Rice OsYUC12 / 13 / 14 polygene mutant as well as encoding protein and application thereof

Abstract

The invention discloses a rice OsYUC12 / 13 / 14 polygene mutant as well as an encoding protein and application thereof. By simultaneously destroying biological functions of the encoding protein of three genes OsYUC12 / 13 / 14, the length of rice grains can be remarkably increased, and the content of rice apparent amylose can be reduced; the gene OsYUC12, the gene OsYUC13 and the gene OsYUC14 are knocked out at a fixed point at the same time through the CRISPR / Cas9 technology, a function deletion mutant is obtained, the length of rice grains is remarkably increased, the appearance quality of rice is improved, meanwhile, the apparent amylose content is remarkably reduced, and the eating quality of the rice can be improved; the new function of the rice YUCCA gene family OsYUC12 / 13 / 14 mutant provided by the invention provides a candidate gene for plant breeding, and also provides a potential application value in the aspect of improving the rice yield and quality.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biological breeding, and specifically relates to a rice OsYUC12 / 13 / 14 multi-gene mutant and its encoded protein and application. Background Art

[0002] my country is the world's largest rice producer. As my country's main food crop, rice production and quality are related to national food security and people's health. Improving rice production and quality plays a significant role in ensuring my country's food security and people's living needs.

[0003] Rice yield is composed of three factors: the number of panicles per plant, the number of grains per panicle, and grain weight. Grain weight is controlled by grain shape, which includes three factors: grain length, grain width, and grain thickness. Grain shape not only affects rice yield, but also affects rice processing quality and appearance quality. Grain length, as one of the three factors of grain shape, plays an important role in regulating rice yield and quality.

[0004] With the continuous development of society, modern molecular breeding technology is also emerging. Compared with traditional breeding, modern molecular breeding has many advantages such as high selection efficiency and short breeding cycle. In order to effectively carry out modern molecular breeding and accelerate variety selection, it is necessary to locate and explore the functions of genes regulating target traits. Therefore, discovering and utilizing genes related to regulating rice grain length is of great significance to the breeding and improvement of high-yield and high-quality rice.

[0005] To date, there are no reports on the functional loss of rice OsYUC12 / 13 / 14 and its application in rice breeding. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a rice OsYUC12 / 13 / 14 mutant gene.

[0009] In order to solve the above technical problems, the present invention provides the following technical solution: a rice OsYUC12 / 13 / 14 mutant gene, wherein: the rice OsYUC12 / 13 / 14 mutant gene is any one of the following:

[0010] (i) OsYUC12 has an insertion of a base (A) between nucleotides 594-595 from the ATG start of its wild-type gene, OsYUC13 has an insertion of a base (T) between nucleotides 604-605 from the ATG start of its wild-type gene, and OsYUC14 has an insertion of a base (T) between nucleotides 604-605 from the ATG start of its wild-type gene, which is OsYUC12 / 13 / 14-1;

[0011] (ii) OsYUC12 had an insertion of a base (A) between 594-595 nucleotides from the ATG start of its wild-type gene, OsYUC13 had an insertion of a base (A) between 603-604 nucleotides from the ATG start of its wild-type gene, OsYUC14 had a deletion of 596-615 nucleotides from the ATG start of its wild-type gene, and OsYUC12 / 13 / 14-2.

[0012] As a preferred embodiment of the rice OsYUC12 / 13 / 14 mutant gene of the present invention, the nucleotide sequence of the OsYUC12 / 13 / 14-1 mutant gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the OsYUC12 / 13 / 14-1 mutant gene is shown in SEQ ID NO: 2.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a protein for translating the OsYUC12 / 13 / 14 mutant gene, comprising: the amino acid sequence of the OsYUC12 wild-type protein is shown in SEQ ID NO: 4, the amino acid sequence of the OsYUC13 wild-type protein is shown in SEQ ID NO: 5, and the amino acid sequence of the OsYUC14 wild-type protein is shown in SEQ ID NO: 6.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a recombinant vector, comprising: the recombinant vector comprises the OsYUC12 / 13 / 14 mutant gene.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a rice mutant containing an OsYUC12 / 13 / 14 mutant gene, comprising:

[0016] Selection of gene target sequence: Screen the target sequence based on the full length of CDS of OsYUC12 / 13 / 14 gene;

[0017] Construction of CRISPR / Cas9 expression vector: site-directed editing of OsYUC12 / 13 / 14 genes, and transformation of the obtained recombinant vector into Agrobacterium;

[0018] Agrobacterium-mediated genetic transformation: Prepare Agrobacterium infection solution, infect rice callus tissue, then culture the infected rice callus tissue, and obtain transgenic mutant plants by single plant sequencing and identification.

[0019] As a preferred embodiment of the method of the present invention, the nucleotide sequence of the target sequence OsYUC12-SG of OsYUC12 is shown as SEQ ID NO: 8, the nucleotide sequence of the target sequence OsYUC13-SG of OsYUC13 is shown as SEQ ID NO: 9, and the nucleotide sequence of the target sequence OsYUC14-SG of OsYUC14 is shown as SEQ ID NO: 10.

