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CN108503700B - Rice grain protein and its encoding gene and application - Google Patents

Rice grain protein and its encoding gene and application Download PDF

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CN108503700B
CN108503700B CN201810610171.4A CN201810610171A CN108503700B CN 108503700 B CN108503700 B CN 108503700B CN 201810610171 A CN201810610171 A CN 201810610171A CN 108503700 B CN108503700 B CN 108503700B
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rice
grain
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nucleotide sequence
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CN108503700A (en
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陈亮
林雅容
朱义旺
王�锋
陈睿
梅法庭
范美英
郭新睿
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Xiamen University
Institute of Biotechnology of Fujian Academy of Agricultural Science
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Institute of Biotechnology of Fujian Academy of Agricultural Science
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Abstract

本发明提供了水稻Os01g66970.1蛋白及其编码基因与应用，该蛋白具有如SEQ ID NO.2所示的氨基酸序列，或为SEQ ID NO.2所示的氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的具有同等功能的氨基酸序列；该编码基因具有如SEQ ID NO.1所示的核苷酸序列，或与SEQ ID NO.1所示的核苷酸序列杂交的核苷酸序列。本发明还公开了利用该基因培育大粒水稻品种的方法。即通过基因编辑方法对该基因进行编辑，从而获得大粒水稻品种；其次，本发明粒型调控基因调控效果好，与野生型相比，水稻粒长明显增加，可以显著提高水稻产量，在水稻杂种优势利用中具有广阔的应用前景。The present invention provides rice Os01g66970.1 protein and its encoding gene and application. The protein has the amino acid sequence shown in SEQ ID NO.2, or the amino acid sequence shown in SEQ ID NO.2 after one or more amino acid residues The amino acid sequence with the equivalent function formed by the substitution, deletion or addition of the base; the coding gene has the nucleotide sequence shown in SEQ ID NO.1, or hybridizes with the nucleotide sequence shown in SEQ ID NO.1 nucleotide sequence. The invention also discloses a method for cultivating large-grain rice varieties by using the gene. That is, the gene is edited by a gene editing method to obtain a large-grain rice variety; secondly, the grain shape regulation gene of the invention has a good regulation effect. Compared with the wild type, the grain length of the rice is significantly increased, and the rice yield can be significantly improved. It has broad application prospects in the utilization of advantages.

Description

Rice grain type protein and coding gene and application thereof
Technical Field
The invention relates to the technical field of gene editing, in particular to a rice grain type protein Os01g66970.1 and a coding gene and application thereof.
Background
As rice (Oryza sativa. L) is one of the most important food crops in the world, increasing the yield thereof with the decrease of the cultivated land area and the increase of the population has a very important strategic significance for solving the future food safety problem. The grain shape (including grain length, grain width, aspect ratio and grain thickness) of rice is not only one of the important indexes affecting rice yield, but also an important factor affecting the appearance quality, commodity quality and processing quality of rice. Therefore, the research on the genetic mechanism of rice grain shape has important guiding significance for cultivating high-yield and high-quality rice varieties.
The completion of rice whole genome sequencing and the continuous improvement of genetic linkage maps lay the foundation for the positioning of rice grain shape QTL, the cloning and the functional analysis of important genes. However, the genetic control mechanism of the trait of the granuloma is still imperfect due to the complexity of the granuloma inheritance itself and the limitations of the research methods. Therefore, the clone and function research of the rice grain type related gene have important significance for determining the molecular mechanism for regulating and controlling the grain type in the rice and the application of the molecular mechanism in cross breeding.
In recent years, the rapid development of genome editing technology provides a highly effective auxiliary tool for the research of rice gene function and the improvement of genetic breeding. The method utilizes a repair mechanism that site-specific nuclease causes double strand break on a plant genome DNA sequence to stimulate cell self homologous recombination and non-homologous end joining, and completes targeted modification on the plant genome by randomly adding or deleting the number of bases of a target sequence, thereby realizing site-specific editing of a target DNA sequence. On rice, the technology realizes the knockout of a plurality of negative regulatory genes so as to obtain the improvement of favorable agronomic traits and has a powerful propulsion effect on the breeding process of rice molecules.
Disclosure of Invention
The invention aims to provide rice grain type protein Os01g66970.1 and a coding gene and application thereof.
