WO2020156367A1 - 提高水稻产量和/或稻瘟病抗性的方法及其所用蛋白质 - Google Patents

提高水稻产量和/或稻瘟病抗性的方法及其所用蛋白质 Download PDF

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WO2020156367A1
WO2020156367A1 PCT/CN2020/073414 CN2020073414W WO2020156367A1 WO 2020156367 A1 WO2020156367 A1 WO 2020156367A1 CN 2020073414 W CN2020073414 W CN 2020073414W WO 2020156367 A1 WO2020156367 A1 WO 2020156367A1
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rice
ray1
protein
sequence
target
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PCT/CN2020/073414
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French (fr)
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李莉
李懿星
张大兵
邱牡丹
王天抗
宋书锋
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湖南杂交水稻研究中心
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Priority claimed from CN201910107261.6A external-priority patent/CN111534536B/zh
Priority claimed from CN201910107277.7A external-priority patent/CN111518181B/zh
Application filed by 湖南杂交水稻研究中心 filed Critical 湖南杂交水稻研究中心
Priority to US17/427,570 priority Critical patent/US11692200B2/en
Publication of WO2020156367A1 publication Critical patent/WO2020156367A1/zh

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Definitions

  • the invention relates to the field of biotechnology breeding, in particular to a method for improving rice yield and/or rice blast resistance and its protein.
  • Rice is an important food crop that provides staple food for more than half of the world's population.
  • Plant type plays an important role in rice yield, quality, resistance and light energy utilization efficiency. Its composition includes plant height, tiller number, tiller angle and panicle type, among which panicle type is one of the key factors determining rice yield. Therefore, digging into the genes related to rice panicle branching development and clarifying the mechanism of rice panicle branching is of great significance for shaping rice panicle shape and increasing rice yield.
  • Rice blast is the main disease of rice caused by Magnaporthe oryzae (asexual generation: Pyricularia oryzae) infection. It occurs worldwide and can cause a loss of rice yield of 11% to 30%. In severely affected fields, The yield loss is as high as 80%, and even the pellets are not harvested, which seriously affects the yield and quality of grain.
  • the discovery of rice blast resistance genes can provide new genetic resources for the cultivation of rice blast resistant rice.
  • the technical problem to be solved by the present invention is how to improve rice yield and/or rice blast resistance.
  • the present invention first provides a method for cultivating target rice.
  • the method for cultivating target rice includes the following steps: inhibiting the activity of the RAY1 protein in the starting rice to obtain the target rice; compared with the starting rice, the target rice shows increased yield and/or larger grains /Or increased resistance to rice blast and/or increased plant height and/or lengthened stem nodes; the RAY1 protein is a protein composed of the amino acid sequence shown in SEQ ID No. 1 in the sequence table.
  • the inhibition of the activity of the RAY1 protein in the rice may be the inhibition of all or part of the activity of the RAY1 protein in the rice.
  • the increase in yield may be an increase in the yield of a single rice plant; the increase in grain size may be an increase in the length of the grain.
  • the increase in the yield of the rice per plant can be embodied in the increase in the ear length of the rice and/or the increase in the total number of grains per ear and/or the increase in the number of branches at a time.
  • the inhibition of the activity of the RAY1 protein in rice can be achieved by losing the function of the gene encoding the RAY1 protein.
  • the coding gene of the RAY1 protein can be 1) or 2) as follows:
  • the function of the gene encoding the RAY1 protein can be lost by any method in the prior art to cause deletion mutation, insertion mutation or base conversion mutation in the gene, thereby causing loss of gene function.
  • the loss of the function of the gene encoding the RAY1 protein may be the loss of all or part of the function of the gene encoding the RAY1 protein.
  • methods such as chemical mutagenesis, physical mutagenesis, RNAi, gene-directed editing, homologous recombination, etc. can be used to lose the function of the gene encoding the RAY1 protein.
  • the entire coding gene of the RAY1 protein can be used as the target, and the various elements that regulate the expression of the RAY1 protein coding gene can be used as the target, as long as the loss of gene function can be achieved.
  • the first exon, the second exon, the third exon and/or the fourth exon of the coding gene of RAY1 can be used as targets.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • Sequence and related systems Clustered regularly interspaced short palindromic repeats/CRISPR associated, CRISPR/Cas9 system
  • CRISPR/Cas9 technology is adopted, and the target sequence involved is TCGTCGAGAGCTACGAGAT, and the coding gene of the sgRNA (guide RNA) used is shown in SEQ ID No. 4 in the sequence table.
  • the present invention uses a recombinant vector pYLCRISPR/Cas9-MT-RAY1 capable of expressing guide RNA and Cas9.
  • the recombinant vector pYLCRISPR/Cas9-MT-RAY1 is to replace the fragment between the two BsaI restriction sites of the vector pYLCRISPR/Cas9-MTmono with a DNA fragment containing a specific sgRNA encoding gene and a U3 promoter and maintain pYLCRISPR/Cas9 -Recombinant vector obtained by unchanged other nucleotides of MTmono, specifically the DNA molecule shown in SEQ ID No.
  • the above method is applicable to any rice, such as: rice japonica (Oryza sativa subsp. japonica) or rice indica (Oryza sativa subsp. indica), as long as it contains the above target sequence.
  • rice japonica Oryza sativa subsp. japonica
  • rice indica Oryza sativa subsp. indica
  • the present invention also protects the application of substances that inhibit the activity of RAY1 protein in any of the following (1)-(5): (1) increase rice yield; (2) increase rice resistance to rice blast Resistance; (3) Increase the plant height of rice; (4) Increase the internode length of rice; (5) Increase the grain size;
  • the RAY1 protein is composed of the amino acid sequence shown in SEQ ID No. 1 in the sequence table protein.
  • the inhibition of the activity of the RAY1 protein can be the inhibition of all or part of the activity of the RAY1 protein.