[0020] As a preferred embodiment of the method of the present invention, the Agrobacterium is one of GV3101 Agrobacterium and EHA105 Agrobacterium.

[0021] As a preferred embodiment of the method of the present invention, the method comprises: the culture medium formula of the rice callus is 30 g / L sucrose, 4.4 g / L MS culture medium, 50 g / L sorbitol, 1.4% agar, 3 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 50 mg / L carbenicillin and 50 mg / L hygromycin B.

[0022] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the rice OsYUC12 / 13 / 14 mutant gene in reducing the amylose content and improving the taste quality of rice.

[0023] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the rice OsYUC12 / 13 / 14 mutant gene in increasing rice yield.

[0024] Beneficial effects of the present invention:

[0025] 1. The present invention discloses the application of rice OsYUC12 / 13 / 14 genes in changing rice grain shape. Rice OsYUC12 / 13 / 14 is site-specifically edited using CRISPR / Cas9 technology, and rice callus is transformed by an Agrobacterium-mediated method to obtain transgenic rice. Compared with unedited wild-type materials, the grain length of transgenic rice is increased. The genes and genetic engineering technical means provided by the present invention have great application value in increasing rice yield without affecting other agronomic traits of rice.

[0026] 2. Compared with single gene mutation, the simultaneous mutation of the three genes OsYUC12 / 13 / 14 in the present invention can synergistically increase the length of rice grains and improve the appearance quality of rice. At the same time, the apparent amylose content is significantly reduced, which can improve the taste quality of rice.

[0027] 3. The new functions of the OsYUC12 / 13 / 14 mutants of the present invention provide candidate genes for plant breeding and have potential application value in improving rice yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0029] Figure 1 This is the gene structure diagram of OsYUC12 / 13 / 14 in Example 1.

[0030] Figure 2 This is a comparison chart of the predicted encoded proteins in Example 1.

[0031] Figure 3 This is the sequencing peak diagram of the wild type and gene-edited materials in Example 4.

[0032] Figure 4 This is a comparison diagram of plant types in Example 5.

[0033] Figure 5 This is a graph of grain length and grain width in Example 5.

[0034] Figure 6 This is the content of amylose and protein in Example 5. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0036] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention, so the present invention is not limited to the specific embodiments disclosed below. Secondly, the "one embodiment" or "embodiment" referred to herein refers to a specific feature, structure or characteristic that may be included in at least one implementation of the present invention. The "in one embodiment" that appears in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

[0037] Sources of raw materials used in the present invention: RNA rapid extraction (R701) and reverse transcription system reagents were purchased from Nanjing Novozyme; CRISPR / Cas vector construction kit BGK032 was purchased from Biotech; DH5α (DL1001) and EHA105 (AC1010) strains were purchased from Weidi Biotech; sucrose (A502792-0500), sorbitol (A610491-0500), and sodium hypochlorite solution (JC21WA7002) were all purchased from Sangon Biotech; MS medium (M519), agar (A111), and 2,4-D (D309) were all purchased from PhytoTech LABS Company; 6-benzylaminopurine (01-110), naphthylacetic acid (86-87-3), carbenicillin (4800-94-6), hygromycin B (31282-04-9) were purchased from Solarbio Biological Company; plant tissue culture antibacterial agent PPM (71806-DC-001) was purchased from Plant Cell Technology Company; acetosyringone (2478-38-8) and tween20 (V900548) were purchased from Sigma Company; tryptone (LP0042B) and yeast extract (LP0021B) were purchased from OXOID Biological Company;

[0038] The instruments used in the embodiments of the present invention are: the PCR amplification instrument is S1000TM ThermalCycler produced by BIORAD; the real-time fluorescence quantitative PCR instrument is CFX96 Real-Time System produced by BIORAD; the illumination incubator GXZ type is produced by Ningbo Jiangnan Instrument Factory; the constant temperature oscillator IS-RSDA is produced by IncuShaker.

[0039] Unless otherwise stated, the experimental methods disclosed in the present invention all adopt conventional techniques in molecular biology and related fields in the art.

[0040] Example 1 Cloning of OsYUC12, OsYUC13 and OsYUC14 genes

[0041] By inputting the gene locus numbers (LOC_Os02g17230, LOC_Os11g10140 and LOC_Os11g10170) of OsYUC12, OsYUC13 and OsYUC14 into the National Rice Data Center (https: / / www.ricedata.cn / gene / ), the genomic DNA sequences of the corresponding genes and the protein sequences encoded by the genes were obtained. The nucleotide sequence of the wild-type gene of OsYUC12 is shown in SEQ ID NO: 1, the nucleotide sequence of the wild-type gene of OsYUC13 is shown in SEQ ID NO: 2, the nucleotide sequence of the wild-type gene of OsYUC14 is shown in SEQ ID NO: 3, and the gene structures of OsYUC12, 13 and 14 are shown in Figure 1 The amino acid sequence of the wild-type protein of OsYUC12 is shown in SEQ ID NO: 4, the amino acid sequence of the wild-type protein of OsYUC13 is shown in SEQ ID NO: 5, the amino acid sequence of the wild-type protein of OsYUC14 is shown in SEQ ID NO: 6, and the comparison diagram of the predicted encoded proteins is shown in Figure 2 shown.

