A rice mutant arc3 with high resistance to brown planthopper and its application

Abstract

The invention discloses a rice highly resistant brown planthopper mutant arc3 and its application, and the preparation method thereof specifically comprises the following steps: screening the sgRNA target sequence of the rice gene Os03g50150; designing primers as shown in SEQ ID NO.2 and SEQ ID NO.3 according to the sgRNA target sequence; mixing the primers and performing annealing treatment to form double-stranded DNA; first constructing the double-stranded DNA fragment onto a pOs-sgRNA vector, and then transferring it onto a pH-Ubi-cas9 vector; transferring the vector into rice tissue for induction and obtaining a rice resistant brown planthopper mutant arc3 plant through genetic transformation. In the invention, the expression or activity of the Os03g50150 gene or the active fragment of the Os03g50150 gene in rice is reduced; and the resistance of rice to brown planthopper is significantly improved.

Description

Technical Field

[0001] The invention belongs to the field of crop genetics, and particularly relates to a rice highly resistant brown planthopper mutant arc3 and an application thereof. Background Art

[0002] Rice (Oryza sativa) is one of the important food crops in my country, and the safe production of rice is closely related to my country's food security. ) is the most serious pest that harms rice production in my country. The rice production loss caused by brown planthoppers accounts for about 30% of all diseases and pests each year, and the direct production loss is more than one million tons. Brown planthoppers use their piercing and sucking mouthparts to feed on the sap of rice or wild rice phloem, consuming a large amount of nutrients from the plant, which easily causes the rice plant to wilt. In agricultural production, it is commonly known as the "louse burn" phenomenon, causing rice production to decrease or even fail. At the same time, brown planthoppers are also a migratory pest. Affected by the southwest monsoon, brown planthoppers migrate to my country from March to May every year. Because the biotype and number of the migrating insect source are difficult to control, brown planthoppers are also hidden, explosive and destructive. In addition, brown planthoppers can also spread two viral diseases, grassy dwarf disease and toothed leaf dwarf disease, causing indirect damage. At present, chemical pesticides are mainly used to control brown planthoppers in agricultural production. The use of chemical pesticides can kill brown planthoppers in a short time, but pesticide residues not only pollute the environment, but also kill the natural enemies of brown planthoppers, induce brown planthoppers to develop pesticide resistance, and make brown planthoppers more difficult to control. Therefore, discovering endogenous brown planthopper resistance genes in rice, clarifying their molecular mechanisms and conducting breeding practices are more economical and effective methods to prevent brown planthopper damage, which is of great significance to ensuring food security.

[0003] In rice, among the cloned brown planthopper resistance genes, Bph14, Bph18, Bph26, and Bph9 all belong to the NBS-LRR resistance gene. Among them, Bph14 and Bph9 are two genes with relatively clear insect resistance functions and molecular mechanisms. Bph14 is the first cloned rice brown planthopper resistance gene. This gene encodes a typical CC-NBS-LRR protein. The Bph14 gene is highly expressed in the thin wall cells near the leaf sheath, root, and leaf vascular bundle of rice. Transgenic plants and near-isogenic lines of Bph14 are resistant to brown planthoppers, inhibiting the feeding, growth, and development of brown planthoppers (Du et al., 2009). Hu et al. (2017) found that the CC domain, NBS-ARC domain, and CC-NBS domain of the Bph14 gene alone have the same resistance to brown planthoppers as the full-length, while the LRR domain alone cannot produce resistance to brown planthoppers. At the same time, the resistance domain can form a homologous complex with the full-length domain of Bph14, which can interact with the transcription factors OsWRKY46 and OsWRKY72 and inhibit the degradation of these two transcription factors. OsWRKY46 and OsWRKY72 can regulate the expression of downstream receptor-like protein kinases and callose synthesis genes to regulate rice resistance to brown planthoppers (Hu et al., 2017). The Bph9 gene encodes an NBS-LRR protein containing two NBS domains. It is located in the intracellular membrane system and can trigger immune responses such as cell death. The near-isogenic lines of Bph9 activated the salicylic acid and jasmonic acid signaling pathways in the body, and the resistance mechanism to brown planthoppers was shown to be avoidance and antibiotics (Zhao et al., 2016); Wang et al. (2021) found that the CC domain, NBS domain, and LRR domain of Bph9 protein all have the characteristics of interacting with themselves and other domains, and the LRR domain has the function of recognizing the feeding signal of brown planthoppers, which can form polymers between molecules and trigger downstream defense responses. This study further revealed the molecular mechanism of CC-NBS-NBS-LRR protein regulating rice resistance to brown planthoppers (Wang et al., 2021). It can be seen that so far, the reports on NBS-LRR proteins in the process of rice resistance to brown planthoppers are all positively regulating rice resistance to brown planthoppers and contain typical CC-NBS-LRR structures, while the functions and molecular mechanisms of other types of resistance proteins in rice resistance to brown planthoppers need to be further analyzed.