Hair brushMing rice grain type protein Os01g66970.1 from rice (A)Oryza Sativa) Has an amino acid sequence shown as SEQ ID NO. 2; or an amino acid sequence having equivalent functions, which is formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence.
The coding gene of the rice grain type protein Os01g66970.1 has a nucleotide sequence shown as SEQ ID NO. 1; or generated by adding, substituting or deleting one or more bases in the nucleotide sequence shown in SEQ ID NO.1, and encodes the nucleotide sequence for controlling rice grain type protein Os01g66970.1.
The invention also aims to provide the application of the rice grain type related protein or the coding gene in controlling the rice grain type and improving the rice yield. The large-grain rice variety is cultivated by knocking out the genes, and the large-grain rice variety is realized through a CRISPR/Cas9 system. The sgRNA target sequence for knocking out the gene consists of a nucleotide sequence shown in SEQ ID NO. 6; namely 5'-GGGTGGGGATGGGGATGGACGGG-3'.
The invention also provides a primer pair for detecting the transgenic plant with the knocked-out gene, wherein the primer pair consists of JC-F and JC-R; wherein the JC-F consists of a nucleotide sequence shown in SEQ ID NO. 4; the JC-R
Consists of a nucleotide sequence shown as SEQ ID NO. 5; namely:
JC-F:5’-AAATTCCATCAAAAAGCAGA- 3’(SEQ ID NO .4)；
JC-R:5’-ACAACCAAAAATAACATCGG- 3’(SEQ ID NO .5)。
compared with the prior art, the beneficial results of the invention are as follows:
the invention provides a method for obtaining a large-grain rice variety by targeting a rice grain type related gene Os01g66970.1 through a gene editing method for cultivating the large-grain rice variety; (2) the grain type regulatory gene has good regulatory effect, and compared with the wild type, the grain length and the grain weight of the rice are increased, so that the yield of the rice can be obviously improved, and the rice has wide application prospect in the utilization of rice heterosis.
Drawings
FIG. 1 shows the sequence analysis and gene editing target design of Os01g66970.1 gene of rice. Black boxes indicate exons, black lines indicate introns, and white boxes indicate untranslated regions. The Cas9 recognition sequence is located at 85 bp of the first exon.
FIG. 2 Cas 9-mediated acquisition of different genotypes of Os01g66970.1. WT is a wild-type sequence, Arabic numerals indicate the number of bases that are deleted or added (minus indicates base deletion and plus indicates base addition), and lowercase letters indicate added bases.
FIG. 3 phenotype investigation of different genotypes of the Os01g66970.1 gene. WT is a gang you 8515 wild type control plant, 970-4 and 970-5 are mutant plants with different genotypes.
FIG. 4 shows grain type and grain weight analysis of Os01g66970.1 gene mutant. WT is a gang you 8515 wild type control plant, 970-4 and 970-5 are mutant plants with different genotypes.
FIG. 5 analysis of Os01g66970.1 gene mutant related yield traits. WT is a gang you 8515 wild type control plant, 970-4 and 970-5 are mutant plants with different genotypes.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
The test materials used in the following examples were all commercially available products unless otherwise specified.
Example 1: cloning of Os01g66970.1 Gene of Rice
According to the Os01g66970.1 gene information recorded in the rice data center, a full-length primer sequence F is designed to be 5 'ATGGCGCGCACCGAGA 3' and R is designed to be 5 'CTATGTACAGTTGAGCTTCACGAGG 3'. And amplifying the Os01g66970.1 genome sequence by using the genomic DNA of Okayou 8515 as a template.
Example 2: sequence analysis and gene editing target design of rice Os01g66970.1 gene
The rice Os01g66970.1 gene is shown as a sequence SEQ ID NO. 1. Sequence analysis shows that the gene contains 7 exons, namely, the 1 st-195 th exon (the first exon), the 1040 th-1111 th exon (the second exon), the 1375 th-1442 th exon (the third exon), the 1594 th-1741 th exon (the fourth exon), the 1847 th-1930 th exon, the 2358 th-2444 th exon and the 3409 th-3609 th exon (the seventh exon) in the sequence table.