[0042] Example 2 Construction of OsYUC12 / 13 / 14 gene editing vector

[0043] 1. Design of gene editing target sequence

[0044] The gene coding region sequences were obtained by inputting the gene locus numbers (LOC_Os02g17230, LOC_Os11g10140 and LOC_Os11g10170) of OsYUC12, OsYUC13 and OsYUC14 into the rice genome database RGAP (http: / / rice.uga.edu / index.shtml).

[0045] According to the results of homology comparison analysis of the coding sequences of the OsYUC12 / 13 / 14 genes, a degenerate leader sequence was designed as shown in SEQ ID NO.7, which can guide the Cas9 protein to simultaneously recognize the target sequences in the three genes OsYUC12, OsYUC 13 and OsYUC14. The target sequence of OsYUC12 is shown in SEQ ID NO.8, the nucleotide sequence of the target sequence OsYUC13-SG of OsYUC13 is shown in SEQ ID NO: 9, and the nucleotide sequence of the target sequence OsYUC14-SG of OsYUC14 is shown in SEQ ID NO: 10, so as to simultaneously edit the genes.

[0046] 2. Vector construction and Agrobacterium transformation and preservation

[0047] Based on the leading sequence, the following primer sequences were designed and synthesized:

[0048] SG-F: tgattgCCATAGTTGTTCGTAGCCCG (as shown in SEQ ID NO: 11);

[0049] SG-R: aaacCGGGCTACGAACAACTATGG (as shown in SEQ ID NO: 12);

[0050] The uppercase nucleotides of SG-F and SG-R are the forward and reverse complementary sequences of the leading sequence, respectively; the lowercase nucleotides are the CRISPR / Cas vector sequence DNA. These two primers are used to form a fragment with sticky ends through base complementation, and then the target fragment is connected to the vector through the reaction solution in the kit.

[0051] The ligation reaction solution was transformed into Escherichia coli DH5a by heat shock at 37°C, cultured inverted on kanamycin medium overnight, and single clones were selected. The plasmid was extracted by shaking and sent to a sequencing company for sequencing. Correct sequencing indicated that the recombinant vector CRISPR / Cas9-OsYUC12 / 13 / 14 was positive.

[0052] The positive recombinant vector CRISPR / Cas9-OsYUC12 / 13 / 14 was transformed into Agrobacterium EHA105 by heat shock at 42°C, cultured inverted overnight on rifampicin and kanamycin medium, and single clones were selected and shaken and stored at -80°C for later use; the stored bacteria were engineered bacteria of the CRISPR / Cas9-OsYUC12 / 13 / 14 gene editing vector.

[0053] The reaction procedure of PCR amplification is shown in Table 1, and the reaction system is shown in Table 2.

[0054] Table 1

[0055]

[0056] Table 2

[0057]

[0058] Example 3 Obtaining transgenic rice

[0059] 1. Induction of callus

[0060] Hull the green fresh rice seeds that are not fully mature (with the increase of rice seed maturity, the conversion efficiency and induction rate of callus tissue will decrease), place the hulled seeds in 75% ethanol and wash them for 1 minute, then place the seeds in 25% sodium hypochlorite solution containing 1-2 drops of tween20, wash for 30 minutes, and perform subsequent operations on a sterile clean bench. Wash with sterile water 2-3 times, 10 minutes each time. The sterilized seeds are sown on MSD1 (pH5.8, MS media, 4.4g / L; Sucrose, 30g / L; 2,4-D, 2.0mg / L; Agar, 0.8%) medium and cultured in a 28℃ light incubator (uninterrupted light). After about half a month, the healthy callus tissue is peeled off with sterilized tweezers and transferred to the freshly prepared MSD medium. After about a week, the subsequent infection steps can be carried out.

[0061] 2. Agrobacterium-mediated genetic transformation

[0062] Inoculate Agrobacterium with OsYUC12 / 13 / 14 gene editing vector into the corresponding resistant liquid culture medium, culture in a constant temperature incubator overnight, dilute the bacterial solution to 0.1-0.2 with TY+AS (pH5.5, Yeast Extra 3g / L, Tryptone5g / L, Acetos-yringone 0.2M) culture solution, and shake in a 25°C incubator for 2-4 hours away from light. Mix the callus on MSD1 with the above bacterial solution and incubate for about 0.5h, shaking manually 2-3 times during the period. Take out the incubated callus and place it on sterile paper, absorb the excess culture solution and dry it thoroughly. The dried callus was transferred to MSD2 (pH 5.8, Sucrose 30 g / L, MS Media 4.4 g / L, Sorbitol 50 g / L, Agar 1.4%, 2,4-D 2 mg / L, Acetosyringone 0.2 M) medium and cultured at 22° C. in the dark for 2-3 days.

