CN117587033A - Gene DUF630/632 for controlling rice yield and quality traits, protein and application - Google Patents

Abstract

The invention discloses a gene DUF630/632 for controlling rice yield and quality traits, a protein and application thereof, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the CDS sequence is shown as SEQ ID NO. 2. According to the invention, a CRISPR/Cas9 gene editing technology is utilized to knock out rice endosperm specific expression genes, two homozygous mutant rice endosperms with different mutation types show obvious opaque chalky phenotype, the grain length and thousand grain weight of the mutant seeds are obviously increased, the total starch content is obviously reduced, the total protein content is obviously increased, the fat content and the soluble sugar content are obviously increased, and the physicochemical properties of starch are obviously changed. The invention is helpful for elucidating the molecular regulation and control mechanism of rice seed development, enriching the biological functions of DUF630/632 structural domain genes, and providing new important gene reserves, germplasm resources and theoretical basis for genetic improvement of rice yield and quality.

Description

Gene DUF630/632 for controlling rice yield and quality traits, protein and application

Technical Field

The invention belongs to the technical fields of genetic engineering and genetic breeding, and particularly relates to a gene DUF630/632 for controlling rice yield quality traits, a protein and application thereof.

Background

High yield and high quality are a permanent goal of rice breeding, yield determines the temperature saturation problem of people, and quality affects the living standard of people. The grain shape is an important character affecting the yield and appearance quality of rice, and the elucidation of the genetic control mechanism is helpful for increasing the yield per unit of rice and improving the quality of rice. Starch is the main component of rice endosperm and its structure and content directly affect the yield and quality of rice. Chalky affects rice appearance quality, processing quality, polished rice yield and market value. The related genes for controlling the quality traits of the rice yield are excavated more, and the molecular mechanism of the related genes is clarified, so that the related genes have important scientific value and production guidance significance for the genetic improvement of the rice yield quality.

In eukaryotes, many families of genes encoding proteins with ambiguous functions are identified as functionally unknown Domains (DUFs) and are conserved, many DUFs have important biological functions. The structure and function of some unknown functional Domains (DUFs) or uncharacterized protein families (UncharacterizedProteinFamilies, UPFs) in the Pfam database are analyzed by a computational structural genomics method, however, only a few genes with the structural domains of the rice DUF630/632 have been identified in detail, and play an important role in regulating and controlling biological processes such as leaf morphology, plant height, tillering, stomata development, grain filling, seed development and the like. Rolled and erect leaf (REL 2) encodes a plasma membrane-localized protein involved in the regulation of biological processes such as leaf curl uprightness, follicular cell development, and auxin synthesis transport (REL 2, AGene EncodingAn Unknown Function Protein which Contains DUF630 and DUF632 Domains Controls Leaf Rolling in Rice. Rice,2016, 9:37); rice dwarfand low tillering 10 (OsDLT 10) may influence rice plant height and tillering by regulating shoot development through WUS-CLV pathway auxin homeostasis (Rice dwarfand low tillering (OsDLT 10) regulates tiller number by monitoring auxin homeostasis.plant Science,2020, 297:110502); rice stomata developmental defect 1 (RSD 1) regulates stomatal density and dehydration processes, possibly contributing to cultivation of drought-resistant crops (RSD 1 Is Essential for Stomatal Patterning andFiles in Rice. Front in Plant Science,2020, 11:600021); the gating-defective andgrain width (FGW 1) encodes a DUF630/632 domain protein, and together regulate rice grain shape and grain Filling by interacting with 14-3-3 and GF14f proteins (FGW 1, a protein containing DUF and DUF632 domains, regulates grain size and Filling in Oryza sativa L. Through careful analysis, the genes are all located at the same gene locus (LOC_Os10g 41310/Os10g 0562700) on the 10 th chromosome of rice, which shows that the genes have multiple biological functions.

However, the DUF630/632 structural domain genes are still freshly reported to be involved in regulating and controlling the yield and quality traits, so that more rice DUF630/632 structural domain genes are necessary to be excavated and biological functions of the genes are analyzed, new important gene reserves and germplasm resources are provided for genetic improvement of rice yield and quality, a scientific theoretical basis is laid for cultivating more excellent rice varieties, and reference is provided for similar researches of other crops.

Disclosure of Invention

The invention aims to provide a gene DUF630/632 for controlling rice yield and quality traits, protein and application thereof, which provide new important gene reserves, germplasm resources and theoretical basis for rice yield and quality genetic improvement and can also provide reference for other crop similar researches.

In order to achieve the aim, the invention provides a gene DUF630/632 for controlling rice yield and quality traits, which is 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; the rice yield quality traits include: the endosperm is transparent; changing the particle shape; the grain length and thousand grain weight are reduced; the total starch content in the seeds is increased; reduced total protein, fat and soluble sugar content; the starch viscosity value in the seed increases.

Another object of the present invention is to provide a protein encoded by the gene DUF630/632, the amino acid sequence of which is shown as SEQ ID NO. 3.

