroids, or tannins.

[0011] The present invention has the following beneficial effects: (1) This application cloned a gene that controls chalkiness and globulin in rice using bioinformatics and reverse genetics methods. Glb1 This gene is highly specifically expressed in the rice endosperm during development, and mutants can be obtained by gene knockout. glb1-3 Genes and mutants glb1-6 Genes. Compared with the wild type, the mutant showed a highly significant increase in chalky grain rate and chalkiness, abnormally ellipsoidal or spherical complex starch grains, highly significant decreases in grain thickness and thousand-grain weight, highly significant increase in total protein content, highly significant decrease in globulin content, highly significant decrease in amylose content, highly significant increase in taste value, and increased content of most secondary metabolites.

[0012] (2) This application verifies Glb1Genes play an important role in regulating biological pathways such as the biosynthesis of secondary metabolites, ABC transport, and amino acid synthesis, and can provide important gene reserves, germplasm resources, and theoretical basis for the genetic improvement of rice quality and yield. Attached Figure Description

[0013] Figure 1 This invention relates to genes that regulate chalkiness and globulin in rice. Glb1 Organizational expression pattern diagram.

[0014] Figure 2 This invention is for knocking out rice Glb1 Identification diagrams of gene and mutant genotypes and phenotypes.

[0015] Figure 3 Wild type and mutant of this invention glb1 Comparative analysis chart of rice quality determination.

[0016] Figure 4 Wild type and mutant of this invention glb1 Comparative scanning electron microscopy analysis of complex starch granules in the endosperm of mature seeds.

[0017] Figure 5 Wild type and mutant of this invention glb1 Clustering heatmap of differential metabolites.

[0018] Figure 6 Wild type and mutant of this invention glb1 Differential metabolite pathway enrichment map. Detailed Implementation

[0019] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0020] Example 1: Rice chalkiness and globulin genes Glb1 Organizational expression patterns This invention first uses bioinformatics analysis to discover rice seeds that specifically highly express chalky white protein and globulin. Glb1 Gene, Glb1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1. Glb1 The CDS sequence is shown in SEQ ID NO. 2.Figure 1 As shown, the rice of the present invention Glb1 A graph showing the expression levels of a gene in different rice tissues. This gene is highly expressed during the early grain-filling stage, and its expression level gradually decreases as the seed matures, but it is almost not expressed in the roots, stems, leaves, and leaf sheaths. Therefore, this indicates... Glb1 The gene is highly specifically expressed in the rice grain-filling endosperm. Glb1 Gene promoters have important significance and value in the research of gene improvement of rice quality, and their promoter sequences are shown in SEQ ID NO.7.

[0021] Example 2: Rice Glb1 Gene knockout, mutant genotype and phenotype identification 1. Construction and transformation of gene knockout vectors A CRISPR / Cas9 gene knockout plasmid construction kit was purchased from a biotechnology company. The vector used was VK005-01, and the prokaryotic resistance was kanamycin. Hygromycin resistance was used to detect positive plants. Rice chalkiness and globulin genes were selected using the CRISPRdirect website through the National Rice Data Center website. Glb1 The first exon contains 20 bp of the target site, and AGG is the PAM sequence.

[0022] Primer sequences are shown in the attached table: Table 1 lists the primers involved in this invention.

[0023] After receiving the kit, centrifuge the test tubes before use to avoid leaving solution residue on the tube walls, and then conduct the experiment. The specific experimental procedures are as follows: (1) Preparation of dimer: After mixing the reagents in Table 2, heat at 95℃ for 3 minutes, cool to room temperature, and let stand at 16℃ for 5 minutes.

[0024] Table 2. Reaction system for dimer preparation.

[0025] (2) Attaching the dimer to the support: Connect the reagents in Table 3 by incubating at 16°C overnight.