[0063] 3. Screening

[0064] The yellow or brown callus on MSD2 was washed with MSD liquid (PH 5.8, Sucrose 30g / L, MS Media 4.4g / L, Carbenicillin 400mg / L, Plant Preservative Mixture 1ml / L) for 3 times, 10 minutes each time. The dried callus was transferred to MSD3 (pH 5.8, Sucrose 30g / L, MS Media 4.4g / L, Agar 0.8%, 2,4-D 2mg / L, Carbenicillin 250mg / L, Plant Preservative Mixture 1ml / L, Hygromycin B 50mg / L) medium, and the incubator was adjusted to 28°C, with uninterrupted light, and the culture plate was replaced every 15 days. During the process of replacing the medium, the callus with bacteria and brown color was discarded.

[0065] 4. Rooting and sprouting

[0066] About one month later, the callus will proliferate new small callus. When it grows to 3-5 mm, the callus is transferred to MSD4 (pH5.8, Sucrose 30g / L, MS Media4.4g / L, Sorbitol50g / L, Agar 1.4%, N-(Phenylmethyl)-9H-purin-6-amine 3mg / L, NAA 0.5mg / L, Carbenicillin 50mg / L, Hygromycin B 50mg / L) medium. The medium is replaced every 2 weeks. After culturing at 28°C with uninterrupted light for about 3 weeks, green spots will appear on the callus. At this time, the green callus is transferred to rooting medium (pH5.8, Sucrose 30g / L, MS Media4.4g / L, Agar 1.4%, Carbenicillin 50mg / L, Hygromycin B 50mg / L), 25-30℃, 8 hours of darkness and 16 hours of light, culture on the culture medium until rooting, transfer the rooted transgenic plants to soil for growth, and finally obtain transgenic materials.

[0067] Example 4 Identification of mutation positions of gene editing materials

[0068] The DNA of young leaves of transgenic and unedited ZH11 was extracted from each plant using the CTAB method. The editing site sequences were amplified using the following primers:

[0069] OsYUC12-seq-F (the nucleotide sequence is shown in SEQ ID NO: 13), OsYUC12-seq-R (the nucleotide sequence is shown in SEQ ID NO: 14), OsYUC13-seq-F (the nucleotide sequence is shown in SEQ ID NO: 15), OsYUC13-seq-R (the nucleotide sequence is shown in SEQ ID NO: 16), OsYUC14-seq-F (the nucleotide sequence is shown in SEQ ID NO: 17), and OsYUC14-seq-R (the nucleotide sequence is shown in SEQ ID NO: 18).

[0070] DNA was extracted from the leaves of the gene-edited transgenic material and the wild-type ZH11, and the extracted DNA was specifically amplified and sequenced using the above primers; the sequenced DNA sequence of the gene-edited transgenic material was compared with the sequenced DNA sequence of the wild-type ZH11, and the gene mutation site and mutation type were determined based on the sequencing peak graph. The sequence with a single peak graph and base insertion, deletion or substitution compared with the wild type was selected as the homozygous mutant material with successful gene editing; if the sequencing peak graph had mixed peaks or double peaks, it might be a heterozygous mutant. The sequencing results are as follows Figure 3 As shown. Homozygous mutants with insertions or deletions that were not multiples of 3 bases were selected to obtain the following two gene-edited OsYUC12 / 13 / 14 homozygous mutant materials:

[0071] OsYUC12 / 13 / 14-1 (OsYUC12 inserts a base (A) between 594-595 nucleotides from the ATG start of its wild-type gene, OsYUC13 inserts a base (T) between 604-605 nucleotides from the ATG start of its wild-type gene, and OsYUC14 inserts a base (T) between 604-605 nucleotides from the ATG start of its wild-type gene) and OsYUC12 / 13 / 14-2 (OsYUC12 inserts a base (A) between 594-595 nucleotides from the ATG start of its wild-type gene, OsYUC13 inserts a base (A) between 603-604 nucleotides from the ATG start of its wild-type gene, and OsYUC14 is missing nucleotides 596-615 from the ATG start of its wild-type gene).

[0072] Example 5 Phenotypic Analysis of Gene Editing Materials

[0073] OsYUC11 / 12 / 13 / 14 is a CRISPR / Cas9 gene editing material constructed by knocking out the OsYUC11 (wild-type nucleotide sequence as shown in SEQ ID NO: 19) site on the basis of OsYUC12 / 13 / 14.

[0074] Gene editing site: TTCCTATG-CTGCGCAAA, mutation type: TTCCTATGaCTGC GCAAA, the other OsYUC12,13.14 mutation types are consistent with OsYUC12 / 13 / 14-2 mutation types.

[0075] Based on the leading sequence, the following primer sequences were designed and synthesized:

[0076] SG-F: tgattgTTCCTATGCTGCGCAAA (nucleotide sequence shown in SEQ ID NO: 20);

[0077] SG-R: aaacTTTGCGCAGCATAGGAA (nucleotide sequence shown in SEQ ID NO: 21);

[0078] The vector construction is consistent with Example 2

[0079] The pure mutant material OsYUC13 / 14 was obtained by hybridizing OsYUC12 / 13 / 14 with the wild-type material ZH11 and separating the offspring; the pure mutant material OsYUC12 was obtained by hybridizing OsYUC12 / 13 / 14 with the wild-type material ZH11 and separating the offspring; the pure mutant material OsYUC14 was obtained by hybridizing OsYUC12 / 13 / 14 with the wild-type material ZH11 and separating the offspring; the two transgenic materials OsYUC12 / 13 / 14-1 and OsYUC12 / 13 / 14-2 obtained in Example 2 are wild-type materials ZH11, and the plant type diagram is shown in the figure. Figure 4 shown.