  • the increase in rice yield may be to increase the yield per plant of rice; the increase in rice yield per plant may be embodied in increasing the ear length and/or the total number of grains per ear and/or the number of branches at a time;
  • the increase in grain size can be the increase in grain length.
  • the substance that inhibits the RAY1 protein can be any one of the following (1)-(3): (1) a specific sgRNA, the target sequence of the specific sgRNA is TCGTCGAGAGCTACGAGAT; (2) the code (1) DNA molecules of specific sgRNA; (3) a vector for expressing the specific sgRNA described in (1).
  • the coding gene of the specific sgRNA is shown in SEQ ID No. 4 in the sequence list.
  • the vector for expressing specific sgRNA is the recombinant vector pYLCRISPR/Cas9-MT-RAY1.
  • the recombinant vector pYLCRISPR/Cas9-MT-RAY1 is to replace the fragment between the two BsaI restriction sites of the vector pYLCRISPR/Cas9-MTmono with a DNA fragment containing a specific sgRNA encoding gene and a U3 promoter and maintain pYLCRISPR/Cas9 -Recombinant vector obtained by unchanged other nucleotides of MTmono; specifically, replacing the fragment between the two BsaI restriction sites of vector pYLCRISPR/Cas9-MTmono with the DNA molecule shown in SEQ ID No. 5 in the sequence list owned.
  • the rice is a rice japonica rice variety (Oryza sativa subsp. japonica) or a rice indica rice variety (Oryza sativa subsp. indica).
  • the japonica rice variety may be Oryza Sativa L.spp.japonica.
  • the present invention provides a protein RAY1.
  • the protein RAY1 provided by the present invention is a protein composed of the amino acid sequence shown in SEQ ID No. 1 in the sequence table.
  • the protein shown in SEQ ID No. 1 consists of 443 amino acid residues.
  • the present invention also provides a gene encoding protein RAY1.
  • the gene encoding protein RAY1 provided by the present invention is as follows 1) or 2):
  • the coding region is the DNA molecule shown in SEQ ID No. 3 in the sequence list.
  • SEQ ID No. 3 in the sequence list consists of 1332 nucleotides, and encodes the protein shown in SEQ ID No. 1 in the sequence list.
  • the present invention also provides a specific sgRNA whose target sequence in the rice genome is: TCGTCGAGAGCTACGAGAT.
  • the present invention also provides a recombinant plasmid containing the coding gene of Cas9 protein, the coding gene of sgRNA and the U3 promoter; the target sequence of the sgRNA is: TCGTCGAGAGCTACGAGAT.
  • Figure 1 is a gel electrophoresis diagram of PCR amplified RAY1 cDNA full-length sequence.
  • Figure 2 is a map of the intermediate vector pYLgRNA-U3.
  • Figure 3 shows 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 diagram of the intermediate vector pYLgRNA-U3-RAY1 expression cassette.
  • Figure 5 is a vector map of the genome editing vector pYLCRISPR/Cas9-MTmono.
  • Fig. 6 is an electrophoresis diagram of the result of PCR detection of monoclonal colonies transformed into E. coli with recombinant vector pYLCRISPR/Cas9-MT-RAY1.
  • Figure 7 shows the type of RAY1 mutation and the type of amino acid encoded after the mutation.
  • Figure 8 shows the phenotype comparison between the L-46 rice plant and Nipponbare NIP; where A is the plant height and plant type; B is the panicle and primary branch; C is the panicle length and stem internode length.
  • Figure 9 shows the statistical results of agronomic traits of the L-46 rice plant and Nipponbare NIP.
  • Figure 10 shows the comparison and statistical results of rice traits between the L-46 rice plant and Nipponbare NIP rice.
  • Figure 11 shows the statistical results of total plot weight and total weight per plant of strain L-46 rice plants and Nipponbare NIP.
  • Figure 12 shows the identification results of rice blast inoculation at the seedling stage of the L-46 rice plant and Nipponbare NIP; among them, ZA18, ZB10, ZB13, ZB20, ZC2, ZC10, and ZG1 are the physiological races of rice blast.
  • Figure 13 shows the relative expression levels of rice blast resistance-related genes OsPR1a, OsPR10, and PBZ1 in rice plants of L-46, L-47 and L-48 and Nipponbare NIP.
  • the expression vector pYLgRNA-U3 is in the literature "Shi Jiangwei, Li Yixing, Song Shufeng, Qiu Mudan, Deng Yao, Li Li. CRISPR/Cas9 directed editing of rice panicle development Osal genes. Hybrid Rice (HYBRID RICE), 2017, 32(3): 74 -78.”, the public can obtain it from the Hunan Hybrid Rice Research Center. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.
  • Nipponbare The rice variety Nipponbare (NIP) is listed in the literature "MP, A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant. 2015Aug 3; 8(8): 1274-84.doi:10.1016/ As disclosed in j.molp.2015.04.007.Epub 2015 Apr 24.”, the public can obtain it from Hunan Hybrid Rice Research Center. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.
  • RAY1FL-F ATGGAGATGCACGAGTGCTG
  • RAY1FL-R ATGGAGATGCACGAGTGCTG
  • PCR amplification was performed.
  • the amplified product is a DNA fragment with a size of about 1300 bp, and the result is shown in Figure 1.
  • the DNA fragment was 1332 bp in length and had the nucleotide sequence shown in SEQ ID No. 3 in the sequence listing, which was named RAY1.
  • the genomic DNA of RAY1 gene is 1659 bp in length, containing 4 exons and 3 introns, and its nucleotide sequence is shown in SEQ ID No. 2 in the sequence list.
  • the CDS region of the RAY1 gene determine the sequence of the 20th base upstream of NGG (N is any base) as A, and use the sequence consisting of 19 bases immediately downstream of "A" as the candidate target site (due to the intermediate vector
  • the transcription start base of the pYLgRNA-U3 promoter is A, which is the same as the 20th base upstream of NGG. Therefore, the remaining 19 base sequences are considered as target sites to be selected), and the target site sequence is obtained: TCGTCGAGAGCTACGAGAT. It is located on the third exon of the gDNA of RAY1 gene, specifically the DNA molecule shown in SEQ ID No. 2 864-882 in the sequence list, that is, the DNA molecule shown in SEQ ID No. 3 653-671 in the sequence list DNA molecule.