Another object of the invention is to provide a mutant duf/632-22 or duf/630-23 gene for controlling rice yield and quality traits, wherein the nucleotide sequence of the mutant duf/632-22 gene is shown as SEQ ID NO.4, and the CDS sequence is shown as SEQ ID NO. 5; the nucleotide sequence of the duf630/632-23 gene is shown as SEQ ID NO.7, and the CDS sequence is shown as SEQ ID NO. 8; the rice yield quality traits include: chalky endosperm; changing the particle shape; the grain length and thousand grain weight are increased; the total starch content in the seeds is reduced; elevated total protein, fat and soluble sugar content; the starch viscosity value in the seed is reduced.

Another object of the present invention is to provide the encoded protein of the mutant duf630/632-22 or duf630/632-23 gene, wherein the amino acid sequence of the encoded protein of the mutant duf630/632-22 is shown as SEQ ID NO. 6; the amino acid sequence of the coded protein of the mutant duf/632-23 is shown as SEQ ID NO. 9.

Another object of the present invention is to provide the use of the gene DUF630/632, the encoded protein of the gene DUF630/632, the gene of the mutant DUF/632-22 or DUF/632-23 or the encoded protein of the gene of the mutant DUF/632-22 or DUF/632-23 for regulating the quality traits of rice yield.

The gene DUF630/632 for controlling the rice yield and quality traits, the protein and the application of the invention have the following advantages:

the DUF630/632 gene regulates the quality character of rice yield, the rice endosperm of a mature seed of flower 11 (ZH 11) in a normal wild japonica rice variety is transparent, the CRISPR/Cas9 gene editing technology is utilized to knock out the specific expression DUF630/632 gene of the rice endosperm, two homozygous mutants DUF/632-22 (deleted G base) and DUF/632-23 (inserted T base) of different mutation types show the apparent opaque chalky phenotype character, the grain length and thousand grain weight of the mutant seeds are obviously increased, the total starch content is obviously reduced, the total protein content is obviously increased, the fat content and the soluble sugar content are obviously increased, and the physical and chemical properties of starch are obviously changed. The invention is helpful for elucidating the molecular regulation and control mechanism of rice seed development, enriching the biological functions of DUF630/632 structural domain genes, providing new important gene reserves, germplasm resources and theoretical basis for the genetic improvement of rice yield and quality, and providing reference for the similar research of other crops.

Drawings

FIG. 1 shows the prediction of the structural domain of the encoded protein of the rice DUF630/632 gene and the construction of a phylogenetic tree.

FIG. 2 shows the expression pattern of rice DUF630/632 gene tissue; a is an expression level diagram of DUF630/632 genes in different tissues of rice; b is GUS staining chart of DUF630/632 gene in different rice tissues.

FIG. 3 is a bar graph showing the expression levels of the DUF630/632 gene in different rice tissues.

FIG. 4 is a graph showing the expression level of DUF630/632 gene in different rice tissues.

FIG. 5 is a diagram of the phenotype identification of the knockout DUF630/632 and mutants.

FIG. 6 shows the measurement of physicochemical indexes of wild type and mutant rice quality.

FIG. 7 is a vector diagram of the gene knockout vector VK005-01 used in the present invention.

FIG. 8 is a vector diagram of the GUS staining vector pCAMBIA-1305.1 used in the present invention.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 Rice DUF630/632 protein Domain prediction and phylogenetic Tree construction

The predicted analysis of the domains was performed using the SMART website (http:// SMART. Embl-heidelberg. De /), with the input of the protein sequences corresponding to the DUF630/632 genes, as shown in FIG. 1A, which shows that the DUF630/632 proteins have DUF630 and DUF632 domains between 1-59 and 280-623 amino acids, respectively.

Downloading protein sequences corresponding to DUF630/632 genes in a national paddy data center, placing the protein sequences into BLASTP of NCBI website for homology search, downloading and storing different species of protein sequences into Fasta format, performing multiple alignment of amino acids by Clustal (http:// www.clustal.org) program, and then constructing phylogenetic tree by bootstrapping method by MEGA (version 5.0) software. As shown in the B of figure 1, the result shows that the protein is relatively conserved in plants such as wart grain wild rice, coconut, oil palm, brachypodium distachyon, wheat, pineapple and the like, and has relatively close homology relationship with nitrate regulatory protein 2 and frizzled upright leaf type proteins. Wherein the red filled small red boxes represent the DUF630/632 proteins of the invention.

The nucleotide sequence of the DUF630/632 gene is shown as SEQ ID NO.1, the CDS sequence is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein of the DUF630/632 gene is shown as SEQ ID NO. 3.

Example 2 expression pattern of DuF630/632 Gene tissue of Rice and GUS staining

1. Rice DUF630/632 gene tissue expression pattern

The expression level of DUF630/632 in different development periods of different tissues of rice is detected by using the RiceXPro website https:// riccexpro.