[0080] The above materials were subjected to phenotypic analysis, and the results are shown in Table 1.

[0081] Table 1

[0082]

[0083] The particle length and particle width of the above materials are as follows Figure 5 As shown, the amylose content and protein content are Figure 6 The results showed that compared with the wild-type materials, the mature grain length of the OsYUC12 / 13 / 14-1 and OsYUC12 / 13 / 14-2 mutant materials increased significantly, the grain width did not change significantly, the amylose content decreased, and the protein content did not differ significantly; while the grain width of the OsYUC11 / 12 / 13 / 14 mutant materials was significantly reduced compared with the other materials.

[0084] The gene and genetic engineering technology provided by the present invention can significantly increase the grain length of rice without affecting other agronomic traits of rice, and has great application value.

[0085] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

[0086] synthetic construct

[0087] Sequence Listing

[0088] SEQ ID NO.1

[0089] Artificial sequence

[0090] SEQ ID NO.1

[0091] ATGGAGGAGGTGGTTGTTCTCATCATCGGTGCGGGGCCGGCAGGCCTAGC

[0092] GACGGCAGCATGCCTCACATTGCAACATGTCGCCTATGCCATTATTGAGC

[0093] GTGAGTCATGCACCGCATCGTTATGGCGCCACCGTACCTATGACCGCCTC

[0094] AAATTGCACCTCGCCAAGGAATTCTGCGAGTTGCCACACATGGCGTATCC

[0095] CTCTGGCACGCCGACATACGTCCCCAGGGAGAGTTTTGTGGAATACCTAG

[0096] ACAGCTACACTGACCGATTTGGGATCCAACCAAGGTACGATACCTCTGTC

[0097] GAGTCAGCAACATATGATCAAGGAAAGAAGCATTGGGCTGTGCTAGCAC

[0098] AGGACACTGACACCGGTGTTGTAGCAAGACTCACTGCACGGTTCCTCATC

[0099] ATGGCCACCGGTGAGAAAAGCGCAGCAAGCATTCCACTAGTCCCTGGCC

[0100] TCGCCGGGTTCGAGGGAGAAGCTATCCACTCATCAGCGTACAAATCCGG

[0101] GAATGGCTATACTGGGAAGAGTGTGCTTGTTGTTGGTGCCGGGAACTCTG

[0102] GCATGGAGATCGCCTACGACCTTGCCACACATGGCGCCCACACCTCCATA

[0103] GTTGTTCGCAGCCCGGTACACATAATGACAAAAGAACTAATACGTTTTGG

[0104] GATGACAATGGTGCAAAATCTAGGCCTATCAGTGACAATTGTGGACCCTC

[0105] TTCTTGTAATGGCAGCAAAATTAATTTTTTGGGACTTGTCAAAGCATGGA

[0106] ATTATGAGGCCTAAAATGGGCCCTCTCTTACTGAAGTCACAAACTGGCAA

[0107] ATCTGCTGTCATTGATGTCGGCACTGCAAAATTAATCACTAGAGGAGTTA

[0108] TCGACGTGCTCGAAGGAATATTGAAGATAAATGCAAACAATGTTGAATTT

[0109] CATTGTGGGAGGCAAATTCCATTTGATGCCATTGTGTTTGCCACGGGCTA

[0110] CAAAAGCACAGTTAATACATGGCTAAAGAATGGTGAAAGCATGTTTAGA

[0111] AATGATGGTTTTCCCAAGAAAAAATTTCCAAATCATTGGAGAGGTGAAA

[0112] ATGGGCTCTATTGTGCTGGGTTTGCAAGGAGAGGATTGGTCAGTATTGCC

[0113] ATGGATGCCAAGAATATTGTTGATGACATCAGAGCCACCATGTATCAAGT

[0114] GTCTTGCTAA

[0115] SEQ ID NO.2

[0116] ATGGAGGCTGTGGAGGAGGTGGTCGTTCTCATCGTCGGTGCGGGGCCAG

[0117] CAGGCCTGGCAACAGCAGCGTGCCTCGCACAGCGCCATGTGCCCTATGTC

[0118] ATTGTCGAGCGTGAGTCCTGCACCGCGTCGCTGTGGCGCCACCGTGCCTA

[0119] TGACCGACTCAAGCTGCACCTCGCCAAGGAGTTCTGTGAGCTGCCACACA

[0120] TGGCGTATCCTGTAGGCACGCCAACATACATCCCTAGGGATATGTTTGTG

[0121] GAGTACCTAGATAGCTACACTGACCAGTTTGGGATACGACCAAGGTACC

[0122] ACACTGCTATCGAGTCAGCAATATATGATGGAGGTAAGAACCGTTGGGCT

[0123] GTGCTAGCACGGGACACGGACACTAGTGTTGTGACAAGGCTCACCGCAC