  • the target site adapter primer sequence is as follows:
  • RAY1-Cas9-F GGCA TCGTCGAGAGCTACGAGAT
  • RAY1-Cas9-R AAAC ATCTCGTAGCTCTCGACGA
  • RAY1 target site adapter primers RAY1-Cas9-F and RAY1-Cas9-R with ddH2O Dilute the RAY1 target site adapter primers RAY1-Cas9-F and RAY1-Cas9-R with ddH2O to a concentration of 10 ⁇ M. Take 10 ⁇ L to 80 ⁇ L of deionized water to a final volume of 100 ⁇ L, mix well and heat shock at 90°C After 30s, move to room temperature to complete annealing, obtain RAY1 target site adapter, labeled RAY1-Cas9.
  • the RAY1 intermediate vector was sequenced and confirmed, and the results showed that the RAY1 intermediate vector had 19 bases more than the pYLgRNA-U3 vector plasmid, and the 19 bases were the RAY1 target site sequence (as shown in the box in Figure 3). This indicates that the RAY1 target site sequence has been successfully constructed into the pYLgRNA-U3 vector plasmid, and the intermediate vector is named pYLgRNA-U3-RAY1.
  • the amplified product was detected by gel electrophoresis, and it was determined that it was a DNA molecule about 550 bp in size (as shown in Figure 4). The amplification result was consistent with expectations.
  • the amplified product was recovered and purified, and named as the RAY1 intermediate vector expression cassette.
  • the expression cassette includes an sgRNA encoding gene and a U3 promoter, wherein the sgRNA target sequence is TCGTCGAGAGCTACGAGAT, and the sgRNA encoding gene is shown in SEQ ID No. 4 in the sequence table.
  • the gene editing vector pYLCRISPR/Cas9-MTmono (shown in Figure 5) and the RAY1 intermediate vector expression cassette were digested and ligated to obtain the final vector for RAY1 gene directed editing.
  • the PCR amplified product was subjected to gel electrophoresis, and the electrophoresis result (shown in Figure 6) showed that the RAY1-cas9-2 monoclonal colony could amplify a band with a size of 550 bp, which was consistent with expectations.
  • the sequencing results showed that the DNA fragment shown in SEQ ID No. 5 in the sequence list successfully replaced the DNA fragment between the two BsaI restriction sites on the gene editing vector pYLCRISPR/Cas9-Mtmono.
  • Example 3 Using recombinant plasmids to cultivate target rice
  • the recombinant vector pYLCRISPR/Cas9-MT-RAY1 transforms rice Nipponbare
  • the RAY1 gene-directed editing recombinant vector pYLCRISPR/Cas9-MT-RAY1 was transformed into rice Nipponbare callus, and positive mutants were screened and identified.
  • mutants were detected by PCR and sequenced to obtain 3 types of homozygous mutants, named RAY1-46, RAY1-47 and RAY1-48. Sequencing results show (as shown in Figure 7) that mutant RAY1-46 has a deletion of 11 bases at positions 660-670 of the CDS of RAY1 gene, a frame shift at position 220 of the amino acid sequence of RAY1 protein, and a frame shift at position 430.
  • mutant RAY1-47 inserts a base T between the 668th and 669th positions of the CDS of the RAY1 gene, a frameshift occurs at the 223rd position of the RAY1 protein amino acid sequence, and the translation stops at the 434th position;
  • Mutant RAY1-48 has a deletion of 2 bases between the 668th and 671th positions of the CDS of the RAY1 gene, a frameshift occurs at the 223rd position of the RAY1 protein amino acid sequence, and translation stops at the 433rd position.
  • mutant RAY1-46, mutant RAY1-47 and mutant RAY1-48 harvest the T1 generation seeds of mutant RAY1-46, mutant RAY1-47 and mutant RAY1-48 respectively, and plant T1 generation seeds and seedlings
  • the T1 generation rice line with no foreign vector and genetically stable was screened out.
  • the T1 rice lines of mutant RAY1-46, mutant RAY1-47 and mutant RAY1-48 after screening were designated as L-46, L-47 and L-48, respectively.
  • Rice lines L-46, L-47 and L-48 and wild-type rice Nipponbare (control) were planted in different plots. Each plot was planted with 32 rice plants with an area of 1.7 square meters.
  • mutant T1 generation plants and wild-type plants were compared. The results are shown in Table 1 and 8-10, compared to the wild-type plant, a mutant T 1 progenies of internode elongation is more significant; body height T 1 generation of mutant plants mature, ear length, a The number of branches and the total number of grains per panicle increased to varying degrees; the grain length of mutant T 1 generation plants also increased significantly.
  • Rice mutant lines L-46, L-47 and L-48 were inoculated with rice blast physiological races ZA18, ZB10, ZB13, ZB20, ZC2, ZC10, and ZG1, respectively, and the resistance spectrum of rice blast was determined. At the same time, wild-type rice Nipponbare and rice blast highly susceptible variety co39 were set as controls.
  • the specific method is as follows: use 5 ⁇ gelatin solution to configure different physiological races into a spore suspension of 5 ⁇ 10 4 spores/ml, and spray the spore suspension evenly on rice seedlings at the two-leaf and one-core or three-leaf stage with a spray gun Then, the inoculated rice seedlings were cultured in the dark for 24 hours, and then transferred to an alternating light and dark environment (12 hours of light, 12 hours of darkness) for culture, where the cultured environment temperature was 27°C and relative humidity was 90% . Each mutant line and control were inoculated with 10 rice plants, and the experiment was repeated three times. One week later, the disease was investigated (see Table 3 for judging criteria). Calculate the disease index of mutant and wild-type rice plants based on the disease.