As shown in FIG. 2A, FIG. 3 and FIG. 4, the results indicate that DUF630/632 is highly expressed in terms of grouted seed specificity, with the highest expression in the developing endosperm at 5 days post-flowering.

2. GUS staining

The promoter sequence of the DUF630/632 gene was recombined into the pCAMBIA-1305.1 expression vector by an In-fusion recombinase system (http:// bioinfo. Com/fusion /) (see FIG. 8), the pCAMBIA-1305.1 expression vector was first digested with KpnI and SpeI to linearize it, the wild-type flower 11 (ZH 11) genomic DNA was amplified by using primers (see GUS of Table 1), the cut gel was recovered by electrophoresis, the PCR product was recombined into the pCAMBIA-1305.1 expression vector by an In-fusion recombinase system, it was confirmed that no base mutation occurred by sequencing, and the constructed vector was transferred into the Agrobacterium strain (Agrobacterium tumefaciens) by a hot-shock method.

TABLE 1 all primer tables of the invention

Taking different fresh tissues (roots, stems, leaves, leaf sheaths and endosperm of different development periods) of wild GUS transgenic positive rice materials taking medium flower 11 (ZH 11) as a receptor, cutting the roots, stems, leaves and leaf sheaths into small sections, removing the shells of part of seeds, cutting part of seeds into centrifuge tubes with proper size by a blade without removing the shells, adding X-Glu staining solution, using tinfoil paper to avoid light, placing into a 37 ℃ incubator for staining, and properly adjusting the specific time according to the staining degree. After dyeing is completed, the dye liquor is discarded, decolorized with alcohol and then photographed by a scanner.

As shown in FIG. 2B, GUS staining was performed on different tissues of the pDOF 630/632 GUS positive plants, and the results showed that the DUF630/632 gene was highly specifically expressed in the grouting endosperm, especially in seeds at 5 days after flowering, the expression level of the gene was gradually decreased with gradual maturation of the seeds, and hardly expressed in roots, stems, leaves, leaf sheaths.

The results show that the DUF630/632 gene is highly specifically expressed in rice grouting endosperm.

EXAMPLE 3DUF630/632 Gene editing and mutant phenotype identification

1. Construction and transformation of Gene knockout vectors

Relevant kits were purchased from Beijing-only Shang Lide Biotechnology Co., ltd for construction of CRISPR/Cas9 gene knockout plasmid, the vector used was designated VK005-01 (see FIG. 7), and prokaryotic resistance was kanamycin and hygromycin resistance was used to detect positive plants. And selecting 20bp of the DUF630/632 gene as a target site through a CRISPRdirect website by utilizing a national rice data center website, wherein TGG is a PAM sequence. The CAG linker was added to the 5 '-end of the F primer (see Target in Table 1) of the Target sequence to be Target-F, and the AAC linker was added to the 5' -end of the R primer (see Target in Table 1) to be Target-R. The specific experimental operation steps are as follows:

(1) Preparation of dimers

The dimer reaction system (25. Mu.L) contained: 5. Mu.L Target-F (10. Mu.M), 5. Mu.L Target-R (10. Mu.M) and 15. Mu.L ddH ₂ O, mixing, slowly cooling at 95 deg.C for 3min and 95 deg.C to 25 deg.C, for example, -1 deg.C/20 s, or naturally cooling the sample tube in 95 deg.C water to room temperature, and standing at 16 deg.C for 5min.

(2) The dimer being bound to a support

The reaction system (10. Mu.L) in which the dimer was attached to the carrier contained: 1 μL Cas9/gRNAVector, 1 μL dimer of step (1), 1 μL Solution1, 1 μL Solution2, and 6 μ L H ₂ O。

Reaction conditions: the ligation was carried out overnight at 16 ℃.

(3) Transformation

Adding the product obtained in the step (2) into DH5a competent cells, uniformly mixing, ice-bathing for 30min, heat-shocking at 42 ℃ for 90s, standing on ice for 2min, adding 500 mu L of antibiotic-free LB, placing in a constant-temperature shaking table at 37 ℃ for 170rpm, resuscitating for 1h, coating LB plates with kanamycin resistance (K+), and culturing at 37 ℃ in an inverted overnight manner.

(4) Bacterial liquid PCR

The bacterial liquid PCR amplification system (10. Mu.L) comprises: 0.5. Mu.L of Sq-primer (see Table 1), 0.5. Mu.L of Target-R, 1. Mu.L of bacterial liquid/control template, 5. Mu.L of Mix (3G Taq Master Mix for PAGE) and 3. Mu.L of ddH ₂ O. The bacterial liquid PCR amplification procedure was as follows: 95 ℃ for 3min; [98 ℃ for 30s;55 ℃ for 30s;68 ℃ for 30s]A total of 35 reaction cycles; 72 ℃ for 8min;4 ℃ for 10min. The positive clone was picked and sent to the company for sequencing by Sq-primer (see Table 1), and the sequencing result was analyzed by DNAMAN or Snapgene software to construct the Target site Target of the Target gene on the VK005-01 vector.