[0124] AGTTCCTTGTCGTGGCCACTGGCGAGAACAGTGCGGCAAGCATTCCACCA

[0125] GTTCCTGGTCTCACTAGGTTTGAGGGAGAAGCCATTCACTCGTCGGCTTA

[0126] CAAGTCTGGGAGAGCCTATACCGGGAAGAATGTGCTTGTGGTCGGTGCC

[0127] GGGAATTCTGGCATGGAGATCGCCTATGATCTTGCCACGCATGGAGCCCA

[0128] CACCTCCATAGTTGTTCGTAGCCCGATACACATCATGACCAAAGAACTAA

[0129] TACGATTTGGGATGACAGTGGTCCAAAATCTAGGGCTAACAGTGACAAC

[0130] AGCGGATTCTCTTCTTGTGATGGCTGCAAATTTTTATTTTTGGAGACTTGTC

[0131] AAAACATGGTATTACGAGGCCTAAAATTGGTCCTCTTTTACTGAAATCAC

[0132] AAACTGGCCGGTCCGCCGTCATAGATGTAGGGACTGCAAGATTAATCAA

[0133] AGGAGGAGTTATCAAATGTTCCAAGGAATATCGAAGATAAAAACAAAT

[0134] AGTATTGAATTTCCATGGTGGGAAGCAAATTCCTTTTGATGCCATTGTATTT

[0135] GCAACAGGCTACAAAAGCACAGTAAATACATGGCTAAAAAAAATTCAA

[0136] TAATTGCTGGTTGGATTGGGTAA

[0137] SEQ ID NO.3

[0138] ATGAATGCTATGGAGGACGTGGTTGTTCTCATCGTTGGTGCGGGGCCAGC

[0139] AGGCCTAGCAACGGCGGCGTGCCTTGCACAGCGGCATGTGCCATACATC

[0140] ATTGTCGAGCGTGAGTCCAGCACCGCATCGTTGTGGCGCCACCGTGCCTA

[0141] CGACCGCCTCAAGCTACACCTCGCCAAGGAGTTCTGCGAGCTGCCACACA

[0142] TGGCGTATCCGGCGGGCACGCCGACGTACGTTCCTAGGGATATGTTTGTG

[0143] GAGTACCTAGATAGCTATGCTAACCAGTTTGGGATACGGCCGAGGTACCA

[0144] CACTGCTGTTGAGTCAGCAATACATGATAAAGGTAAGAACCAATGGGTC

[0145] GTGCTAGTACGAGACATGGACACTAGTGTTGTGGCAAGGCTTGCTACACA

[0146] GTTCCTTGTTGTGGCCGCTGGCGAGAACAGTGCGGCAAACATTCCACCAA

[0147] TTCCTGGCCTCAGTAGGTTCGAGGGAGAAGCCATTCACTCGTCAGCTTAC

[0148] AAGTCCGGGAGAGCCTATACCGGGAAGAGTGTGCTTGTGGTAGGTGCCG

[0149] GGAATTCTGGCATGGAGATCGCTTATGACCTTGCCACGCATGGAGCCCAC

[0150] ACCTCCATAGTTGTTCGTAGCCCGGTACACATAATGACCAAAGAACTAAT

[0151] ATGGTATGGGATGACAATGGTGCAAAATTTAGGGCTAAATGTGACAGCA

[0152] GTGGACTCTCTTCTTGTAATGGCGGCAAATTTCTATTTCGGAGACTTGTCA

[0153] AAACATGGTATTATGAGGCCTAAAATGGGTCCTCTCTTACTCAAGTCACA

[0154] AACAGGCCGGTCTGCCGTTATTGATGTTGGCACTGCAAGATTAATCAAAG

[0155] GAGGAGTTATCAAAGTGTTCCAAGGAATATCAAAGATAAATACAAACAG

[0156] TGTTGAATTTCATGGTGGGAGGCAAAATTCATTTGACGCCATTGTATTTG

[0157] CGACAGGTTACAAAAGCACAGTTAATGCATGGCTAAAGAATGGTGAAAG

[0158] CATGTTTAAAGACGATGGTTTCCCTAAAAATTATTTTCCAAATCATTGGA

[0159] GAGGTGAAAATGGGTTGTATTGTGCTGGGTTTGCTAGGAGAGGATTGGCC

[0160] GGTATTGCCATGGATGCGAAAAATATTGCAAATGACATCGTGGCCGCCAT

[0161] GGATAAAATGTCATGTTAA

[0162] SEQ ID NO.4

[0163] MEEVVVLIIGAGPAGLATAACLTLQHVAYAIIERESCTASLWRHRTYDRLKL

[0164] HLAKEFCELPHMAYPSGTPTYVPRESFVEYLDSYTDRFGIQPRYDTSVESATY

[0165] DQGKKHWAVLAQDTDTGVVARLTARFLIMATGEKSAASIPLVPGLAGFEGE

[0166] AIHSSAYKSGNGYTGKSVLVVGAGNSGMEIAYDLATHGAHTSIVVRSPVHIM

[0167] TKELIRFGMTMVQNLGLSVTIVDPLLVMAAKLIFWDLSKHGIMRPKMGPLLL

[0168] KSQTGKSAVIDVGTAKLITRGVIDVLEGILKINANNVEFHCGRQIPFDAIVFAT

[0169] GYKSTVNTWLKNGESMFRNDGFPKKKFPNHWRGENGLYCAGFARRGLVSI

[0170] AMDAKNIVDDIRATMYQVSC

[0171] SEQ ID NO.5

[0172] MEAVEEVVVLIVGAGPAGLATAACLAQRHVPYVIVERESCTASLWRHRAYD

[0173] RLKLHLAKEFCELPHMAYPVGTPTYIPRDMFVEYLDSYTDQFGIRPRYHTAIE

[0174] SAIYDGGKNRWAVLARDTDTSVVTRLTAQFLVVATGENSAASIPPVPGLTRF

[0175] EGEAIHSSAYKSGRAYTGKNVLVVGAGNSGMEIAYDLATHGAHTSIVVRSPI

[0176] HIMTKELIRFGMTVVQNLGLTVTTADSLLVMAANFYFGDLSKHGITRPKIGP

[0177] LLLKSQTGRSAVIDVGTARLIKGGVIKVFQGISKIKTNSIEFHGGKQIPFDAIVF