  • disease index ⁇ (number of diseased plants at various levels ⁇ corresponding grade)/(total number of surveyed plants ⁇ 9) ⁇ 100.
  • the results are shown in Tables 4, 5 and Figure 12.
  • the disease index of strains L-46, L-47 and L-48 is much lower than that of wild-type rice Nipponbare and co39. Wild-type rice Nipponbare and co39 compare all the tested rice None of the blast fungus races was resistant, while the rice plants of strains L-46, L-47 and L-48 showed resistance to all the tested blast fungus races.
  • Table 5 The disease index statistics table of mutant T1 generation rice plants and wild-type rice plants
  • the leaf sheaths and leaves of rice plants of rice varieties Nipponbare, strains L-46, L-47 and L-48 were collected to extract total RNA, treated with DNAseDNase I to remove residual DNA, and reverse transcribed into cDNA with oligdT.
  • PR1a-QF/QR PR1a-QF: CGTCTTCATCACCTGCAACT and PR1a-QR: TGTCCATACATGCATAAACACG
  • PR10-QF/QR PR10-QF: CTCATCCTCGACGGCTACTT and PR10-QR: ATCAGGAAGCAGCAATA1-CF
  • PBZ1-QF GGGTGTGGGAAGCACATACA and PBZ1-QR: CCTCGAGCACATCCGACTTT
  • ACTIN was used as the internal reference for detection, and the primers used were ACTIN-QF (ACTIN-QF: TGCTATGTACGTCGCCATCCAG) and ACTIN-QR (ACTIN-QR: AATGAGTAACCACGCTCCGTCA).
  • the invention uses the CRISPR/Cas9 technology to edit the rice RAY1 gene at a specific point.
  • the rice RAY1 gene is knocked out, the protein RAY1 is inactivated, and a new generation of new rice germplasm with significantly improved yield and disease resistance is obtained.
  • the obtained RAY1 fixed-point editing line has increased yield, larger rice grains, increased rice ear length, increased total number of single ears, increased number of branches at a time, and increased resistance to rice blast.
  • the invention can be used to increase rice yield and disease resistance to rice blast, and provides materials for the development of new high-yield disease-resistant varieties.

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Abstract

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/或对稻瘟病的抗性增强和/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。

Description

提高水稻产量和/或稻瘟病抗性的方法及其所用蛋白质 技术领域
本发明涉及生物技术育种领域，具体涉及一种提高水稻产量和/或稻瘟病抗性的方法及其所用蛋白质。
背景技术
水稻(Oryza sativa)作为重要的粮食作物，为世界上一半以上的人口提供主食。为填补人口增长与耕地减少导致的巨大粮食缺口，20世纪80年代，科学家提出了水稻超高产育种理论，即理想株型与杂种优势利用相结合。株型对水稻产量、品质、抗性以及光能利用效率有着重要作用，其构成包括植株高度、分蘖数目、分蘖角度以及穗型等，其中穗型是决定水稻产量的关键因素之一。因此深入挖掘水稻穗分枝发育相关基因，阐明水稻穗分枝机制，对于水稻穗型塑造及提高水稻产量有着重要意义。
稻瘟病是由Magnaporthe oryzae(无性世代：Pyricularia oryzae)侵染引起的水稻主要的病害，在世界范围内普遍发生，对水稻产量造成的损失可达11％～30％，在严重发病的田块，产量损失高达80％，甚至颗粒无收，严重影响粮食产量与质量。发掘稻瘟病抗基因可为培育抗稻瘟病水稻提供新的基因资源。
发明公开
本发明所要解决的技术问题是如何提高水稻的产量和/或稻瘟病抗性。
为了解决上述技术问题，本发明首先提供了一种培育目的水稻的方法。
本发明所提供的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/或对稻瘟病的抗性增强和/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。
