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CN-121991976-A - Application of E3 ubiquitin ligase OsRING18 in rice stripe virus resistance

CN121991976ACN 121991976 ACN121991976 ACN 121991976ACN-121991976-A

Abstract

The invention relates to an E3 ubiquitin ligase coding gene and application thereof in rice stripe virus resistance. The invention uses CRISPR/Cas9 gene editing system to knock-out OsRING gene from wild ZH11 rice to obtain homozygous OsRING function-deleted mutant rice line, which shows that compared with wild type Japanese ZH11 rice, osRING gene function-deleted mutant rice line shows stronger disease resistance.

Inventors

  • SUN ZONGTAO
  • YANG ZIHANG
  • ZHANG HEHONG
  • LI YANJUN
  • WEI ZHONGYAN
  • CHEN JIANPING

Assignees

  • 宁波大学

Dates

Publication Date
20260508
Application Date
20241108

Claims (10)

  1. 1. An application of a rice E3 ubiquitin ligase encoding gene OsRING18 in rice breeding of Rice Stripe Virus (RSV) resistant, wherein the nucleotide sequence of OsRING gene is shown in SEQ ID NO. 1.
  2. 2. The use of claim 1, wherein the amino acid sequence of OsRING gene is shown as SEQ ID NO. 2.
  3. 3. The use as claimed in claim 1, wherein the functional deletion of the rice E3 ubiquitin ligase encoding gene OsRING is achieved by a CRISPR/Cas9 gene editing system, and the target sequence of the CRispr/Cas9 gene editing system is shown as SEQ ID NO. 3.
  4. 4. A preparation method of a transgenic plant resistant to rice stripe virus comprises the following steps: 1) Constructing a rice OsRING gene knockout vector; According to OsRING sequences shown in SEQ ID No.1, designing primers in a CRISPR-GE website, cloning a gene sgRNA fragment from rice, recovering glue after PCR amplification, respectively connecting target sequences to a U3 promoter and a gRNA scafold of a pYLsgRNA-OsU vector, connecting the U3 promoter, the target sequences and the gRNA scafold into a sgRNA expression cassette by using an overlap PCR technology by taking the two products obtained by PCR as templates, mixing the gRNA expression cassette product with an uncleaved PYLCRISPR/Cas9Pubi-H plasmid, performing enzyme digestion at 37 ℃ by using BsaI endonuclease, adding 1.5 mu L of 10X DNA LIGASE Buffer and 35U of T4 ligase connection, selecting positive clones, sequencing by biological company, and confirming construction of an expression vector YL-Hu-OsRING; 2) Genetic transformation of rice, agrobacterium transformation and callus induction culture: The callus induction comprises the steps of selecting mature rice seeds, strictly sterilizing the seeds, inducing the callus, sterilizing the seeds in 75% ethanol for 1min, washing the seeds with sterile water, sterilizing the seeds in 30% sodium hypochlorite for 20min, washing the seeds with sterile water for 5-6 times, placing the sterilized seeds on an induction culture medium, culturing the seeds in a 28 ℃ illumination incubator for 3 weeks, transferring the formed callus to a secondary culture medium by using sterile forceps, and continuously culturing the callus for 1 week under the same condition; Transforming and culturing agrobacterium tumefaciens, namely transforming a plasmid containing a target vector into a agrobacterium tumefaciens strain GV3101 by an electric shock method, mixing 5 mu L of plasmid with 100 mu L of agrobacterium tumefaciens competent cells, performing 220V electric shock transformation in a 4 ℃ precooled electrode cup, performing shake culture on a non-resistant LB liquid medium for 3 hours, coating on an LB solid medium containing Kan and Rif, and performing dark culture at 28 ℃ for 2 days until single colony appears; Screening and infecting agrobacterium, namely identifying agrobacterium by using a PCR reaction, selecting positive clones, culturing in a shaking incubator at a constant temperature of 28 ℃ overnight, selecting a proper amount of callus, placing in a sterile triangular flask, adding agrobacterium suspension, infecting at room temperature for 20min, shaking from time to time, pouring out bacterial liquid after infecting, placing sterile filter paper on the callus to suck excessive bacterial liquid, transferring to a solid co-culture medium paved with the sterile filter paper, and culturing in the dark at 26 ℃ for 3 days; The callus is cultured and rooted, namely, the co-cultured callus is washed by sterile water containing carbenicillin, dried in a super clean bench for about 30min, transferred to a screening culture medium containing hygromycin B, cultured in the dark at 28-30 ℃ for 3-4 weeks, and transferred to a rooting culture medium when seedlings grow to 2-3cm and have obvious root systems, and cultured under the sterile illumination at 28-30 ℃; 3) Identification of positive OsRING.sup.18 loss-of-function mutant rice: The total DNA of wild ZH11 rice and OsRING gene function-deleted mutant rice is extracted by CTAB method, amplified by PCR reaction, sequenced by biological company, compared and analyzed by DNAMAN software, and two OsRING gene function-deleted mutant rice strains named osring18#5 and osring18#8 are identified after primer is shown as SEQ ID NO.6-7 and compared with wild ZH11 rice sequence.
  5. 5. The method according to claim 4, wherein the induction medium comprises 24.1g/L of N6 medium and 2mg/L of 2,4-D, PH =5.8.
  6. 6. The method according to claim 4, wherein the secondary medium comprises 24.1g/L of N6 medium, 2mg/L of 2,4-D, 50mg/L of hygromycin, 300mg/mL of cephalosporin, and pH=5.8.
  7. 7. The method of claim 4, wherein the rooting medium comprises 1/2MS 39.45g/L, 0.5mg/L NAA, 50mg/L hygromycin and pH=5.8.
  8. 8. The method according to claim 4, wherein the co-culture medium comprises 24.1g/L of N6 medium, 2mg/L of 2,4-D, 200. Mu. Mol/L of acetosyringone, and pH=5.2.
  9. 9. A method for improving rice stripe virus resistance comprises the step of knocking out OsRING gene in wild ZH11 rice by using a CRISPR/Cas9 gene editing system, wherein the nucleotide sequence of the OsRING gene is shown as SEQ ID NO. 1.
  10. 10. A method for identifying rice for resistance to rice stripe virus, comprising the steps of: Extracting total DNA of wild ZH11 rice and rice to be detected by using a CTAB method, carrying out PCR reaction amplification, sequencing by a biological company, comparing and analyzing by using DNAMAN software, comparing the amplified product sequences of the rice to be detected and the wild ZH11 rice, and confirming the mutation condition of the rice OsRING gene to be detected, wherein the primer is shown as SEQ ID No. 6-7.