[0178] ATGYKSTVNTWLKKNSIIAGWIG

[0179] SEQ ID NO.6

[0180] MNAMEDVVVLIVGAGPAGLATAACLAQRHVPYIIVERESSTASLWRHRAYD

[0181] RLKLHLAKEFCELPHMAYPAGTPTYVPRDMFVEYLDSYANQFGIRPRYHTA

[0182] VESAIHDKGKNQWVVLVRDMDTSVVARLATQFLVVAAGENSAANIPPIPGL

[0183] SRFEGEAIHSSAYKSGRAYTGKSVLVVGAGNSGMEIAYDLATHGAHTSIVVR

[0184] SPVHIMTKELIWYGMTMVQNLGLNVTAVDSLLVMAANFYFGDLSKHGIMR

[0185] PKMGPLLLKSQTGRSAVIDVGTARLIKGGVIKVFQGISKINTNSVEFHGGRQN

[0186] SFDAIVFATGYKSTVNAWLKNGESMFKDDGFPKNYFPNHWRGENGLYCAG

[0187] FARRGLAGIAMDAKNIANDIVAAMDKMSC

[0188] SEQ ID NO.7

[0189] CCATAGTTGTTCGTAGCCCG

[0190] SEQ ID NO.8

[0191] CCTCCATAGTTGTTCGCAGCCCG

[0192] SEQ ID NO.9

[0193] CCTCCATAGTTGTTCGTAGCCCG

[0194] SEQ ID NO.10

[0195] CCTCCATAGTTGTTCGTAGCCCG

[0196] SEQ ID NO.11

[0197] tgattgCCATAGTTGTTCGTAGCCCG

[0198] SEQ ID NO.12

[0199] aaacCGGGCTACGAACAACTATGG

[0200] SEQ ID NO.13

[0201] TATCCACTCATCAGCGTACA

[0202] SEQ ID NO.14

[0203] AGCTAGAAAGTGAGAACAAGTC

[0204] SEQ ID NO.15

[0205] GGGAGAAGCCATTCACTCGT

[0206] SEQ ID NO.16

[0207] GGTCATGATGTGTATCTGC

[0208] SEQ ID NO.17

[0209] ATGGACACTAGTGTTGTGGCAA

[0210] SEQ ID NO.18

[0211] GAAAGATACAACATGACAATTTCGG

[0212] SEQ ID NO.19

[0213] ATGGAGAAGGCTCTAGTTCTGATTGTTGGAGCAGGCCCATCTGGTCTTGC

[0214] AACAGCGGCGTGCCTTGGCCAGCTCTCTATACCCTATGTTATTATCGAGC

[0215] GTGAGGATTGTACCGCGTCGCTGTGGCGTAAGCACACATATGATCGCCTC

[0216] AAGCTTCACCTCGCAAAGGAGTTCTGCGAGATGCCCCACATGCCATATCC

[0217] GGAAGACACACCAACCTACATCCCCAAGATTCAATTTTTGAGGTATATGG

[0218] ATGATTATGTGGAGCATTTCAACATTTGTCCCAAATTTAACTCATCTGTTG

[0219] AGTCATGTTTGTATGATGATGTGCAAAAATATTGGGTTGTCACAACACAT

[0220] GACCAAGTGAATGGCATGGTGTCCAAGTATGCAGCTAGGTTCTTAGTTGT

[0221] AGCAAGTGGTGAAAATAGTGCAGGGAACATTCCTAGCATCCCTGGACTA

[0222] GAGGATTTCTCTGGCCATGTCATACACTCATCAAGCTTTAGGTCAGCAGA

[0223] TTCCTATGCTGCGCAAAGGGTGCTTGTTGTTGGATGTGGAAACTCAGGCA

[0224] TGGAGATTGCCTATGATCTTTCAAGCCATGGAGCAAATACCTCCATAGTC

[0225] ATCCGCAGCCCTCTGCATGTGATGACAAAGGAGCTGATTCACATGGGGAT

[0226] GAAACTTGCAAGCTGGAGCCTCCCAGTGAAATTTGTTGATTTTATTCTTGT

[0227] GGTCCTTGCATATCTCTGGTTTGGCAATCTCTCAAAGTATGGCATCGTGA

[0228] GGCCCAATAAGGGGCCATTGCTTCTTAAGGCAAATACTGGTCGGTCAGCA

[0229] GTAATAGATGTTGGCACTGTGGAGTTAATAAAGAAAGGGGATATCAAGG

[0230] TCTTTGGTACAATATCTTGCATCAAGGGAAATGTTGTTGAATTTGATGAT

[0231] GGGAAAGAAAGTTACTTTGATGCTATTGTTTTTGCTACAGGCTACACAAG

[0232] TACTGCAAACAACTGGCTTAAGAATGGTGAGGATATGATGAACAAAGAA

[0233] GGCATGCCCAAGAAGGACTTCCCAAATCACTGGAAAGGTTCAAATGGAC

[0234] TCTACTGTGTTGGGTTTGCGAGGAGAGGATTATCTGGTATTGCTCATGAC

[0235] GCCAAAAACGTTGCTAATGACGTCAAGGCCTTCCTAGATTTGATGGCACC

[0236] ATTCTAA

[0237] SEQ ID NO.20

[0238] tgattgTTCCTATGCTGCGCAAA

[0239] SEQ ID NO.21

[0240] aaacTTTGCGCAGCATAGGAA

Claims