(5) Agrobacterium-mediated transformation

The plasmids with correct sequence are selected and sent to Jiangsu hundred grid gene company for agrobacterium transformation and dip-dyeing experiments, and EHA105 (Agrobacterium tumefaciens) for agrobacterium is screened by using hygromycin resistance selection medium with medium flower 11 as a receptor.

2. Extraction of leaf DNA (SDS method)

(1) Solution preparation

1M Tris-HCl preparation: using 121.1g Tris base in 800mL ddH ₂ In O, the pH value is adjusted to 8.0 by concentrated HCl, and the volume is fixed to 1L.

0.5M EDTA formulation: 186.1g EDTA in 600mL ddH ₂ In O, naOH is used for regulating the pH value to 8.0, and water is added for constant volume to 1L.

Preparing SDS extract: 100mL of 1M Tris-HCl (pH 8.0), 50mL of 0.5M EDTA (pH 8.0), 29.25g of NaCl and 12g of SDS, and 800mL of ddH were added ₂ O, dissolving at 65 ℃ and fixing the volume to 1L.

KAC preparation: 29.5mL glacial acetic acid, KOH pH4.8, and constant volume to 100mL.

Chloroform-isoamyl alcohol (V: v=24:1): 500mL of chloroform and 21mL of isoamyl alcohol were used.

(2) DNA extraction

Cutting the picked blades into pieces, putting the pieces into a 2mL centrifuge tube, putting steel balls into the pieces, freezing the pieces by liquid nitrogen, and grinding the pieces into powder by using a proofing machine; 600 μl of SDS extract preheated at 65deg.C was added, and after mixing, the mixture was put into a water bath at 65deg.C for half an hour, and the mixture was shaken twice. Adding 150 mu LKAC, shaking, standing at-20deg.C for half an hour; 750 μl chloroform-isoamyl alcohol (V: v=24:1) was added and shaken for half an hour; centrifuging at 12,000rpm for 10min; aspirate 400 μl of supernatant into a new 1.5mL new centrifuge tube; adding pre-cooled 800 mu L of alcohol, shaking uniformly, and standing at-20 ℃ for half an hour; centrifuging at 12,000rpm for 10min, slowly discarding supernatant, and volatilizing the liquid; 100. Mu.L ddH was added ₂ O, after the precipitate is dissolved, the precipitate is ready for use.

3. PCR amplification (KOD-FX method)

The KOD-FX method PCR amplification system (50. Mu.L) comprises: 5. Mu.L of template DNA, 5. Mu.L of primer (Hyg-F/R each 2.5. Mu.L or seq-F/R each 2.5. Mu.L) (10. Mu.M each), 25. Mu.LBuffer, 10. Mu.L dNTPs and 1. Mu.L KOD-FX using 4. Mu.LddH ₂ O makes up 50. Mu.L. The KOD-FX PCR amplification procedure is shown in Table 2 below.

TABLE 2KOD-FX PCR amplification procedure

Note that: * T of step 3 _m The value of 55℃can be adjusted, and the extension time at 68℃in step 4 can be adjusted according to the size of the amplified fragment, so that the amplification rate of KOD-FX is 1kb/min.

4. Identification of transgenic positive plants and genotypes

Positive seedlings were detected with hygromycin Hyg-F/R (see Hyg in table 1), sequencing primers Seq-F/R (see Seq in table 1) were designed near the knocked-out target sites, PCR amplification was performed to sequence fragments including the target sequences, and the genotype and mutation type of the transgenic individual was determined from alignment with the wild-type target sequences and peak patterns of the mutation sites were observed.

The PCR amplification system (50. Mu.L) contained: 5 μLDNA/control template, 5 μLHyg-F/R, 10 μdNTPs (2 μmol/L), 25 μLbuffer, 1 μLKODFX and 4. Mu.LddH ₂ O. The PCR amplification procedure was as follows: 95 ℃ for 3min; [98 ℃ for 30s;55 ℃ for 30s;68 ℃ for 30s]A total of 35 reaction cycles; 72 ℃ for 8min;4 ℃ for 10min.