上述方法中，所述抑制出发水稻中RAY1蛋白的活性可为抑制出发水稻中RAY1蛋白的全部活性或部分活性。
上述方法中，所述产量增加可为单株水稻产量增加；所述籽粒变大可为籽粒长度增加。
上述方法中，所述单株水稻产量增加可体现在水稻的穗长变长和/或单穗总粒数增多和/或一次枝梗数量增加。
上述方法中，所述抑制出发水稻中RAY1蛋白活性可通过使所述RAY1蛋白的编码基因的功能丧失实现。
所述RAY1蛋白的编码基因可为如下1)或2)：
1)序列表中SEQ ID No.2所示的DNA分子；
2)序列表中SEQ ID No.3所示的DNA分子。
上述方法中，使所述RAY1蛋白的编码基因的功能丧失，可采用现有技术 中的任何方式，以使基因产生缺失突变、插入突变或碱基变换突变，进而使基因的功能丧失。
上述方法中，使所述RAY1蛋白的编码基因的功能丧失可为使所述RAY1蛋白的编码基因的全部功能丧失或部分功能丧失。
上述方法中，使RAY1蛋白编码基因功能丧失，可采取化学诱变、物理诱变、RNAi、基因定点编辑、同源重组等方法。
无论采取哪种方法，既可将RAY1蛋白的整个编码基因作为靶标，又可将调控RAY1蛋白编码基因表达的各个元件作为靶标，只要能实现基因功能丧失即可。如可以将RAY1的编码基因的第1外显子、第2外显子、第3外显子和/或第4外显子作为靶标。
上述基因组定点编辑中，可采用锌指核酸酶(Zinc finger nuclease，ZFN)技术、类转录激活因子效应物核酸酶(Transcription activator-like effectornuclease，TALEN)技术或成簇的规律间隔的短回文重复序列及其相关系统(Clusteredregularly interspaced short palindromic repeats/CRISPR associated，CRISPR/Cas9 system)技术，以及其它能实现基因组定点编辑的技术。
本发明的具体实施例中采用了CRISPR/Cas9技术，其中涉及的靶序列为TCGTCGAGAGCTACGAGAT，所使用的sgRNA(向导RNA)的编码基因如序列表中SEQ ID No.4所示。
进一步具体的，本发明中使用了能表达向导RNA和Cas9的重组载体pYLCRISPR/Cas9-MT-RAY1。所述重组载体pYLCRISPR/Cas9-MT-RAY1为用包含有特异sgRNA编码基因和U3启动子的DNA片段替换载体pYLCRISPR/Cas9-MTmono的两个BsaⅠ酶切位点之间的片段并保持pYLCRISPR/Cas9-MTmono的其它核苷酸不变得到的重组载体，具体为用序列表中SEQ ID No.5所示的DNA分子替换掉载体pYLCRISPR/Cas9-MTmono的两个BsaⅠ酶切位点之间的片段得到的。上述方法适用于任何水稻，如：水稻粳稻品种(Oryza sativa subsp.japonica)或水稻籼稻品种(Oryza sativa subsp.indica)，只要含有上述靶序列即可。本发明列举的例子是水稻日本晴(Oryza Sativa L.spp.japonica)。
为了解决上述技术问题，本发明还保护抑制RAY1蛋白活性的物质在如下(1)-(5)中任意一种中的应用：(1)提高水稻的产量；(2)提高水稻对稻瘟病的抗性；(3)提高水稻的株高；(4)增加水稻的茎节间长；(5)提高籽粒大小；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。
上述应用中，所述抑制RAY1蛋白的活性可为抑制RAY1蛋白的全部活性或部分活性。
上述应用中，所述提高水稻产量可为提高水稻的单株产量；所述提高水稻单株产量可体现在提高水稻的穗长和/或单穗总粒数和/或一次枝梗数量；所述提 高籽粒大小可为提高籽粒的长度。
上述应用中，所述抑制RAY1蛋白的物质可为如下(1)-(3)任意一种：(1)特异sgRNA，所述特异sgRNA的靶标序列为TCGTCGAGAGCTACGAGAT；(2)编码(1)所述特异sgRNA的DNA分子；(3)表达(1)所述特异sgRNA的载体。
上述应用中，所述特异sgRNA的编码基因如序列表中SEQ ID No.4所示。
上述应用中，所述表达特异sgRNA的载体为重组载体pYLCRISPR/Cas9-MT-RAY1。所述重组载体pYLCRISPR/Cas9-MT-RAY1为用包含有特异sgRNA编码基因和U3启动子的DNA片段替换载体pYLCRISPR/Cas9-MTmono的两个BsaⅠ酶切位点之间的片段并保持pYLCRISPR/Cas9-MTmono的其它核苷酸不变得到的重组载体；具体为用序列表中SEQ ID No.5所示的DNA分子替换掉载体pYLCRISPR/Cas9-MTmono的两个BsaⅠ酶切位点之间的片段得到的。
上述应用中，所述水稻为水稻粳稻品种(Oryza sativa subsp.japonica)或水稻籼稻品种(Oryza sativa subsp.indica)。所述水稻粳稻品种可为水稻日本晴(Oryza Sativa L.spp.japonica)。
为了解决上述技术问题，本发明又提供了一种蛋白质RAY1。
本发明所提供的蛋白质RAY1为序列表中SEQ ID No.1所示的氨基酸序列组成的蛋白质。
其中，SEQ ID No.1所示的蛋白质由443个氨基酸残基组成。
为了解决上述技术问题，本发明还提供了一种编码蛋白质RAY1的基因。
本发明所提供的编码蛋白质RAY1的基因为如下1)或2)：
1)序列表中SEQ ID No.2所示的DNA分子；
2)编码区如序列表中SEQ ID No.3所示的DNA分子。
其中，序列表中SEQ ID No.3由1332个核苷酸组成，编码序列表中SEQ ID No.1所示的蛋白质。
为了解决上述技术问题，本发明还提供一种特异sgRNA，其在水稻基因组中的靶序列为：TCGTCGAGAGCTACGAGAT。
为了解决上述技术问题，本发明还提供一种重组质粒，含有Cas9蛋白的编码基因、sgRNA的编码基因和U3启动子；所述sgRNA的靶序列为：TCGTCGAGAGCTACGAGAT。
附图说明
图1为PCR扩增RAY1cDNA全长序列凝胶电泳图。
图2为中间载体pYLgRNA-U3的图谱。
图3为pYLgRNA-U3-RAY1测序序列与中间载体pYLgRNA-U3序列比对图。
图4为中间载体pYLgRNA-U3-RAY1表达盒扩增电泳检测图。
图5为基因组编辑载体pYLCRISPR/Cas9-MTmono载体图谱。
图6为PCR检测重组载体pYLCRISPR/Cas9-MT-RAY1转化大肠杆菌的单克隆菌落的结果电泳图。
图7为RAY1突变类型及突变后编码的氨基酸类型。