1. A rice OsYUC12 / 13 / 14 mutant gene, characterized in that: The rice OsYUC12 / 13 / 14 mutant gene is any one of the following: (i) inserting an A base between nucleotides 594-595 of the start codon ATG of the wild-type gene of OsYUC12, inserting a T base between nucleotides 604-605 of the start codon ATG of the wild-type gene of OsYUC13, and inserting a T base between nucleotides 604-605 of the start codon ATG of the wild-type gene of OsYUC14; (ii) An A base was inserted between nucleotides 594-595 of the start codon ATG of the wild-type gene of OsYUC12, an A base was inserted between nucleotides 603-604 of the start codon ATG of the wild-type gene of OsYUC13, and nucleotides 596-615 of the start codon ATG of the wild-type gene of OsYUC14 were deleted.

2. The rice OsYUC12 / 13 / 14 mutant gene according to claim 1, characterized in that: The nucleotide sequence of the OsYUC12 wild-type gene is shown in SEQ ID NO: 1, the nucleotide sequence of the OsYUC13 wild-type gene is shown in SEQ ID NO: 2, and the nucleotide sequence of the OsYUC14 wild-type gene is shown in SEQ ID NO:

3.

3. The protein of the rice OsYUC12 / 13 / 14 mutant gene according to claim 1 or 2, characterized in that: The amino acid sequence of the OsYUC12 wild-type protein is shown in SEQ ID NO:4, the amino acid sequence of the OsYUC13 wild-type protein is shown in SEQ ID NO:5, and the amino acid sequence of the OsYUC14 wild-type protein is shown in SEQ ID NO:

6.

4. A recombinant vector, characterized in that: The recombinant vector comprises the mutant gene (i) or (ii) according to claim 1.

5. A method for preparing a rice mutant containing the mutant gene according to claim 1, characterized in that: include, Selection of gene target sequence: Screen the target sequence based on the full length of CDS of OsYUC12 / 13 / 14 gene; Construction of CRISPR / Cas9 expression vector: site-directed editing of OsYUC12 / 13 / 14 genes, and transformation of the obtained recombinant vector into Agrobacterium; Agrobacterium-mediated genetic transformation: Prepare Agrobacterium infection solution, infect rice callus tissue, then culture the infected rice callus tissue, and obtain transgenic mutant plants by single plant sequencing and identification.

6. The method according to claim 5, characterized in that: The nucleotide sequence of the target sequence OsYUC12-SG of OsYUC12 is shown in SEQ ID NO: 8, the nucleotide sequence of the target sequence OsYUC13-SG of OsYUC13 is shown in SEQ ID NO: 9, and the nucleotide sequence of the target sequence OsYUC14-SG of OsYUC14 is shown in SEQ ID NO:

10.

7. The method according to claim 5, characterized in that: The Agrobacterium is one of GV3101 Agrobacterium and EHA105 Agrobacterium.

8. The method according to claim 5, characterized in that: The culture medium formula of the rice callus tissue is 30 g / L sucrose, 4.4 g / L MS culture medium, 50 g / L sorbitol, 1.4% agar, 3 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 50 mg / L carbenicillin and 50 mg / L hygromycin B.

9. Use of the rice OsYUC12 / 13 / 14 mutant gene as claimed in claim 1 in reducing the content of amylose and improving the taste quality of rice.

10. Use of the rice OsYUC12 / 13 / 14 mutant gene as claimed in claim 1 in increasing rice yield.

Images