The Os01g66970.1 gene of the experimental variety Ongyou 8515 designs a Cas9 recognition site at a first exon 85 bp position, as shown in figure I. The recognition target sequences are respectively: (sgRNA) GGGTGGGGATGGGGATGGACGGG.
Example 3: construction of targeting vector and genetic transformation of rice
The experiment adopts a gene editing technology as a third generation gene editing technology CRISPR/Cas9, the used vector is an artificially synthesized recombinant plasmid pCXUN-Cas9-gRNA (circular vector), the framework vector pCXUN-Cas9 is a common linear vector ordered on the market, (the experiment is purchased from Beijing Weishanglide Biotech Co., Ltd.), the full length of a Cas9 protein sequence is 4206 base pairs, and 1401 amino acids are coded. Its expression is driven by the Ubiquitin promoter, and the gRNA is driven by the U6 promoter. The gRNA fragment was synthesized by primer synthesis and ligated to the U6 promoter by DNA ligase to form the recombinant plasmid pCXUN-Cas9-gRNA (circular vector).
In the invention, the vector pCXUN-Cas9-gRNA is used for transforming calluses of japonica rice, indica rice and glutinous rice as receptor materials for genetic transformation, the genetic transformation is realized by an agrobacterium-mediated method, and a new red rice variety is created by screening, differentiating and rooting of resistant calluses, wherein Xiushui 134 and Gaoyou 8515 are introduced as an example, and the specific steps are as follows:
1. the mature rice seeds were husked and sterilized and inoculated in an induction medium (NB medium +2, 4-D2 mg. L)-1pH5.8), culturing at 28 deg.C for 7 days in dark, removing radicle, culturing for 7 days, transferring embryogenic callus to new induction medium, and subculturing every 15 days. The granular callus with natural dispersion, bright yellow color and diameter of about 3mm after three subcultures can be used for agrobacterium transformation.
2. Marking a small amount of recombinant agrobacterium liquid on YEB solid cultureNutrient medium (containing kanamycin 50 mg.L-1And rifampicin 50 mg. L-1pH5.8), and culturing at 28 deg.C in dark for 48h for activation; taking single colony on the activated plate for transferring and cutting bacteria, culturing at 28 deg.C for two days in dark, and culturing with AAM medium (containing 100. mu.M.L)-1Acetosyringone, pH 5.2), washing and re-suspending the bacteria, adjusting the concentration of the bacteria liquid to OD600nm = 1.5-2.0, and standing for 1h to obtain the agrobacterium suspension.
3. Soaking the embryogenic callus obtained in the step 1 in the agrobacterium suspension obtained in the step 2, standing for 30min, airing the callus on sterile filter paper, and inoculating the callus on a co-culture medium (NB medium +2, 4-D2 mg. L)-1+ Acetylsyringone 100. mu.M.L-1pH5.8), and cultured at 25 ℃ for 3 days in the dark.
4. And (3) selecting the callus co-cultured in the step (3), placing the callus in a wide-mouth culture bottle, washing the wide-mouth culture bottle with sterile water for 5 times, and shaking the wide-mouth culture bottle for several times each time until filamentous thalli are not seen in the water. The final use of the medicine contains 250 mg.L-1Standing with sterile water for 1 hr, air drying the callus on sterile filter paper, inoculating to screening culture medium (NB culture medium +2, 4-D2 mg. L)-1+ carbenicillin 250 mg. L-1 + hygromycin 50mg·L-1pH5.8), dark culture at 28 ℃ is carried out, 1 transfer is carried out every two weeks, and about three weeks is needed for the growth of the tumor-like bright yellow resistant callus from the browned and shriveled callus.
5. Transferring the resistant callus obtained in step 4 to a differentiation medium (NB medium +2, 4-D2 mg. L)-1+KT 10mg·L-1 + NAA 0 .4 mg·L-1pH 5.8), the callus started turning green after 2 weeks, shoots were grown after 3 weeks, and roots were grown afterwards. Transferring the seedling to rooting culture medium (1/2 MS culture medium, pH 5.8), washing the culture medium on the root after the seedling grows to root, and transplanting to experimental base.