图8为株系L-46水稻植株与日本晴NIP的表型比较；其中，A为株高及株型；B为穗及一次枝梗；C为穗长及茎节间长。
图9为株系L-46水稻植株与日本晴NIP农艺性状统计结果。
图10为株系L-46水稻植株与日本晴NIP稻谷性状比较及统计结果。
图11为株系L-46水稻植株与日本晴NIP的小区总重与单株总重统计结果。
图12为株系L-46水稻植株与日本晴NIP的苗期的稻瘟病接种鉴定结果；其中ZA18、ZB10、ZB13、ZB20、ZC2、ZC10、ZG1为稻瘟病生理小种。
图13为稻瘟病抗性相关基因OsPR1a、OsPR10、PBZ1在株系L-46、L-47和L-48水稻植株中与日本晴NIP中的相对表达量。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均可从商业途径得到。
表达载体pYLgRNA-U3在文献“史江伟，李懿星，宋书锋，邱牡丹，邓尧，李莉.CRISPR/Cas9定点编辑水稻穗发育Osal基因.杂交水稻(HYBRID RICE)，2017，32(3)：74-78.”中公开过，公众可从湖南杂交水稻研究中心所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
表达载体pYLCRISPR/Cas9-MTmono在文献“史江伟，李懿星，宋书锋，邱牡丹，邓尧，李莉.CRISPR/Cas9定点编辑水稻穗发育Osal基因.杂交水稻(HYBRID RICE)，2017，32(3)：74-78.”中公开过，公众可从湖南杂交水稻研究中心所获得获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
水稻品种日本晴(NIP)在文献“MP，A Robust CRISPR/Cas9System for Convenient,High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant.2015Aug 3；8(8):1274-84.doi:10.1016/j.molp.2015.04.007.Epub 2015Apr 24.”中公开过，公众可从湖南杂交水稻研究中心所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
稻瘟病菌(Magnaporthe oryzae)生理小种ZA18、ZB10、ZB13、ZB20、ZC2、ZC10和ZG1在文献：“Characterization of molecular identity and pathogenicity of rice blast fungus in Hu nan province of China.Plant Disease,2017,101(4)：557-561.”中公开过，公众可从湖南杂交水稻研究中心所获得，该生物材料只为重复本发明的相关实验所用， 不可作为其它用途使用。
实施例1、RAY1蛋白编码基因的克隆及分析
以水稻日本晴cDNA为模板，RAY1FL-F(ATGGAGATGCACGAGTGCTG)与RAY1FL-R(ATGGAGATGCACGAGTGCTG)为引物，进行PCR扩增。扩增产物为一条大小约1300bp的DNA片段，结果如图1所示。经序列测定，该DNA片段长度为1332bp，具有序列表中SEQ ID No.3所示的核苷酸序列，将其命名为RAY1。其编码一个由443个氨基酸组成的蛋白质RAY1，其氨基酸序列如序列表中SEQ ID No.1所示。RAY1基因基因组DNA全长1659bp，含有4个外显子和3个内含子，其核苷酸序列如序列表中SEQ ID No.2所示。
实施例2、水稻RAY1基因靶标位点的选择及敲除载体的构建
一、靶序列的设计
在RAY1基因的CDS区域确定NGG(N为任意碱基)上游第20个碱基为A的序列，将紧邻“A”的下游19个碱基组成的序列作为待选靶位点(由于中间载体pYLgRNA-U3启动子转录起始碱基为A，与NGG上游第20个碱基相同，因此，视剩余 19个碱基序列为待选靶位点)，获得靶位点序列：TCGTCGAGAGCTACGAGAT。其位于RAY1基因gDNA的第3外显子上，具体为序列表中SEQ ID No.2第864-882位所示的DNA分子，即序列表中SEQ IDNo.3第653-671位所示的DNA分子。
二、重组质粒的构建
1、中间载体pYLgRNA-U3-RAY1的构建
(1)RAY1靶位点接头引物设计及合成
靶位点序列确定后，在靶序列正义链5’前加GGCA，反义链5’前加AAAC，得到靶位点接头引物。靶位点接头引物序列如下：
RAY1-Cas9-F： GGCATCGTCGAGAGCTACGAGAT
RAY1-Cas9-R： AAACATCTCGTAGCTCTCGACGA
(2)RAY1靶位点接头的制备
将RAY1靶位点接头引物RAY1-Cas9-F和RAY1-Cas9-R用ddH2O稀释成浓度为10μM的母液，各取10μL至80μL去离子水中至终体积为100μL，充分混匀后90℃热激30s，移至室温完成退火，获得RAY1靶位点接头，标记为RAY1-Cas9。
(3)RAY1中间载体的构建
将1μL pYLgRNA-U3载体质粒(如图2所示)、1μL 10×T4 DNA Ligase Buffer、1μL靶位点接头RAY1-Cas9、1μL BsaⅠ限制性内切酶和0.5μL 10×T4DNA Ligase混合均匀，用PCR仪进行反应，反应条件为：37℃5min，20℃5min，5个循环，获得RAY1中间载体。对RAY1中间载体进行测序确认，结 果显示：RAY1中间载体比pYLgRNA-U3载体质粒多出19个碱基，该19个碱基为RAY1靶位点序列(如图3框体所示)。这表明RAY1靶位点序列已经成功构建入pYLgRNA-U3载体质粒中，将该中间载体命名为pYLgRNA-U3-RAY1。
2、重组载体pYLCRISPR/Cas9-MT-RAY1的构建
(1)RAY1中间载体表达盒的扩增
以中间载体pYLgRNA-U3-RAY1为模板，Uctcg-B1(TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
)和gRcggt-BL(AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC)为引物进行PCR扩增，得到扩增产物。将扩增产物进行凝胶电泳检测，确定其为一条大小约550bp DNA分子(如图4所示)，该扩增结果与预期一致。回收纯化该扩增产物，并将其命名为RAY1中间载体表达盒。该表达盒包含sgRNA编码基因和U3启动子，其中sgRNA靶标序列为TCGTCGAGAGCTACGAGAT，sgRNA编码基因如序列表中SEQ ID No.4所示。