[0004] NBS-LRR genes play an important role in rice resistance to brown planthoppers, but research on rice resistance to brown planthoppers mainly focuses on typical CC-NBS-LRR genes. Liu Yunfei et al. (2014) believed that there are 545 resistance genes in rice, of which 402 genes encode CC-NBS-LRR resistance proteins, and the remaining genes encode other types of resistance proteins, but the role of resistance genes with other structures such as CC-NBS-ARC and NBS-ARC in rice resistance to brown planthoppers has not yet been reported. Summary of the invention

[0005] The invention provides a method for resisting brown planthopper in rice, comprising the steps of reducing the expression or activity of Os03g50150 gene or active fragment of Os03g50150 gene in rice.

[0006] The method for reducing the expression or activity of the Os03g50150 gene or the active fragment of the Os03g50150 gene in rice includes inhibiting the expression or activity of the Os03g50150 gene or its active fragment by gene mutation, gene knockout, gene interruption, or Crispr technology.

[0007] The present invention also provides a method for preparing a rice brown planthopper-resistant mutant arc3, ​​which specifically comprises the following steps:

[0008] Screening sgRNA target sequence: Screening the sgRNA target sequence of rice gene Os03g50150, wherein the sgRNA target sequence is shown in SEQ ID NO.1;

[0009] Design primers: design primers as shown in SEQ ID NO.2 and SEQ ID NO.3 according to the sgRNA target sequence;

[0010] Constructing a vector: After the primers are mixed and annealed, double-stranded DNA is formed; first, the double-stranded fragment is constructed onto the pOs-sgRNA vector, and then transferred to the pH-Ubi-cas9 vector;

[0011] For detailed information about the carrier, please refer to Zhang, J., Zhou, Z., Bai, J., Tao, X., Wang, L., Zhang, H., & Zhu, JK (2020). Disruption of MIR396e and MIR396 improves rice yield under nitrogen-deficient conditions. National science review, 7(1), 102–112. https: / / doi.org / 10.1093 / nsr / nwz142;

[0012] The vector was transferred into rice tissues for induction and genetic transformation to obtain rice brown planthopper-resistant mutant arc3 plants;

[0013] The genetic transformation method can be obtained by referring to the method described in the literature (Hiei Y, Ohta S, Komari T, Kumashiro T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6 (2): 271-282.) and improving it.

[0014] The use of the inhibitor of the Os03g50150 gene or its active fragment is for resisting brown planthopper.

[0015] The overexpression of the Os03g50150 gene is used to enhance the sensitivity of plants to brown planthoppers.

[0016] Previous studies have shown that OsEIL1, a transcription factor in the plant hormone ethylene signal transduction pathway, can negatively regulate rice resistance to brown planthoppers (Ma et al., 2020). By analyzing the transcriptome data of the oseil1 insect-resistant mutant, we found that a CC-NBS-ARC type resistance gene Os03g50150 (named OsARC3 below) was significantly downregulated and that the OsEIL1 transcription factor could bind to the promoter of the OsARC3 gene and directly activate OsARC3 expression.

[0017] At the same time, the structural analysis of OsARC3 protein revealed that OsARC3 is a special type of R protein, which lacks the LRR domain at its carboxyl end and is not a typical NBS-LRR protein; the expression spectrum analysis of OsARC3 gene revealed that this gene responds to the feeding of brown planthoppers and also responds to the treatment of various hormones, such as methyl jasmonate, gibberellin, and brassinolide, all of which are involved in the process of rice resistance to brown planthoppers. Further insect resistance identification results showed that the overexpression of OsARC3 gene significantly enhanced the sensitivity of rice to brown planthoppers, while the CRISPR / cas9 knockout of OsARC3 gene significantly enhanced the resistance of rice to brown planthoppers.