To investigate the potential biological functions of rice DUF630/632, the structural features of the gene were first analyzed, the whole genome length was 6192bp, the CDS length was 2061bp, the coding was 686 amino acids, including 5 exons, the initiation codon sequence was ATG, and the termination codon sequence was TAA (A of FIG. 5). A20 bp sequence with better specificity is selected on the first exon of the gene as a knockout target site, and TGG is used as a PAM sequence. The experimental operation is carried out according to the construction instruction of the Beijing-only Shang Lide Biotechnology Co kit VK005-01 vector, the vector (plasmid or bacterial liquid) with successful sequencing is sent to Jiangsu Baige Gene technology Co for agrobacterium-mediated transformation, the Zhonghua 11 (ZH 11) of the japonica rice variety is taken as a transgenic receptor, and the result analysis is carried out as a wild type control at the same time. Taking T0 generation transgenic single plant test tube plantlet, and then carrying out hygromycin gene detection to determine whether the seedling is positive. And (3) extracting leaf DNA after the test tube plantlet grows stably, designing a sequencing primer near the knockout target site for sequencing by a PCR amplification company, and determining the genotype and mutation type of the transgenic individual plant according to comparison with a wild type target sequence and observation of a peak diagram of a mutation site. Homozygotes of different mutation modes are detected in the T0 generation single plant, then the homozygote mutation types with stable inheritance are planted in the next year, all relevant experiments are carried out after the T1 generation is obtained, and compared with the wild type, one G base is deleted from duf/632-22, and one T base is inserted into duf/632-23 (B of FIG. 5). The frame shift mutations generated by these mutations lead to premature termination and changes in amino acid sequence. Finally, two independent homozygous mutant families duf/632-22 and duf/632-23 are selected for phenotype identification and all subsequent related experiments.

The nucleotide sequence of the mutant duf630/632-22 gene is shown as SEQ ID NO.4, the CDS sequence of the mutant duf/632-22 gene is shown as SEQ ID NO.5, the amino acid sequence of the mutant duf/632-22 gene is shown as SEQ ID NO.6, the nucleotide sequence of the mutant duf/632-23 gene is shown as SEQ ID NO.7, the CDS sequence of the mutant duf/632-23 gene is shown as SEQ ID NO.8, and the amino acid sequence of the mutant duf/632-23 gene is shown as SEQ ID NO. 9.

5. Phenotype identification of mature seed of transgenic plant

Mature seeds of the harvested wild-type ZH11 and homozygous mutants duf/632-22, duf/632-23 were placed in a 45℃oven oven dried to constant weight. Removing husks with a brown rice machine to obtain brown rice, removing aleurone layer with a rice polisher, grinding into polished rice, comparing phenotype of seeds, and photographing and recording in a scanner. 200 seeds with full grains are randomly selected, the grain length and grain width are measured by a ten thousand-depth seed automatic tester (SC-A1 type, hangzhou ten-thousand-depth detection technology company), and the thousand-grain weight is calculated. The grain thickness was measured using a vernier caliper and 10 grains or more per sample. Results are expressed as mean ± SD (standard deviation).

Several representative wild-type ZH11 and mutant duf630/632T1 generation mature seed brown rice and polished rice were randomly selected for phenotype comparison and photographed, respectively, and wild-type endosperm ZH11 was found to be transparent, while the different homozygous mutant types duf/632-22, duf630/632-23 exhibited distinct chalky phenotypes (C, D of FIG. 5); at the same time, the mutation of the gene changes the seed grain shape, the grain length and thousand grain weight of the mutant seeds are extremely obviously increased (E, H and K of 5 of fig. 5), and the grain width and grain thickness are not obviously different (F, I and J of 5 of fig. 5) (scale is 1 cm).

In conclusion, the DUF630/DUF632 gene not only regulates the grain shape and grain weight, thereby affecting the rice yield, but also regulates the accumulation of rice endosperm storage substances, thereby affecting the rice quality.

6. Scanning electron microscope observation of rice endosperm composite starch granule morphology structure

The method comprises the steps of selecting mature and dried complete seeds of wild ZH11 and mutants duf/632-22 and duf/632-23 respectively, shelling the seeds by a brown rice machine to obtain brown rice, gently breaking the middle position of the brown rice by tweezers to naturally break the brown rice, and sending a sample to a southwest crop gene resource development of Sichuan agricultural university and observing and photographing by using a scanning electron microscope (GeminiSEM 300) of a national key laboratory scanning electron microscope.

As shown in G of FIG. 5 (scale: 2 μm), the cross-section of endosperm of ZH11 and duf630/632-22, duf/632-23 was observed by scanning electron microscope, and the starch particles of wild endosperm cells were irregularly polygonal and closely arranged, but the mutant starch particles were loosely arranged, large gaps were formed between the particles, and part of the starch particles became smaller, and were spherical or ellipsoidal in shape. The results show that the DUF630/DUF632 gene regulates rice endosperm starch biosynthesis, and that mutation of the gene can affect the formation of composite starch granules, resulting in an opaque chalky phenotype and thus affecting rice quality.

Example 4 wild-type and mutant Rice quality determination

1. Determination of total starch content

The following procedure was carried out using the Megazyme kit according to the kit handbook and the experimental methods in reference 1 (OsPK 2 encodes a plastidic pyruvate kinase involved in rice endosperm starch synthesis, compound granule formation and grain filing.plant Biotechnol J.2018Nov;16 (11): 1878-1891) or in reference 2 (doctor paper, cai Yicong, "map cloning and functional research of the Rice chalky Gene OsPK 2", china national academy of agricultural sciences, 2018):

