阅读说明：本技术 提高水稻产量的方法及其所用蛋白质 (Method for increasing rice yield and protein used by same ) 是由 李莉 李懿星 张大兵 邱牡丹 王天抗 宋书锋 于 2019-02-02 设计创作，主要内容包括：本发明公开了一种提高水稻产量的方法及其所用蛋白质。本发明提供了一种培育目的水稻的方法,包括如下步骤：抑制出发水稻中RAY1蛋白的活性,得到目的水稻；所述目的水稻与所述出发水稻相比,表现出产量增加和/或籽粒变大和/或植株株高增加和/或茎节间变长；所述RAY1蛋白为序列表中SEQ ID No.1所示的氨基酸序列组成的蛋白质。本发明利用CRISPR/Cas9技术,定点编辑水稻RAY1基因,通过移码突变,敲除了水稻了RAY1基因,使蛋白RAY1失活,获得了产量明显提高的新一代水稻新种质。(The invention discloses a method for improving rice yield and protein used by the method. The invention provides a method for cultivating target rice, which comprises the following steps: inhibiting the activity of RAY1 protein in the original rice to obtain the target rice; compared with the starting rice, the target rice shows that the yield is increased, the grain is enlarged, the plant height of a plant is increased, and/or the stem internode is lengthened; the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table. The invention utilizes CRISPR/Cas9 technology to edit rice RAY1 gene at fixed point, and knocks out RAY1 gene through frameshift mutation, so that protein RAY1 is inactivated, and a new generation of new rice new germplasm with obviously improved yield is obtained.)

1. A method for breeding target rice, comprising the steps of: inhibiting the activity of RAY1 protein in the original rice to obtain the target rice;

compared with the starting rice, the target rice shows that the yield is increased, the grain is enlarged, the plant height of a plant is increased, and/or the stem internode is lengthened;

the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

2. The method of claim 1, wherein: the yield increase is the increase of the yield of the single-plant rice; the kernel becomes larger and has increased length.

3. The method according to claim 1 or 2, characterized in that: the inhibition of the activity of RAY1 protein in the starting rice is achieved by loss of function of the gene encoding the RAY1 protein.

4. The method of claim 3, wherein: the encoding gene of the RAY1 protein is 1) or 2) as follows:

1) a DNA molecule shown as SEQ ID No.2 in the sequence table;

2) a DNA molecule shown as SEQ ID No.3 in the sequence table.

5. The method of claim 3, wherein: the method for losing the function of the encoding gene of the RAY1 protein is a CRISPR/Cas9 method.

6. The method of claim 5, wherein: the CRISPR/Cas9 method comprises the step of introducing an sgRNA expression vector into the starting rice, wherein the target sequence of the sgRNA is TCGTCGAGAGCTACGAGAT.

7. Use of a substance inhibiting the activity of RAY1 protein in any one of the following (1) to (4):

(1) the yield of the rice is improved;

(2) the plant height of the rice is improved;

(3) increase the internode length of the rice;

(4) the size of the grains is improved;

the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

8. The use according to claim 7: the method is characterized in that: the rice yield is increased by increasing the single-plant yield of rice; the increasing of the grain size is increasing of the length of the grain.

9. Use according to claim 7 or 8, characterized in that: the substance inhibiting RAY1 protein is any one of the following (1) to (3):

(1) a specific sgRNA, wherein the target sequence of the specific sgRNA is TCGTCGAGAGCTACGAGAT;

(2) a DNA molecule encoding the specific sgRNA of (1);

(3) and (2) a vector for expressing the specific sgRNA in (1).

10. Any of the following products;

p1, a protein, which is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table;

p2, a nucleic acid molecule encoding the protein of P1;

p3, the nucleic acid molecule according to P2, characterized in that: the nucleic acid molecule is 1) or 2) as follows:

1) a DNA molecule shown as SEQ ID No.2 in the sequence table;

2) the coding region is a DNA molecule shown as SEQ ID No.3 in the sequence table.

Technical Field

The invention relates to the field of biotechnology breeding, in particular to a method for improving rice yield and protein used by the method.

Background

Rice (Oryza sativa) is an important food crop and provides staple food for more than half of the world's population. In order to fill the huge grain gap caused by population growth and farmland reduction, scientists put forward the super-high yield breeding theory of rice in the 80 th 20 th century, namely the combination of ideal plant type and heterosis utilization. The plant type plays an important role in rice yield, quality, resistance and light energy utilization efficiency, and comprises plant height, tillering number, tillering angle, spike type and the like, wherein the spike type is one of key factors determining the rice yield. Therefore, the method deeply excavates genes related to development of rice panicle branches, clarifies a rice panicle branching mechanism, and has important significance for shaping rice panicle types and improving rice yield.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the yield of rice.

In order to solve the above technical problems, the present invention provides a method for breeding target rice.

The method for cultivating the target rice provided by the invention comprises the following steps: inhibiting the activity of RAY1 protein in the original rice to obtain the target rice; compared with the starting rice, the target rice shows that the yield is increased, the grain is enlarged, the plant height of a plant is increased, and/or the stem internode is lengthened; the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

In the above method, the inhibition of the activity of the RAY1 protein in the original rice may be inhibition of the whole or part of the activity of the RAY1 protein in the original rice.