(2)RAY1定点编辑终载体的构建及转化
利用BsaⅠ限制性内切酶和T4 DNA Ligase，酶切并连接基因编辑载体pYLCRISPR/Cas9-MTmono(如图5所示)和RAY1中间载体表达盒，获得RAY1基因定点编辑终载体。转化大肠杆菌，涂布于含有卡那霉素的平板上，37℃过夜培养。
(3)重组载体pYLCRISPR/Cas9-MT-RAY1的检测
随机挑取步骤(2)中过夜培养的4个单克隆菌落，分别命名为RAY1-cas9-1、RAY1-cas9-2、RAY1-cas9-3和RAY1-cas9-4，利用pYLCRISPR/Cas9-MT载体检测引物SP1(CCCGACATAGATGCAATAACTTC)和SP2(GCGCGGTGTCATCTATGTTACT)对4个单克隆菌落进行PCR检测。PCR扩增产物进行凝胶电泳，电泳结果(如图6所示)表明，RAY1-cas9-2单克隆菌落能扩增出大小为550bp的条带，该结果与预期一致。
提取RAY1-cas9-2单克隆的质粒DNA进行测序。测序结果显示：序列表中SEQ ID No.5所示的DNA片段成功替换掉基因编辑载体pYLCRISPR/Cas9-Mtmono上两个BsaⅠ酶切位点之间的DNA片段。这表明含有U3启动子和sgRNA编码基因的表达盒成功构建到基因编辑载体pYLCRISPR/Cas9-MTmono上，即RAY1的基因组定点编辑载体构建成功，获得重组载体pYLCRISPR/Cas9-MT-RAY1。
实施例3、利用重组质粒培育目的水稻
一、重组载体pYLCRISPR/Cas9-MT-RAY1转化水稻日本晴
利用农杆菌介导转化水稻愈伤组织的方法，将RAY1基因定点编辑重组载体pYLCRISPR/Cas9-MT-RAY1转化水稻日本晴愈伤组织，筛选并鉴定获得阳性突 变体。
二、定点编辑的检测
利用PCR检测阳性突变体，测序获得3种突变类型的纯合突变体，分别命名为RAY1-46、RAY1-47和RAY1-48。测序结果表明(如图7所示)，突变体RAY1-46在RAY1基因CDS的第660-670位上缺失11个碱基，在RAY1蛋白氨基酸序列的第220位出现移码，在第430位翻译终止；突变体RAY1-47在RAY1基因CDS的第668位和第669位之间插入了1个碱基T，在RAY1蛋白氨基酸序列的第223位出现移码，在第434位翻译终止；突变体RAY1-48在RAY1基因CDS的第668位和第671位之间缺失2个碱基，在RAY1蛋白氨基酸序列的第223位出现移码，在第433位翻译终止。
三、表型鉴定
正常培育突变体RAY1-46、突变体RAY1-47和突变体RAY1-48，分别收获突变体RAY1-46、突变体RAY1-47和突变体RAY1-48的T1代种子，种植T1代种子，苗期筛选出没有外源载体且遗传稳定的T1代水稻株系。将筛选后的突变体RAY1-46、突变体RAY1-47和突变体RAY1-48的T1代水稻株系分别记为L-46、L-47和L-48。分小区种植水稻株系L-46、L-47和L-48以及野生型水稻日本晴(对照)，每个小区种植32株水稻，每个小区面积为1.7平方米。
比较突变体T1代植株和野生型植株的表型。结果如表1和图8-10所示，与野生型植株相比较，突变体T 1代植株的茎节伸长更为显著；成熟期突变体T 1代植株的株高、穗长、一次枝梗的数量和单穗总粒数均有不同程度的增加；突变体T 1代植株的籽粒的长度也明显提高。
表1突变体T 1代植株与野生型植株的表型比较
Figure PCTCN2020073414-appb-000001
Figure PCTCN2020073414-appb-000002
注：*p<0.05表示与水稻日本晴比存在显著差异，**p<0.01表示与水稻日本晴比存在极显著差异。
比较突变体T 1代植株和野生型日本晴水稻植株的产量。结果如表2和图11所示，与野生型植株相比较，株系L-46、L-47和L-48的单株产量和小区产量相对于对照均有不同程度的增加。
表2突变体T1代植株与野生型植株的产量比较
Figure PCTCN2020073414-appb-000003
注：*p<0.05表示与水稻日本晴比存在显著差异，**p<0.01表示与水稻日本晴比存在极显著差异。
四、突变体对稻瘟病的抗性鉴定
1、对稻瘟病抗性的初步检测
分别用稻瘟病生理小种ZA18、ZB10、ZB13、ZB20、ZC2、ZC10和ZG1接种水稻突变体株系L-46、L-47和L-48，进行稻瘟病抗谱测定。同时设置野生型水稻日本晴和稻瘟病高感品种co39作为对照。具体方法如下：分别用5‰的明胶溶液将不同生理小种配置成5×10 4个孢子/毫升的孢子悬浮液，用喷枪均匀把孢子悬浮液喷洒于两叶一心或三叶期的水稻幼苗的叶表面，然后将接种后的水稻幼苗暗培养24小时，再转入光暗交替(12小时光照，12小时黑暗)环境中进行培养，其中培养的环境温度为27℃，相对湿度为90％。每个突变株系和对照均接种10株水稻植株，实验重复三次。一周后调查发病情况(评判标准见表3)。根据发病情况计算突变体和野生型水稻植株的病情指数。
水稻苗期叶片病情指数公式为：病情指数＝∑(各级病株数×相应级数)/(调查总株数×9)×100。
表3水稻植株发病情况评判标准
Figure PCTCN2020073414-appb-000004
Figure PCTCN2020073414-appb-000005
结果如表4、5和图12所示，株系L-46、L-47和L-48的病情指数远远低于野生型水稻日本晴和co39，野生型水稻日本晴和co39对检测的所有稻瘟病菌小种均无抗性，而株系L-46、L-47和L-48的水稻植株对检测的所有稻瘟病菌小种均表现出抗性。
表4突变体T1代水稻植株与野生型水稻植株发病情况调查结果
Figure PCTCN2020073414-appb-000006
Figure PCTCN2020073414-appb-000007
表5突变体T1代水稻植株与野生型水稻植株的病情指数统计表
Figure PCTCN2020073414-appb-000008
2、株系L-46、L-47和L-48中稻瘟病抗性相关基因OsPR1a、OsPR10、PBZ1的表达特性分析