accurately weighing 50mg of standard sample, wild ZH11 and refined rice flour of mutants duf/632-22 and duf/632-23 by using a balance, repeating for three times, and placing into a 50mL centrifuge tube; adding 1mL of water, shaking, and boiling in boiling water bath for 10min; taking out, cooling to room temperature, adding 4mL of 2MKOH, and oscillating for about half an hour; 16mL of 1.2M sodium acetate at pH3.8 and 200. Mu.L of 3000U/mL amyloglucosidase were added; placing into a water bath kettle at 60 ℃ for boiling for 45min, oscillating twice, and then fixing the volume to 100mL; transfer 1mL to a new 1.5mL centrifuge tube; centrifuging at 4000rpm for 10min; sucking out 100 μl of supernatant to 10mL of test tube, adding 3mL of OPOD (glucose assay reagent), taking out and mixing after 20min in water bath at 37deg.C; determination of OD Using DU800 Spectrophotometer ₅₁₀ All samples were measured within 1 hour using 100. Mu.L of a reaction solution of 0.1M sodium acetate buffer and 3mLGOPOD under the same conditions as the blank for zeroing. The preparation of glucose standard solutions of different concentrations is shown in Table 3.

TABLE 3 preparation of glucose Standard solutions at different concentrations

By OD ₅₁₀ The glucose content was calculated and then the total starch content was scaled (0.9 x glucose content was defined as total starch content, 12% for sample moisture) and the average was taken as total starch content in triplicate.

2. Determination of amylose content

Amylose content measurement was performed according to the agricultural division designation NY-147-88, and the following procedure was performed with reference to (document 2):

(1) Accurately weighing 50mg of wild ZH11 and mutant duf/632-22, duf/632-23 refined rice flour and standard samples which have passed through a 100-mesh sieve by a balance, putting the obtained mixture into a 50mL volumetric flask, taking care that all the powder is placed at the bottom of the volumetric flask, and biologically repeating each sample three times;

(2) Adding 500 mu L of absolute ethyl alcohol, and slightly and uniformly mixing;

(3) Adding 4.5 mM LNaOH (1 mol/mL) along the wall, and boiling in boiling water for 10min;

(4) Deionized water is used for fixing the volume to 50mL;

(5) After shaking evenly, 500. Mu.L of sample and 5mL of water are sucked into a clean test tube;

(6) Sequentially adding 100 mu L of acetic acid solution (1 mol/mL), 200 mu L of KI-I2 and 4.2mL of deionized water, vigorously shaking and mixing, and standing for 20min;

(7) The amylose content of the standard samples used for drawing the standard curve was 1.5%, 10.4%, 16.2%, 26.5%, respectively, and the absorbance at 620nm was measured (DU 800 spectrophotometer, BECKMAN) and the amylose content was calculated.

3. Determination of total protein content

Weighing 200mg of wild ZH11 and mutant duf630/632-22 and duf630/632-23 refined rice flour respectively, repeating biologically three times, putting the rice flour into the bottom of a 100mL digestion tube, and adding 5mLH2SO4; starting the digestion furnace, putting a sample into the digestion furnace when the temperature is up to 290 ℃, digesting for 20min, taking out the sample, shaking the sample gently, and putting the sample into the digestion furnace again; raising the temperature of the digestion furnace to 320 ℃, shaking the digestion furnace for several times when the mouth of the digestion furnace smokes, keeping the temperature for 1h, taking out the digestion furnace to cool to room temperature, adding 1mL of hydrogen peroxide, and uniformly mixing the digestion furnace and the hydrogen peroxide; placing the digestion tube into a digestion furnace at 320 ℃ for 10min, observing whether the sample becomes clear, taking out, cooling to room temperature, and adding ddH2O to constant volume to 100mL; the total nitrogen content was measured by using a KJELTEC2300 full-automatic Kjeldahl apparatus from FOSS, and then converted into protein content (conversion factor 5.95), and the total protein content in polished rice was calculated by repeating 3 times per sample and taking the average value.

4. Determination of total fat content

The experimental method refers to the national standard GB2906-82 residual method and uses a Soxhlet extractor.

Cleaning the reusable glass cup, putting the glass cup into a baking oven at 105 ℃ to be dried to constant weight, taking the glass cup out for about two hours, putting the glass cup into a dryer to be cooled to room temperature, rapidly weighing the glass cup, and recording data; 2g each of the wild ZH11 and mutant duf630/632-22, duf630/632-23 fine rice flour (dried to constant weight and cooled to room temperature) was precisely weighed with a balance, all samples were repeated three times biologically, and then placed in paper cups for extraction; adding 100mL of petroleum ether into an extraction cup, immersing a paper cup and a sample into the extraction cup, setting the extraction temperature to 150 ℃, keeping the extraction temperature for 1h to enable the sample to be completely dissolved, then leaching for 4-6h, raising the sample to be completely separated from the solvent after the leaching is finished, opening a condenser, controlling the flow rate, and leaching the sample for 2h; petroleum ether is recovered, the glass cup is placed into a baking oven at 105 ℃, and the weight of the glass cup and the sample in the glass cup is accurately weighed after the glass cup is dried to constant weight.