In the above method, the increase in yield may be an increase in yield of individual rice; the kernel enlargement may be a kernel length increase.

In the method, the yield increase of the single-plant rice can be reflected by the fact that the ear length of the rice is lengthened, the total grain number of the single ear is increased and/or the number of branches per time is increased.

In the above method, the inhibition of the activity of RAY1 protein in the starting rice may be achieved by the loss of function of the gene encoding the RAY1 protein.

The encoding gene of the RAY1 protein can be 1) or 2) as follows:

1) a DNA molecule shown as SEQ ID No.2 in the sequence table;

2) a DNA molecule shown as SEQ ID No.3 in the sequence table.

In the above method, the loss of function of the gene encoding the RAY1 protein can be achieved by any method known in the art, such as deletion mutation, insertion mutation or base change mutation of the gene, and further the loss of function of the gene.

In the above method, the loss of function of the gene encoding the RAY1 protein may be the loss of function of all or part of the gene encoding the RAY1 protein.

In the above-mentioned methods, the gene encoding the RAY1 protein is disabled by chemical mutagenesis, physical mutagenesis, RNAi, gene site-directed editing, homologous recombination, or the like.

In any case, the entire gene encoding the RAY1 protein may be targeted, or each element regulating the expression of the gene encoding the RAY1 protein may be targeted, as long as the loss of gene function can be achieved. For example, exon 1, exon 2, exon 3 and/or exon 4 of the gene encoding RAY1 may be targeted.

In the above-mentioned genome site-directed editing, Zinc Finger Nuclease (ZFN) technology, Transcription activator effector-like nuclease (TALEN) technology, clustered regularly spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats/CRISPR associated, CRISPR/Cas9system) technology, and other technologies capable of realizing genome site-directed editing can be adopted.

In the specific embodiment of the invention, the CRISPR/Cas9 technology is adopted, wherein the target sequence involved is TCGTCGAGAGCTACGAGAT, and the coding gene of the sgRNA (guide RNA) used is shown as SEQ ID No.4 in the sequence table.

More specifically, the invention uses a recombinant vector pYLCRISPR/Cas9-MT-RAY1 capable of expressing a guide RNA and Cas 9. The recombinant vector pYRCISPR/Cas 9-MT-RAY1 is a recombinant vector obtained by replacing a fragment between two Bsa I enzyme cutting sites of the vector pYRCISPR/Cas 9-MTmono with a DNA fragment containing a specific sgRNA coding gene and a U3 promoter and keeping other nucleotides of the pYRCISPR/Cas 9-MTmono unchanged, and is specifically obtained by replacing a fragment between two Bsa I enzyme cutting sites of the vector pYRCISPR/Cas 9-MTmono with a DNA molecule shown in SEQ ID No.5 in a sequence table. The above method is applicable to any rice, such as: japonica rice (Oryza sativa subsp. japonica) or indica rice (Oryza sativa subsp. indica) as long as it contains the above target sequence. An example of the present invention is rice Nipponbare (Oryza Sativa L.spp.japonica).

In order to solve the above technical problems, the present invention also protects the use of a substance inhibiting the activity of RAY1 protein in any one of the following (1) to (4): (1) the yield of the rice is improved; (2) the plant height of the rice is improved; (3) increase the internode length of the rice; (4) the size of the grains is improved; the RAY1 protein is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

In the above application, the inhibition of the activity of the RAY1 protein may be the inhibition of the whole activity or part activity of the RAY1 protein.

In the application, the rice yield can be increased by increasing the yield of each plant of rice; the improvement of the yield of each plant of the rice can be embodied in the improvement of the ear length of the rice and/or the total grain number of each ear and/or the number of branches per time; the increasing the grain size may be increasing the length of the grain.

In the above application, the substance inhibiting the RAY1 protein may be any one of the following (1) to (3): (1) a specific sgRNA, wherein the target sequence of the specific sgRNA is TCGTCGAGAGCTACGAGAT; (2) a DNA molecule encoding the specific sgRNA of (1); (3) and (2) a vector for expressing the specific sgRNA in (1).

In the application, the coding gene of the specific sgRNA is shown as SEQ ID No.4 in a sequence table.

In the application, the vector for expressing the specific sgRNA is a recombinant vector pYLCRISPR/Cas9-MT-RAY 1. The recombinant vector pYLCRISPR/Cas9-MT-RAY1 is a recombinant vector obtained by replacing a fragment between two Bsa I enzyme cutting sites of the vector pYLCRISPR/Cas9-MTmono with a DNA fragment containing a specific sgRNA coding gene and a U3 promoter and keeping other nucleotides of pYLCRISPR/Cas9-MTmono unchanged; in particular to a DNA molecule shown in SEQ ID No.5 in a sequence table replacing a fragment between two Bsa I enzyme cutting sites of a vector pYLCRISPR/Cas 9-MTmono.