[0026] Table 3 Reaction system of dimer-linked carriers

[0027] (3) Transformation: The product from (2) was added to DH5α competent cells and mixed well. The cells were then incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and placed on ice for 2 min. 500 μL of antibiotic-free LB was then added, and the cells were placed in a 37℃ constant temperature shaker at 170 rpm for 1 h of recovery. After recovery, the kanamycin resistance (K) was measured. + LB plates were incubated overnight at 37°C inverted.

[0028] Positive clones were selected and sent to the company for Sq-primer sequencing (sequences are shown in Table 1, and bacterial PCR results are shown in Tables 4 and 5). The sequencing results were analyzed using DNAMAN or SNAP software to determine whether the target gene site Target was constructed into the VK005-01 vector.

[0029] Table 4. Bacterial PCR Reaction System

[0030] Table 5. PCR reaction procedure for bacterial culture

[0031] Agrobacterium-mediated transformation steps: Select plasmids with correct sequencing and send them to a biotechnology company for Agrobacterium transformation and infection experiments, using Zhonghua 11 as the recipient and EHA105 Agrobacterium. （ Agrobacterium tumefaciens was screened using hygromycin resistance selective medium.

[0032] 2. Extraction of DNA from leaves (SDS method) (1) Reagent preparation 1M Tris - HCl: 121.1g Tris base and 800mL ddH2O, adjust the pH to 8.0 with concentrated HCl, and bring the volume to 1L.

[0033] 0.5M EDTA: 186.1g EDTA and 600mL ddH2O, adjust the pH to 8.0 with NaOH, and add water to a final volume of 1L.

[0034] SDS extraction solution: 100 mL 1M Tris-HCl (pH 8.0), 50 mL 0.5M EDTA (pH 8.0), 29.25 g NaCl and 12 g SDS, dissolved in 800 mL ddH2O at 65℃, and brought to a final volume of 1 L.

[0035] KAC: 29.5 mL glacial acetic acid, adjust pH to 4.8 with KOH, and bring the volume to 100 mL.

[0036] Solvent ratio 24:1:500mL chloroform and 21mL isoamyl alcohol.

[0037] (2) DNA extraction Cut the collected leaves into small pieces and place them in a 2mL centrifuge tube. Add steel balls and freeze in liquid nitrogen. Grind the mixture into powder using a sample grinder. Add 600μL of SDS extraction solution preheated to 65℃, mix well, and then place in a 65℃ water bath for half an hour, shaking twice during the process. Add 150μL of KAC, shake well, and then place in a -20℃ incubator for half an hour. Add 750μL of 24:1 solution and shake on a shaker for half an hour. Centrifuge at 12,000rpm for 10 minutes. Transfer 400μL of the supernatant to a new 1.5mL centrifuge tube. Add 800μL of pre-cooled ethanol, shake well, and then place in a -20℃ incubator for half an hour. Centrifuge at 12,000rpm for 10 minutes, slowly discard the supernatant, and allow the liquid to evaporate. Add 100μL of ddH2O and wait for the precipitate to dissolve before using.

[0038] 3. PCR amplification PCR amplification and sequencing were performed using the KOD-FX method. The amplification system and procedure are shown in Tables 6 and 7. Table 6. KOD-FX method amplification system

[0039] Table 7. PCR reaction procedure for bacterial culture

[0040] Note: The Tm value of 55℃ in step 3 can be adjusted; the extension time of 68℃ in step 4 can be adjusted according to the size of the amplified fragment; the amplification rate of KOD-FX is 1kb / min.

[0041] 4. Identification of transgenic positive plants and their genotypes Positive seedlings were detected using hygromycin Hyg-F / R (amplification system and procedure are shown in Tables 8 and 9, sequence is shown in Table 1). Sequencing primers Seq-F / R (see Table 1) were designed near the knockout target site for PCR amplification of a 263bp fragment including the target sequence, which was then sent to the company for sequencing. The genotype and mutation type of the transgenic single plant were determined by comparing it with the wild-type target sequence and by observing the peak diagram of the mutation site.