Description

Application of E3 ubiquitin ligase OsRING18 in rice stripe virus resistance Technical Field The invention relates to the technical field of transgenosis and the field of plant disease control, in particular to a rice E3 ubiquitin ligase coding gene OsRING18 and application thereof in resisting rice stripe virus Background Rice stripe disease, a viral disease caused by Rice Stripe Virus (RSV). RSV is a typical representation of the genus leptospirovirus (Tenuivirus) that is transmitted to rice by the plant hopper. The particles of this virus exhibit a non-enveloped, filamentous morphology, with diameters between approximately 3-9 nm. As a class of multi-split negative-sense single-stranded RNA viruses, the genome of RSV consists of four single-stranded RNA fragments, designated RNA1, RNA2, RNA3 and RNA4, respectively, which are 290-510nm, 610nm, 840nm and 2110nm in length, respectively. Among them, the RNA1 fragment has negative sense polarity, and encodes RNA-dependent RNA polymerase (RdRp, RNA-DEPENDENT RNA polymerase), which is a key enzyme for viral replication. The RNA2, RNA3 and RNA4 fragments of RSV employ an antisense coding strategy, each with two Open reading frames (Open READING FRAMES, ORFs) in opposite directions on the Viral strand RNA (Viral RNA, vRNA) and the Viral complementary strand RNA (Viral complementary RNA, vcRNA). Specifically, vRNA2 encodes nonstructural protein P2, vRNA2 encodes glycoprotein Pc2, vRNA3 encodes viral gene silencing inhibitor P3, vRNA3 encodes nucleocapsid protein Pc3, vRNA4 encodes major nonstructural disease-specific S protein P4, and vRNA4 encodes mobile protein Pc4. Plant responses to pathogens are based on the rapid and efficient perception of microorganisms and the coordination of downstream signaling events. Detection of pathogen invasion begins with the recognition of conserved microbial molecules, pathogen-associated molecular patterns (Pathogen-associated molecular patterns, PAMPs), which are predominantly carried out by plant membrane-associated extracellular receptors, which in turn elicit PAMP-triggered immunity (PAMP-TRIGGERED IMMUNITY, PTI). At the same time, phytohormones act as critical system signals, with an important impact on the level of plant resistance. When plant cells interact with microorganisms, hormone levels and hormone cross-interactions can vary significantly. The complex molecular mechanisms that control plant immune responses involve a high degree of proteomic plasticity, with ubiquitination playing a key role as a post-translational protein modification. Ubiquitin is a highly conserved protein modifier with a molecular mass of only 8.5kDa, which, by covalent linkage to the protein of interest, causes the protein of interest to undergo proteasome degradation or alters protein function. Typically, proteins modified by the continuous linkage of ubiquitin residues Lys-48 will be targeted for degradation by the 26S proteasome, a highly conserved proteolytic complex consisting of two subunits. The Ubiquitin-26S protease system (Ubiquitin-26S proteasome system,UPS) involves the sequential action of three enzymes, namely E1 Ubiquitin activating enzyme, E2 Ubiquitin binding enzyme and E3 Ubiquitin ligase, ultimately linking one or more Ubiquitin molecules to a specific protein of interest. Among them, E3 ubiquitin ligases are a key factor determining substrate specificity, and can be divided into four major subfamilies, HECT type, RING type, U-box type and CRLs type according to subunit composition and mechanism of action. In the last decade of research, the scientific community has had a profound understanding of the role of E3 ubiquitin ligase in plant immunity. These studies reveal a key role for E3 ubiquitin ligase in multiple links of plant perception of pathogens, signaling, activation of immune responses, etc. In particular in Arabidopsis, RING-type E3 ubiquitin ligases play a positive role in plant immunity, and sometimes also may play an inhibitory role, which highlights the complexity of E3 ubiquitin ligases in regulating plant immunity. The invention utilizes a CRISPR/Cas9 gene editing system to perform targeted knockout on E3 ubiquitin ligase coding gene OsRING in wild ZH11 rice, successfully identifies two OsRING functional mutant rice strains which are respectively named osring18#5 and osring18#8, and further RSV artificial inoculation experiments find that the two functional mutant rice of OsRING18 shows higher resistance to rice stripe virus compared with the wild ZH11 rice. The invention has practical guiding significance for cultivating transgenic plants resistant to rice stripe disease and southern rice black-streaked dwarf virus, and has important application prospect in the field of plant disease control. Disclosure of Invention The invention relates to a rice E3 ubiquitin ligase coding gene OsRING and a coding protein thereof. In one aspect, the OsRING gene has a nucleotide sequence shown in SEQ ID NO. 1. On the other h