In the above application, the rice is a japonica rice variety (Oryza sativa subsp. japonica) or an indica rice variety (Oryza sativa subsp. indica). The japonica rice variety may be Nipponbare (Oryza sativa L.spp.japonica).

In order to solve the technical problem, the invention also provides a protein RAY 1.

The protein RAY1 provided by the invention is a protein composed of an amino acid sequence shown by SEQ ID No.1 in a sequence table.

Wherein, the protein shown in SEQ ID No.1 consists of 443 amino acid residues.

In order to solve the technical problems, the invention also provides a gene for coding the protein RAY 1.

The gene for coding the protein RAY1 provided by the invention is 1) or 2):

1) a DNA molecule shown as SEQ ID No.2 in the sequence table;

2) the coding region is a DNA molecule shown as SEQ ID No.3 in the sequence table.

Wherein, SEQ ID No.3 in the sequence table is composed of 1332 nucleotides and encodes the protein shown by SEQ ID No.1 in the sequence table.

The invention utilizes CRISPR/Cas9 technology to edit rice RAY1 gene at fixed point, and knocks out RAY1 gene through frame shift mutation, so that protein RAY1 is inactivated, and new generation new rice germplasm with obviously improved yield and disease resistance is obtained. Compared with a wild type control, the obtained RAY1 fixed-point editing line has the advantages of increased yield, enlarged rice grains, increased rice spike length, increased total single spike number, increased number of branches at one time and enhanced resistance to rice blast. Therefore, the invention has important significance for improving the yield and disease resistance of the rice and provides a new material for developing new high-yield disease-resistant varieties.

Drawings

FIG. 1 is a gel electrophoresis diagram of the full-length sequence of PCR amplified RAY1 cDNA.

FIG. 2 is a map of the intermediate vector pYLgRNA-U3.

FIG. 3 is a diagram showing the alignment of the sequencing sequence of pYLgRNA-U3-RAY1 and the sequence of the intermediate vector pYLgRNA-U3.

FIG. 4 is an amplification electrophoresis detection map of the expression cassette of the intermediate vector pYLgRNA-U3-RAY 1.

FIG. 5 is a map of the genome editing vector pYLCRISPR/Cas9-MTmono vector.

FIG. 6 is an electrophoresis diagram showing the result of PCR detection of a single colony of E.coli transformed with the recombinant vector pYLCRISPR/Cas9-MT-RAY 1.

FIG. 7 shows the mutation pattern of RAY1 and the amino acid pattern encoded by the mutation.

FIG. 8 is a phenotypic comparison of line L-46 rice plants with Nipponbare NIP; wherein A is the plant height and the plant type; b is ear and primary branch; c is ear length and internode length.

FIG. 9 shows the statistical results of the agronomic traits of the L-46 rice plant and Nipponbare.

FIG. 10 shows the comparison and statistics of the characteristics of the L-46 rice plant and Nipponbare rice.

FIG. 11 shows the statistics of the total cell weight and the total individual plant weight of the rice plant of strain L-46 and Nipponbare.

FIG. 12 shows the results of the identification of rice blast inoculation of the line L-46 rice plant and Nip at the seedling stage; wherein ZA18, ZB10, ZB13, ZB20, ZC2, ZC10 and ZG1 are physiological races of rice blast.

FIG. 13 shows the relative expression amounts of the rice blast resistance-related genes OsPR1a, OsPR10 and PBZ1 in the rice plants of lines L-46, L-47 and L-48 and NiP.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The expression vector pYLgRNA-U3 is used for editing a RICE ear development Osal gene at a fixed point in a document of Shijiang Wei, Li yi star, Song Shufeng, Qiu peony, Deng Yao, Li. CRISPR/Cas 9. HYBRID RICE (HYBRID RICE), 2017 and 32 (3): 74-78, the biological material is only used for repeating the experiments related to the present invention and is not used for other purposes.

Expression vector pYLCRISPR/Cas9-MTmono in the literature "Shijiang Wei, Li-yi star, Song front, Qiu peony, Du Yao, Li. CRISPR/Cas9 fixed-point editing RICE tasal gene HYBRID RICE (HYBRID RICE), 2017, 32 (3): 74-78, publicly available from the research center for hybrid rice in Hunan, the biomaterial was used only for repeating the experiments related to the present invention, and was not used for other purposes.

Nipponbare (NIP) rice variety "MP, A Robust CRISPR/Cas9System for convention, High-Efficiency Multiplex Genome Editing in Mono cot and dicotplants. mol plant.2015Aug 3; 1274-84.doi 10.1016/j. molp.2015.04.007.Epub2015Apr 24. the biomaterial, which is publicly available from the research center for hybrid rice in Hunan, is used only for repeating the relevant experiments of the present invention and is not used for other purposes.

Physiological races of rice blast fungus (Magnaporthe oryzae) ZA18, ZB10, ZB13, ZB20, ZC2, ZC10 and ZG1 are described in the literature: "Characterization of molecular identity and probability of difference blast in Hunan protocol of China. plant Disease,2017,101 (4): 557 561 ", publicly available from the research center for hybrid rice in Hunan, the biomaterial was used only for repeating the experiments related to the present invention, and was not used for other purposes.