[0042] Table 8 PCR system for transgenic positive plants

[0043] Table 9 PCR Procedures for Transgenic Positive Plants

[0044] like Figure 2 As shown in A, the analysis of rice is performed first. Glb1Basic gene information: The full-length gene is 840 bp, with a CDS of 561 bp, encoding 186 amino acids. A 20 bp sequence with high specificity was selected from the exons of this gene as the knockout target site, and AGG was used as the PAM sequence. Experimental procedures were performed according to the instructions for vector construction using kit VK005-01. Successfully sequenced vectors (plasmids or bacterial cultures) were sent to a biotechnology company for Agrobacterium-mediated transformation. The japonica rice variety Zhonghua 11 (ZH11) was used as the transgenic recipient, and subsequently served as a wild-type control for result analysis. T0 generation transgenic single-plant test-tube seedlings were collected, and hygromycin gene detection was performed to determine if the seedlings were positive. After the test-tube seedlings stabilized, leaf DNA was extracted. Sequencing primers were designed near the knockout target site for PCR amplification, and the sequences were sent to the company for sequencing. The genotype and mutation type of the transgenic single plant were determined by comparing the sequence with the wild-type target sequence and observing the peak diagram of the mutation site. Homozygous mutants with different mutation patterns were detected in T0 generation single plants. Then, in May of the following year, stable homozygous mutant types were planted, and all relevant experiments were conducted. Compared with the wild type, such as... Figure 2 The mutant shown in B is glb1-3 A 10-base deletion (CTACGAGGAG) at the target site results in a mutant. glb1-6 The deletion of one T base at the target site resulted in frameshift mutations in all of the above. These mutations were ultimately selected for phenotypic identification and all subsequent related experiments.

[0045] Among them, mutant glb1-3 The nucleotide sequence of the gene is shown in SEQ ID NO.3, and its CDS sequence is shown in SEQ ID NO.4; mutant glb1-6 The nucleotide sequence of the gene is shown in SEQ ID NO.5, and its CDS sequence is shown in SEQ ID NO.6.

[0046] 5. Phenotypic identification of mature seeds from transgenic plants Harvested wild-type ZH11 and homozygous mutants glb1-3, glb1-6 Mature seeds were dried in a 45°C constant temperature oven until constant weight. Brown rice was obtained by hulling using a brown rice machine, and then milled into polished rice using a rice polishing machine. The phenotypic characteristics of the seeds were then compared and recorded by scanning.

[0047] like Figure 2 As shown in E and F, several representative wild-type ZH11 and mutant strains were randomly selected. glb1-3, glb1-6 Phenotypic comparison and photography of mature seeds from the T1 generation revealed that the mature seeds of the normal wild-type japonica rice variety Zhonghua 11 (WT or ZH11) were colorless and transparent, while the seeds of the three different mutant types were... glb1-3, glb1-6 The seeds exhibited a distinct chalky phenotype, with a highly significant increase in the chalky grain rate and chalkiness. Figure 2 (K, L in the middle).

[0048] 6. Determination of rice yield traits Harvested wild-type ZH11 and mutants glb1-3, glb1-6 Mature seeds were dried in a 45℃ oven until constant weight. Two hundred plump seeds were randomly selected, and their length and width were measured using a Wanshen automatic seed quality analyzer (SC-A1 type, Hangzhou Wanshen Testing Technology Co., Ltd.), and the weight of 1000 seeds was calculated. Seed thickness was measured using vernier calipers, with at least 10 seeds per sample. Results are expressed as mean ± SD (standard deviation).