Example 4: genotype detection and phenotype investigation of Os01g66970.1 gene editor
Extracting the genome DNA of the transformed plant, and performing PCR amplification by using primers CZTF-F (5 'GGGAGATCCAGCTAGAGGTC 3') and CZTF-F (5 'GGAAGGAGGAAGACAAGG 3') to identify the transgenic plant. Further, primers 970-F: 5'AAATTCCATCAAAAAGCAGA3' and 970-R:5'ACAACCAAAAATAACATCGG 3' of the target gene are used for PCR amplification of the genome DNA of the identified transgenic plant, and an Os01g66970.1 gene fragment with a target sequence is obtained and sent to the company for sequencing. The sequencing results are analyzed, and the Os01g66970.1 gene is found to have 5 types of genotypes, wherein three types of editing modes of heterozygous mutation, biallelic modification and homozygous mutation are included. Further observation of the phenotypes of the mutant plants 970-4 and 970-5 revealed that the rice grain length of both mutants increased, the grain width of 970-5 increased to some extent, while the grain width of 970-4 remained essentially unchanged (see FIG. 3). The dry weights of the seeds of the two mutant strains are weighed, and the thousand seed weights of the rice seeds 970-4 and 970-5 are respectively increased by 15.1% and 14.0% compared with those of the wild type Gaoyou 8515 seeds, so that the rice yield is remarkably increased (as shown in figure 4). And other yield-related traits such as rice ear length, primary branch number, secondary branch maturity and grain number per ear, the difference between 970-4 and 970-5 mutant strains and the Gaokou 8515 wild type is not significant, and the traits are basically unchanged.
The Os01g66970.1 gene mutant is obtained by using the CRISPR/Cas9 gene editing technology, and compared with TALEN and ZFN technologies, the Os01g66970.1 gene mutant is simple and convenient in operation process, saves cost, and is operable in common laboratories. The large-grain rice variety obtained by the invention can be screened and removed of the transgenic exogenous fragments through progeny selfing separation, has wider application prospect compared with the prior transgenic breeding, greatly saves the time cost compared with the conventional crossbreeding, and is a novel molecular breeding method for obtaining the large-grain strain.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
SEQUENCE LISTING
<110> university of mansion
Institute of biotechnology, Fujian Academy of Agricultural Sciences
<120> rice grain type protein, and coding gene and application thereof
<130> 11
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 861
<212> DNA
<213> Artificial sequence
<400> 1
atggcgcgca ccgagaaggt tgccggcgat ggctgctccg gtggtggtgg tggtggtgga 60
gaaggtcagg tggaggtgga ggtcggggtg gggatgggga tggacgggaa gggaatgata 120
gagtgccgga tatgccagga ggaaggggat gagggcgcca tggattcccc ctgcgcctgc 180
actggcacgc tcaagttcgc ccacaggaaa tgcatacaaa gatggtgtga caagaagggg 240
aacatcacat gtgaaatctg caaccaggtt tactctccaa attatgtcct ccctccaacc 300
aagtgttgtt cagctgaaat ggacatggat cttaggcaaa gctgggttgg acgaattgat 360
ccccatgatt cgcattttct tgccattgcc attgcagagc agcagctgct gcaagctgaa 420
tttgatgatt gtgtgtcctc aaattcaagt ggtgccacat gctgccgaac tgttgtttta 480
attttgatgt tacttttgct tgtgcgccat gtagttgtgt ttgtgagaga tgttagcatg 540
ctgcaggatg caacagtgtt gtttagcgca actcttcagt tcgcaggatt ctttcttcca 600
tgttatgtta tagcccgttc ttgttatgct tttcaacacc ggaggcgaag acaggtttag 660
gggccacctg aacctggctg gaagcattta ctcaaaacaa ctcaacacca tcacagagag 720
acgagaggaa aagcttgtga agaagtaact caaactccct ttgcttggag cctctgcgaa 780
agatttggct taatttctcc cccaacaaga tgtagttgcc gatttagacc atgggccctc 840
gtgaagctca actgtacata g 861
<210> 2
<211> 285