Fat content = 100 x (sample weight-cup weight)/2 g refined rice flour weight 100%

5. Determination of soluble sugar content

Weighing 100mg of wild ZH11 and mutant duf630/632-22 and duf630/632-23 refined rice flour, repeating biological process three times for each sample, adding 7mL of 80% alcohol into a10 mL centrifuge tube, and shaking; water bath at 80℃for 30min, then centrifugation at 3000rpm for 10min, transferring supernatant in 25mL test tube (pass 1 rinsed with small amount of alcohol, the latter two passes may not be rinsed). 7mL of 80% ethanol is added, water bath is carried out at 80 ℃ for 30min, centrifugation is carried out at 3000rpm for 10min, supernatant is poured out, extraction is carried out three times, and volume is fixed for 25mL. Shaking up and measuring: 0.1mL of the extract was aspirated (0.1 mL of alcohol was added to the blank), 5mL of anthrone sulfuric acid solution was added, the mixture was boiled at 90℃for 15min, and after cooling, 620nm of color was aspirated (0.2 mL of the solution in the tube was shaken well before being transferred to the cuvette by a pipette) (blank control had to be added to the last blank of each cuvette). Wherein, the preparation method of the anthrone sulfuric acid solution (the anthrone sulfuric acid solution is prepared at present) comprises the following steps: 150mg of anthrone was dissolved in 100mL of concentrated sulfuric acid (76 mL of concentrated sulfuric acid was dissolved in 30mL of water); alternatively, 0.9868g of anthrone was dissolved in 80% dilute sulfuric acid (500 mL of concentrated sulfuric acid in 197.5mL of water).

Soluble total sugar (%) = [ (c×v/a)/(w×10) ⁶ )]*100

Wherein C-the amount of sugar (. Mu.g) found from the standard curve; v-total volume of sample extract (mL); a-the amount of sample (mL) taken during color development; w-dry weight of sample (g).

6. Determination of gum consistencies

The method for measuring the gum consistencies is described in GB/T22294-2008, and specifically comprises the following steps:

(1) The polished rice flour of the wild type ZH11 and the mutant duf630/632-22, duf630/632-23 which had passed through a 100-mesh sieve of 100mg was weighed and carefully placed in a10 cm test tube;

(2) Adding 0.2ml of 95% ethanol, adding 2ml of 0.2M KOH solution after vortex oscillation, and vortex oscillation again;

(3) Boiling in boiling water, covering the mouth of the test tube with glass beads, gelatinizing for 8min, and controlling the length of boiled rice flour to be not more than two-thirds of the length of the test tube (which can be controlled by a blower in general);

(4) Taking out and cooling for 5min at room temperature;

(5) Ice water bath for 20min; placing in an illumination incubator at 25 ℃ for standing for 1h;

(6) The length (cm) from the bottom of the tube to the highest point of the rice gel was measured using a ruler, the data was recorded, three biological replicates were set for each sample, and the average was taken.

Sorting the gum consistence: a hard gum consistency, the gum length of which is less than or equal to 40mm; the middle glue thickness is 41mm or less and the glue length is 60mm or less; the soft rubber has the consistency, and the rubber length is more than or equal to 61mm.

7. RVA profile analysis

3g of refined rice flour of wild ZH11 and mutant duf/632-22 are respectively weighed, 25mL of distilled water is added, and the mixture is mixed in an RVA special aluminum box to prepare starch milk with certain concentration. The rice flour viscosity characteristics were measured according to the TechMasterRVA rapid viscosimeter (Perten, swiss) protocol under the following conditions: maintaining at 50deg.C for 1min; rising to 95 ℃ at a speed of 5 ℃/min (9 min); maintaining at 95deg.C for 7min; reducing to 50deg.C (7.5 min) at 6deg.C/min; maintaining at 50deg.C for 4.5min; the stirrer was rotated at 960r/min for the first 10s, and then maintained at 160r/min. Measuring a viscosity curve of the starch paste, and analyzing by using a TCW (TCW) of RVA special test software to obtain 6 characteristic parameters: peak Viscosity (PV), valley viscosity (TV), final Viscosity (FV), break down (PV-TV), return (FV-PV), and paste temperature (peak temperature). Viscosity values are expressed in units of "CPs".

8. Determination of alkali extinction value of Rice grains

The following operations are performed with reference to the NY-147-88 standard:

six grains of polished rice of wild ZH11 and mutants duf/632-22 and duf/632-23 are put into a square box, 10mL of 1.7% KOH solution is added, the mixture is uniformly distributed, and a box cover is covered. Carefully transfer to an oven at 30.+ -. 2 ℃ for 23 hours, take out to observe the decomposition conditions, and then conduct alkali extinction classification according to the following table.