分别采集水稻品种日本晴、株系L-46、L-47和L-48的水稻植株的叶鞘和叶片提取总RNA，利用DNAseDNaseⅠ处理除去残留DNA，利用oligdT将其反转录为cDNA。以此cDNA为模板，分别用引物PR1a-QF/QR(PR1a-QF：CGTCTTCATCACCTGCAACT和PR1a-QR：TGTCCATACATGCATAAACACG)、PR10-QF/QR(PR10-QF：CTCATCCTCGACGGCTACTT和PR10-QR：ATCAGGAAGCAGCAATACGG)和PBZ1-QF/QR(PBZ1-QF：GGGTGTGGGAAGCACATACA和PBZ1-QR：CCTCGAGCACATCCGACTTT)进行qRT-PCR扩增，检测稻瘟病抗性相关基因OsPR1a、OsPR10、PBZ1在日本晴、L-46、L-47和L-48中的表达量。并以ACTIN 为检测内参，所用引物为ACTIN-QF(ACTIN-QF：TGCTATGTACGTCGCCATCCAG)和ACTIN-QR(ACTIN-QR：AATGAGTAACCACGCTCCGTCA)。
结果如图13所示，株系L-46、L-47和L-48的植株中，稻瘟病抗性相关基因OsPR1a、OsPR10、PBZ1的表达量相对于水稻野生型品种日本晴都上调。具体而言，相对于水稻日本晴，OsPR1a、OsPR10、PBZ1在水稻突变株系L46中分别上调8.2倍、10.6倍和2.6倍；在水稻突变株系L47中分别上调7.9倍、11.5倍和2.5倍；在水稻突变株系L48中分别上调8.9倍、10.8倍和3.0倍。这一结果表明RAY1基因负调控稻瘟病抗性相关基因OsPR1a、OsPR10、PBZ1的表达，从而调控水稻植株对稻瘟病的抗病性。
工业应用
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，获得了产量及抗病性明显提高的新一代水稻新种质。获得的RAY1定点编辑株系与野生型对照相比，产量增加、水稻籽粒变大、水稻穗长增加、单穗总数增多、一次枝梗数量增加以及对稻瘟病的抗性增强。本发明可用于提高水稻产量以及对稻瘟病的抗病性，为高产抗病新品种的开发提供了材料。
Figure PCTCN2020073414-appb-000009
Figure PCTCN2020073414-appb-000010
Figure PCTCN2020073414-appb-000011
Figure PCTCN2020073414-appb-000012

Claims (17)

  1. 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；所述目的水稻为A或B：
    A、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；
    B、所述目的水稻与所述出发水稻相比，表现出对稻瘟病的抗性增强；
    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/或对稻瘟病的抗性增强和/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。
  2. 根据权利要求1所述的方法，其特征在于：所述产量增加为单株水稻产量增加；所述籽粒变大为籽粒长度增加。
  3. 根据权利要求2所述的方法，其特征在于：所述抑制出发水稻中RAY1蛋白活性是通过使所述RAY1蛋白的编码基因的功能丧失实现的。
  4. 根据权利要求3所述的方法，其特征在于：所述RAY1蛋白的编码基因为如下1)或2)：
    1)序列表中SEQ ID No.2所示的DNA分子；
    2)序列表中SEQ ID No.3所示的DNA分子。
  5. 根据权利要求3所述的方法，其特征在于：所述使所述RAY1蛋白的编码基因功能丧失的方法为CRISPR/Cas9方法。
  6. 根据权利要求5所述的方法，其特征在于：所述CRISPR/Cas9方法包括向所述出发水稻导入sgRNA表达载体的步骤，所述sgRNA的靶标序列为TCGTCGAGAGCTACGAGAT。
  7. 根据权利要求1所述的方法，其特征在于：所述抑制出发水稻中RAY1蛋白活性是通过使所述RAY1蛋白的编码基因的功能丧失实现的。
  8. 根据权利要求7所述的方法，其特征在于：所述RAY1蛋白的编码基因为如下1)或2)：
    1)序列表中SEQ ID No.2所示的DNA分子；
    2)序列表中SEQ ID No.3所示的DNA分子。
  9. 根据权利要求7所述的方法，其特征在于：所述使所述RAY1蛋白的编码基因功能丧失的方法为CRISPR/Cas9方法。
  10. 根据权利要求9所述的方法，其特征在于：所述CRISPR/Cas9方法包括向所述出发水稻导入sgRNA表达载体的步骤，所述sgRNA的靶标序列为TCGTCGAGAGCTACGAGAT。
  11. 抑制RAY1蛋白活性的物质在如下(1)-(5)中任意一种中的应用：
    (1)提高水稻的产量；
    (2)提高水稻对稻瘟病的抗性；
    (3)提高水稻的株高；
    (4)增加水稻的茎节间长；
    (5)提高籽粒大小；
    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。
  12. 根据权利要求11所述的应用：其特征在于：所述提高水稻产量为提高水稻的单株产量；所述提高籽粒大小为提高籽粒的长度。
  13. 根据权利要求12所述的应用，其特征在于：所述抑制RAY1蛋白的物质为如下(1)-(3)任意一种：
    (1)特异sgRNA，所述特异sgRNA的靶标序列为TCGTCGAGAGCTACGAGAT；
    (2)编码(1)所述特异sgRNA的DNA分子；
    (3)表达(1)所述特异sgRNA的载体。
  14. 根据权利要求11所述的应用，其特征在于：所述抑制RAY1蛋白的物质为如下(1)-(3)任意一种：
    (1)特异sgRNA，所述特异sgRNA的靶标序列为TCGTCGAGAGCTACGAGAT；
    (2)编码(1)所述特异sgRNA的DNA分子；
    (3)表达(1)所述特异sgRNA的载体。
  15. 下述任一种产品；
    P1、一种蛋白质，为序列表中SEQ ID No.1所示的氨基酸序列组成的蛋白质；
    P2、编码P1所述蛋白质的核酸分子；
    P3、根据P2所述的核酸分子，其特征在于：所述核酸分子为如下1)或2)：
    1)序列表中SEQ ID No.2所示的DNA分子；
    2)编码区如序列表中SEQ ID No.3所示的DNA分子。
  16. sgRNA，其在水稻基因组中的靶序列为：TCGTCGAGAGCTACGAGAT。
  17. 重组质粒，所述重组质粒含有Cas9蛋白的编码基因、特异sgRNA的编码基因和U3启动子；所述sgRNA的靶序列为：TCGTCGAGAGCTACGAGAT。
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