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<213> Artificial sequence
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Met Ala Arg Thr Glu Lys Val Ala Gly Asp Gly Cys Ser Gly Gly Gly
1 5 10 15
Gly Gly Gly Gly Glu Gly Gln Val Glu Val Glu Val Gly Val Gly Met
20 25 30
Gly Met Asp Gly Lys Gly Met Ile Glu Cys Arg Ile Cys Gln Glu Glu
35 40 45
Gly Asp Glu Gly Ala Met Asp Ser Pro Cys Ala Cys Thr Gly Thr Leu
50 55 60
Lys Phe Ala His Arg Lys Cys Ile Gln Arg Trp Cys Asp Lys Lys Gly
65 70 75 80
Asn Ile Thr Cys Glu Ile Cys Asn Gln Val Tyr Ser Pro Asn Tyr Val
85 90 95
Leu Pro Pro Thr Lys Cys Cys Ser Ala Glu Met Asp Met Asp Leu Arg
100 105 110
Gln Ser Trp Val Gly Arg Ile Asp Pro His Asp Ser His Phe Leu Ala
115 120 125
Ile Ala Ile Ala Glu Gln Gln Leu Leu Gln Ala Glu Phe Asp Asp Cys
130 135 140
Val Ser Ser Asn Ser Ser Gly Ala Thr Cys Cys Arg Thr Val Val Leu
145 150 155 160
Ile Leu Met Leu Leu Leu Leu Val Arg His Val Val Val Phe Val Arg
165 170 175
Asp Val Ser Met Leu Gln Asp Ala Thr Val Leu Phe Ser Ala Thr Leu
180 185 190
Gln Phe Ala Gly Phe Phe Leu Pro Cys Tyr Val Ile Ala Arg Ser Cys
195 200 205
Tyr Ala Phe Gln His Arg Arg Arg Arg Gln Val Gly Pro Pro Glu Pro
210 215 220
Gly Trp Lys His Leu Leu Lys Thr Thr Gln His His His Arg Glu Thr
225 230 235 240
Arg Gly Lys Ala Cys Glu Glu Val Thr Gln Thr Pro Phe Ala Trp Ser
245 250 255
Leu Cys Glu Arg Phe Gly Leu Ile Ser Pro Pro Thr Arg Cys Ser Cys
260 265 270
Arg Phe Arg Pro Trp Ala Leu Val Lys Leu Asn Cys Thr
275 280 285
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<211> 23
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<213> Artificial sequence
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gggtggggat ggggatggac ggg 23
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aaattccatc aaaaagcaga 20
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acaaccaaaa ataacatcgg 20
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<211> 16
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atggcgcgca ccgaga 16
<210> 7
<211> 25
<212> DNA
<213> Artificial sequence
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ctatgtacag ttgagcttca cgagg 25
<210> 8
<211> 20
<212> DNA
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<400> 8
gggagatcca gctagaggtc 20
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<211> 18
<212> DNA
<213> Artificial sequence
<400> 9
ggaaggagga agacaagg 18
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
aaattccatc aaaaagcaga 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
acaaccaaaa ataacatcgg 20

Claims (5)

1.一种水稻粒型蛋白Os01g66970.1，其特征在于，氨基酸序列如SEQ ID NO .2所示。1. A rice grain protein Os01g66970.1, characterized in that the amino acid sequence is as shown in SEQ ID NO.2. 2.如权利要求1所述的水稻粒型蛋白Os01g66970.1的编码基因，其特征在于，该编码基因核苷酸序列如SEQ ID NO .1所示；或由SEQ ID NO .1所示的核苷酸序列经过添加、取代或缺失一个或几个碱基而生成的，并编码控制水稻粒型蛋白Os01g66970.1的核苷酸序列。2. The coding gene of rice grain protein Os01g66970.1 according to claim 1, wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO.1; or the nucleotide sequence shown in SEQ ID NO.1 The nucleotide sequence is generated by adding, replacing or deleting one or several bases, and encodes the nucleotide sequence that controls the rice grain protein Os01g66970.1. 3.如权利要求2所述的基因在控制水稻粒型上的应用。3. The application of the gene according to claim 2 in controlling rice grain shape. 4.根据权利要求3所述的应用，其特征在于，通过敲除权利要求2所述基因来培育大粒水稻品种。4. The application according to claim 3, wherein large-grain rice varieties are cultivated by knocking out the gene of claim 2. 5.根据权利要求4所述的应用，其特征在于，用于敲除基因的sgRNA靶点序列为5’-GGGTGGGGATGGGGATGGACGGG- 3’。5. The application according to claim 4, wherein the sgRNA target sequence for knocking out the gene is 5'-GGGTGGGGATGGAGGATGGACGGG-3'.
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