TABLE 4 alkali extinction fractionation

9. Gelatinization temperature (DSC) measurement

The gelatinization temperature measurement method is described in reference 3 (doctor paper, li Sanfeng, "map cloning and functional study of rice chalky gene OsBT 1", national academy of agricultural sciences, 2017), and comprises the following steps:

accurately weighing 5mg of wild ZH11 and mutant duf/632-22 refined rice flour which have passed through 100 mesh sieve, respectively, placing into aluminum cuvette, adding 10 μl of double distilled water (ddH) ₂ O) sealing and using differential scanning calorimeterDSC (Different Scanning Calorimeter) the gelatinization temperature is measured, the heating rate is set to 10 ℃/min, and the temperature range is 35-100 ℃.

10. Determination of urea expansion of rice flour

The method for measuring the chain length distribution of the fine structure of amylopectin is described in reference (document 2), and specifically comprises the following steps:

20mg of wild Zhonghua (ZH 11) and refined rice flour of mutant duf/632-22 were weighed respectively, put into a 1.5mL centrifuge tube, 0-9M urea (urea) 1mL (pH was adjusted to 6.0 with glacial acetic acid) was added to each tube, and gelatinized at room temperature for 1 day. Centrifuging at 8000g for 30min, taking out, standing for 1h, and taking a picture. Remarks: all the samples were left in a natural state for 3 months before the experiment.

11. Experimental results

The total starch content in the seeds of the two homozygous mutants duf/632-22 and duf/630-23 was very significantly reduced compared to the wild type ZH11 (A of FIG. 6); mutant amylose content was not significantly different (B of fig. 6); the total protein content mutant was significantly elevated (C of FIG. 6), the soluble sugar content and fat content were significantly elevated (D of FIG. 6 and H of FIG. 6), including the fatty acid components C18:0 and C22:6n3 were significantly elevated, and C16:0 was significantly elevated (I of FIG. 6), indicating that DUF630/632 might also be involved in the lipid metabolic pathway. Both wild type and both mutants were of soft consistency (. Gtoreq.61 mm), and mutants duf630/632-22 were significantly reduced by about 4.6mm compared to ZH11, and mutants duf630/632-23 were significantly reduced by about 6.3mm compared to ZH11 (E of FIG. 6); two mutants were more easily gelatinized in 1.7% KOH solution than the wild type, ZH11 was estimated to be rated 6 (rice grains were partially dispersed and dissolved, fused together with rings; mi Xinyun white, rings disappeared), mutants were rated approximately 7 (rice grains were swollen, rings were incomplete or narrow; rice hearts were white, ring cotton floccules or clouds) (J of FIG. 6); comparison of gelatinization temperatures of wild-type and mutant endosperm starches showed duf630/632-22 endosperm starch T _O Tp is not significantly different from ZH11, but T _C Significantly higher than ZH11 (F of fig. 6); the viscosity characteristics were measured using a Rapid Viscosimeter (RVA) and the results showed that the viscosity profile of the two mutant starches had a similar trend to that of the wild-type ZH11, but the viscosity values of the mutants were always in comparisonThe highest viscosity of duf630/632-22 was only 86.15% of the wild type (G of FIG. 6) at low levels. The solubility of starch granules in urea solution is also an indicator reflecting the physicochemical properties of starch. The polished rice flour of ZH11 and duf630/632-22 was dissolved with urea at different concentrations (0-9M), and studies showed that they were difficult to dissolve in 0-4M urea solution and easy to dissolve in 5-9M urea solution, but the difference was not significant (K of FIG. 6). The above results indicate that the DUF630/632 gene is involved in the formation of multiple biological pathways for rice quality.

While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims (5)

1. A gene DUF630/632 for controlling rice yield and quality characters is 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;

the rice yield quality traits include: the endosperm is transparent; changing the particle shape; the grain length and thousand grain weight are reduced; the total starch content in the seeds is increased; reduced total protein, fat and soluble sugar content; the starch viscosity value in the seed increases.

2. The protein encoded by the gene DUF630/632 according to claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 3.

3. A mutant duf/632-22 or duf/630-23 gene for controlling rice yield and quality characteristics is characterized in that the nucleotide sequence of the mutant duf/632-22 gene is shown as SEQ ID NO.4, and the CDS sequence is shown as SEQ ID NO. 5; the nucleotide sequence of the duf630/632-23 gene is shown as SEQ ID NO.7, and the CDS sequence is shown as SEQ ID NO. 8;

the rice yield quality traits include: chalky endosperm; changing the particle shape; the grain length and thousand grain weight are increased; the total starch content in the seeds is reduced; elevated total protein, fat and soluble sugar content; the starch viscosity value in the seed is reduced.

4. The mutant duf630/632-22 or duf630/632-23 gene encoded protein of claim 3, wherein the amino acid sequence of the encoded protein of mutant duf630/632-22 is as shown in SEQ ID No. 6; the amino acid sequence of the coded protein of the mutant duf/632-23 is shown as SEQ ID NO. 9.

5. Use of the gene DUF630/632 according to claim 1, the encoded protein according to claim 2, the mutant DUF/632-22 or DUF/632-23 according to claim 3 or the encoded protein according to claim 4 for regulating quality traits in rice yield.