[0018] In addition, the present invention also takes OsARC3 gene as the core, and determines the insect-resistant function of OsARC3 gene by analyzing the anti-brown planthopper phenotype of different transgenic plants of OsARC3; determines the molecular nature of OsARC3 by analyzing the expression pattern of OsARC3 gene, the protein structure of OsARC3, subcellular localization and the molecular interaction of different domains; determines the insect-resistant mechanism of OsARC3 by analyzing whether OsARC3 can produce immune response, the stress resistance physiological index of transgenic plants, transcriptome data and yeast two-hybrid screening library. The research results of the present invention will further enrich people's understanding of the role of different types of resistance genes in rice resistance to brown planthoppers, and are expected to provide gene resources and theoretical basis for rice endogenous gene resistance to brown planthoppers breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the effect of an experiment on determining the resistance of rice brown planthopper-resistant mutant arc3 plants to brown planthoppers using the single plant method in the present invention;

[0020] Figure 2 The present invention is a honeydew amount change before and after brown planthopper feeding measured by the honeydew method; wherein A: change in honeydew amount before and after brown planthopper feeding; B: honeydew amount area before and after brown planthopper feeding; ZH11 represents Zhonghua 11 plant; arc3 represents rice brown planthopper-resistant mutant arc3 plant; statistical data after t-test show significant difference and extremely significant difference are represented by * (P<0.05) and ** (P<0.01), respectively.

[0021] Figure 3 This is the KEGG analysis of the transcriptome of arc3 knockout plants in the present invention; the black part in the figure represents up-regulated genes, and the gray part represents down-regulated genes. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. The raw materials described in the present invention can be obtained from the market unless otherwise specified, and the methods described are conventional methods unless otherwise specified.

[0023] Example 1

[0024] Obtaining rice ARC3 transgenic plants

[0025] A specific sg-RNA of OsARC3 (gene number Os03g50150) (sequence as shown in SEQ ID NO.1, specifically 5'-GGGGATCACCTCGTTGTGGGG-3') was designed, and specific primers were designed according to the sequence of the sgRNA (Table 1). After annealing, a double strand was formed and linked into the Crisper (UBI, OsU6) vector, and then transferred into the wild-type rice variety Zhonghua 11 through Agrobacterium-mediated genetic transformation. Several homozygous mutant plants were obtained through molecular biological detection; one of the plants used in this study lacked 7 bases "ACCTCGT" at the coding position of OsARC3 compared with the wild type (as shown in Table 2), and this homozygous mutant plant was named arc3.

[0026] Table 1: OsARC3-specific sgRNA sequence primers

[0027] ARC3-F <![CDATA[5’- TGTG GGGGATCACCTCGTTGTGGGG-3’]]> SEQ ID NO.: 2 ARC3-R <![CDATA[5’- AAAC CCCCATAACGAGGTGATCCCC-3’]]> SEQ ID NO.: 3

[0028] The underlined bases are linkers

[0029] Table 2 Location of base deletions in arc3 plants

[0030] Chinese flower 11 5'-GGGGATCACCTCGTTGTGGGG-3' arc3 5'-GGGGATC————TGTGGGG-3'

[0031] Where “————” indicates the missing base position

[0032] Example 2

[0033] Identification of resistance of rice arc3 plants to brown planthopper:

[0034] Identification of resistance to brown planthopper by single plant method:

[0035] Experimental method: After germination, the seeds of Zhonghua 11 and arc3 plants were sown individually in small plastic pots and grown normally. After about 1 month, when the seedlings will enter the tillering stage, insect resistance identification was carried out. A transparent plastic cover with a height of 40 cm and a diameter of about 8 cm was prepared in advance, with a 6×10 cm ventilation hole on one side of the cover, which was covered with gauze. The seedlings were covered with the cover and the top was covered with gauze.

[0036] Insect resistance identification: When the rice grows to the tillering stage, cover the rice seedlings with a pre-prepared transparent plastic cover with a diameter of 8 cm and a height of 40 cm with ventilation screens, and place about 15 third-instar brown planthopper nymphs in each cover. Set up 8 replicates for each strain, and repeat the insect counting once the next day. Under normal growth and management conditions, after 5-8 days, observe the survival status of the rice seedlings; and record the results. Figure 1 shown.

[0037] Depend on Figure 1It can be seen that when all the flowers 11 died, the arc3 plants were still bright green, indicating that the arc3 rice plants showed obvious resistance to brown planthoppers.