[0049] like Figure 2 As shown in C, D, G, and H, wild-type ZH11 and mutant ZH11 are... glb1-3 There was no significant difference in grain length and width, but the mutants glb1-6 The grain length and width were significantly higher than those of the wild type, possibly due to different mutation types; mutant glb1-3, glb1-6 Both grain thickness and thousand-grain weight were significantly lower than those of the wild type. Figure 2 The I and J in the text indicate the rice chalkiness and globulin genes. Glb1 Genes regulate yield traits.

[0050] Furthermore, we used chemical-physical methods to measure and compare the quality traits of the wild type and the mutant. The experimental results showed that the mutant... glb1-3, glb1-6 The content of globulin and amylose in brown rice decreased significantly, but the mutant... glb1-6 Total protein content and taste value increased significantly ( Figure 3 This indicates that the rice globulin gene... Glb1 Regulating key quality traits may have broad application prospects in quality breeding in the future.

[0051] Example 3: Comparative analysis of scanning electron microscopy structure of starch complex in the endosperm of mature seeds of wild type and mutant. For wild type and mutant glb1-3, glb1-6 Scanning electron microscopy (SEM) analysis of the endosperm complex starch structure in cross-sections of mature rice seeds revealed that wild-type starch granules exhibited dense, irregular polygonal morphology, while mutant granules... glb1-3, glb1-6 It exhibits numerous spherical or ellipsoidal starch granules with gaps, thus producing a distinct chalky phenotype. Figure 4 ).

[0052] Example 4: Comparative analysis of broad-targeted metabolomics in mature seeds of wild type and mutants For wild type and mutant glb1-3, glb1-6Broad-target metabolome sequencing analysis of mature brown rice flour revealed that the mutant strain contained significantly higher levels of alkaloids, amino acids and their derivatives, terpenoids, phenolic acids, organic acids, nucleotides and their derivatives, lignans and coumarins, quinones, steroids, tannins, and other secondary metabolites than the wild type. Figure 5 These substances may be related to rice flavor formation and stress resistance, but further investigation is needed. Differential metabolite pathway enrichment mapping analysis showed... Glb1 Genes may play an important role in regulating biological pathways such as the biosynthesis of secondary metabolites, ABC transport, and amino acid synthesis. Figure 6 ).

Claims

1. A gene regulating chalkiness and globulin in rice Glb1 characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the CDS sequence is shown as SEQ ID NO.

2.

2. The gene of claim 1 Glb1 mutant glb1-3 characterized in that, The mutant glb1-3 The nucleotide sequence of the mutant is shown as SEQ ID NO. 3, and the CDS sequence is shown as SEQ ID NO.

4.

3. The gene of claim 1 Glb1 mutant glb1-6 characterized in that, The mutant glb1-6 The nucleotide sequence of the mutant is shown as SEQ ID NO. 5, and the CDS sequence is shown as SEQ ID NO.

6.

4. The gene of claim 1 Glb1 , the gene mutant of claim 3 glb1-3 or the gene mutant of claim 4 glb1-6 application in regulating rice quality yield selected from any one or more than one of the following: 1) mutant chalky kernel rate and chalkiness; 2) complex starch granule shape; 3) kernel thickness and 1000-grain weight; 4) total protein content; 5) globulin content; 6) amylose content; 7) eating quality value; 8) secondary metabolite content.

5. The mutant gene of claim 3 glb1-3 or the mutant gene of claim 4 glb1-6 application in regulating rice quality and yield, wherein the rice quality and yield is selected from any one or more of the following: 1) increased chalky kernel percentage and chalkiness; 2) abnormal spheroid or spherical shape of complex starch granules; 3) decreased kernel thickness and 1000-grain weight; 4) increased total protein content; 5) decreased globulin content; 6) decreased amylose content; 7) increased eating quality; and 8) increased content of secondary metabolites.

6. Use according to claim 4 or 5, wherein the compound is ###0002### The secondary metabolites include alkaloids, amino acids and their derivatives, terpenoids, phenolic acids, organic acids, nucleotides and their derivatives, lignans and coumarins, quinones, steroids or tannins.

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