[0038] Biological assays before and after feeding of brown planthoppers:

[0039] The insect resistance of arc3 plants was identified by honeydew method; the specific method was based on the literature (Guo HM, Li HC, Zhou SR, Xue HW, Miao XX. 2014. Cis-12-oxo-phytodienoic acid stimulates rice defense response to a piercing-sucking insect. Mol Plant 7(11): 1683-1692.); honeydew is the secretion of brown planthoppers, and its amount can largely reflect the feeding situation of brown planthoppers. We measured the changes in honeydew amount before and after brown planthoppers fed, and found that the amount of honeydew in arc3 plants (arc3) was significantly reduced compared with that in ZH11 (ZH11). Figure 2 ), indicating that arc3 plants showed resistance to brown planthoppers.

[0040] Example 3

[0041] Possible mechanism of resistance of rice arc3 plants to brown planthopper

[0042] To further explore the mechanism of OsARC3 gene's resistance to brown planthoppers, we selected OsARC3 knockout plants and wild-type plants for transcriptome sequencing. KEGG results showed that key genes in the flavonoid biosynthesis pathway were upregulated ( Figure 3 In 2019, Shi Zhenying's team from the Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences analyzed 39 natural rice varieties and found that the increase in flavonoid content in rice materials showed a positive correlation with brown planthopper resistance. We speculate that the mechanism by which the OsARC3 gene regulates rice resistance to brown planthoppers may be achieved through the flavonoid pathway.

[0043] The above embodiments are only preferred specific implementation methods of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent replacements of the technical solutions that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention all fall within the protection scope of the present invention.

[0044] References:

[0045] Liu Yunfei, Wan Hongjian, Li Zhimiao, Ye Qingjing, Wang Rongqing, Ruan Meiying, Yao Zhuping, Zhou Guozhi, Wei Yanping, Yang Yuejian. Structure, function, evolutionary origin and application of plant NBS-LRR disease resistance genes. Molecular Plant Breeding, 2014, 12(02): 377-389.

[0046] Du B, Zhang W, Liu B, Hu J, Wei Z, Shi Z, He R, Zhu L, Chen R, Han B, HeG. Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice. Proc NatlAcad Sci U SA. 2009, 106(52): 22163-221638.

[0047] Hu L,Wu Y,Wu D,Rao W,Guo J,MaY,Wang Z,Shangguan X,Wang H,Xu C,HuangJ,Shi S,Chen R,Du B,Zhu L,He G.The coiled-coil and nucleotide binding domains of Brown Planthopper Resistance14 function in signaling and resistanceagainst planthopper in rice.Plant Cell.2017,29(12):3157-3185.

[0048] Ma F,Yang X,Shi Z,Miao

[0049] Wang Z,Huang J,Nie L,Hu Y,Zhang N,Guo Q,Guo J,Du B,Zhu L,He G,ChenR.Molecular and functional analysis of a brown planthopper resistance proteinwith two nucleotide binding site domains.J Exp Bot.2020,eraa586.

[0050] Zhao Y,Huang J,Wang Z,Jing S,WangY,OuyangY,Cai B,Xin XF,Liu X,ZhangC,PanY,Ma R,Li Q,Jiang W,Zeng Y,Shangguan X,Wang H,Du B,Zhu L,Xu X,Feng YQ,HeSY,Chen R,Zhang Q,He G.Allelic diversity in an NLR gene Bph9 enables rice tocombat planthopper variation.Proc NatlAcad Sci U SA.2016,113(45):12850-12855.

Claims

1. A rice Os arc3 gene brown planthopper resistant mutant arc3, ​​characterized in that: The difference between the wild-type Osarc3 gene of Zhonghua11 and the wild-type Osarc3 gene is the coding position. 5'-GGGGATCACCTCGTTGTGGGG-3' lacks 7 bases ACCTCGT, and the gene number of the Os arc3 gene is Os03g50150.

2. The brown planthopper-resistant mutant arc3 of rice Os arc3 gene according to claim 1, characterized in that: The expression or activity of the Os arc3 gene or its gene active fragment is reduced relative to the wild type of Zhonghua 11.

3. A use of the rice Os arc3 gene brown planthopper-resistant mutant arc3 according to claim 1 for preparing a rice brown planthopper-resistant plant mutant arc3, ​​characterized in that: The plant mutant arc3 is a homozygous mutant plant of the brown planthopper-resistant mutant arc3 of the rice Os arc3 gene.

4. Use of the brown planthopper-resistant mutant arc3 of the rice Os arc3 gene as claimed in claim 1 or the plant mutant arc3 as claimed in claim 3 for breeding brown planthopper-resistant rice.

5. A method for resisting brown planthopper in rice, characterized in that: The method comprises the steps of mutating the wild-type gene of rice Os arc3 into the gene mutant arc3 described in claim 1.

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