Improving rice plant resistance to rice black-streaked dwarf virus using gene editing technology

Abstract

The application provides a method for improving the resistance of rice plants to rice black-streaked dwarf virus by using gene editing technology. By using the method, a rice variety with high resistance to rice black-streaked dwarf virus can be quickly bred, and the loss caused by the disease can be reduced.

Description

Technical fields:

[0001] This invention relates to improving the resistance of rice plants to rice black-streaked dwarf virus using gene editing technology, and belongs to the field of agricultural science and technology. Background technology:

[0002] Rice black-streaked dwarf virus (RBSDV) belongs to the family Reoviridae, genus Fijivirus, and is persistently transmitted by the insect vector, the planthopper. In my country, RBSDV causes rice black-streaked dwarf disease, with infected plants exhibiting dark green, stunted growth, failure to head, or small panicles. Infected fields typically experience yield reductions of 10%-40%, with severely affected fields resulting in complete crop failure. Although there is considerable research on the viral particle morphology, genome sequence, and physicochemical properties of the encoded proteins of RBSDV, reports on the virus's resistance mechanisms in rice are scarce. Due to the lack of resistant varieties in production, disease control primarily relies on chemical pesticides to control the insect vector. Therefore, utilizing CRISPR-Cas9 gene editing technology to create resistant rice materials and cultivate resistant varieties is of great significance for current rice production.

[0003] The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated genes 9) gene editing system is widely found in bacteria and archaea. It is an adaptive immune mechanism developed during long-term evolution, involving RNA-mediated degradation of viral or bacteriophage DNA (Makarova et al., Nat. Rev. Microbiol., 2015, 13: 722-736). Cas9 is a nuclease composed of 1490 amino acids, containing RuvC-like and HNH nuclease functional domains (Dominguez et al., Nat. Rev. Mol. Cell Biol., 2016, 17: 5-15). Cas9 can cleave double-stranded DNA complementary to crRNA, with the cleavage site located 3 nt upstream of the PAM (protospacer adjacent motif, NGG), resulting in double-strand breaks (DSBs) (Gaj et al., Trends Biotechnol., 2013, 31: 397-405). DNA damage-induced DSBs activate two different repair mechanisms within the cell: non-homologous ending-joining (NHEJ) and homologous recombination (HR), to repair the damaged DNA, thereby achieving site-specific genome editing (Wyman and Kanaar, Annu. Rev. Genet., 2006, 40: 363-383). CRISPR-Cas9 technology has been widely used in bacteria, yeast, animals, and plants.

[0004] Because the CRISPR-Cas9 gene editing system targets DNA, it was first applied to resistance studies of DNA viruses in the Geminiviridae family in plant antiviral research. Transient expression of sgRNA-Cas9 constructs targeting the genomic DNA of BSCTV (beet severe curly top virus) and BeYDV (bean yellow dwarf virus) in tobacco inhibited viral accumulation and induced mutations in the target sequence; transgenic tobacco plants overexpressing sgRNA-Cas9 showed immunity to the virus (Ji et al., Nat. Plants, 2015, 1:15144; Baltes et al., Nat. Plants, 2015, 1:15145). Studies of sgRNA target sequences have shown that sgRNAs targeting the IR (intergenic region) of the TYLCV (tomato yellow leafcurl virus) genome replication site are most effective in slowing down or reducing viral DNA accumulation and significantly alleviating disease symptoms in tobacco (Ali et al., Genome Biol., 2015, 16:238). Furthermore, the CRISPR-Cas9 gene editing system targeting the TYLCV CP (coat protein) coding sequence induces viral mutants that can overcome CRISPR-Cas9 gene editing resistance and replicate and spread in plant cells (Alie et al., Sci. Rep., 2016, 6:26912).

[0005] Recent studies have shown that the CRISPR-Cas9 gene editing system can successfully enhance plant resistance to RNA viruses. Potato viruses (PVY) recruit host plants' eIF4E (eukaryotic translation initiation factors) or its isoform eIF(iso)4E using the viral VPg (viral genome-linked protein) protein; their interaction plays a crucial role in viral replication (Robaglia and Caranta, TrendsPlant Sci., 2006, 11:40-45). Using the CRISPR-Cas9 gene editing system, cucumber eIF4E gene mutant plants were constructed, exhibiting resistance to Cucumber vein yellowing virus, Zucchini yellow mosaic virus, and Papayaring spot virus (Chandrasekaran et al., Mol.Plant Pathol., 2016, 17:1140-1153). Similarly, constructing non-transgenic Arabidopsis thaliana eIF(iso)4E gene point mutants using CRISPR-Cas9 gene editing significantly enhanced resistance to TuMV (Turnip mosaic virus) (Pyott et al., Mol. Plant Pathol., 2016, 17: 1276-1288). These studies demonstrate that constructing host factor gene-edited plants involved in viral replication, motility, and pathogenicity via CRISPR-Cas9 is an effective way to enhance viral resistance. Summary of the Invention:

[0006] This invention provides a method for improving the resistance of rice plants to rice black-streaked dwarf virus using gene editing technology. This method can be widely applied in the breeding process of rice resistant to rice black-streaked dwarf virus.

[0007] The present invention provides a method for improving the resistance of rice plants to rice black-streaked dwarf virus through gene editing, which is obtained through the following method:

[0008] 1) Based on the nucleotide sequence of the rice eIF4G gene (LOC_Os07g36940, http: / / rice.uga.edu / cgi-bin / sequence_display.cgi?orf=LOC_Os07g36940.1), a sgRNA sequence targeting eIF4G was designed using CRISPR PLANT tools (http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html);

[0009] 2) Construct the pRGEB32-eIF4G CRISPR / Cas9 gene editing vector;

[0010] 3) T0 generation transgenic rice plants (pRGEB32-eIF4G) were obtained by Agrobacterium-mediated transformation of rice;

[0011] 4) Identification of T0 generation rice plants with eIF4G site gene editing;

[0012] 5) Obtain T2 generation rice eif4g homozygous gene-edited plants that do not carry transgenic components;

[0013] 6) Identification of rice dwarf virus resistance and agronomic traits in T2 generation rice plants with homozygous eif4g gene editing.

[0014] The specific sgRNA sequence targeting the rice eIF4G gene provided by this invention is as follows:

[0015] gagggatttatgtcccagcg.

[0016] Applications of gene editing technology to improve rice plant resistance to rice black-streaked dwarf virus include: breeding rice resistant to rice black-streaked dwarf virus.

[0017] This method can be used to quickly breed rice black-streaked dwarf disease resistant varieties that do not carry genetically modified components, thereby reducing losses caused by rice black-streaked dwarf disease. Attached image description:

[0018] Figure 1 Molecular identification of rice eIF4G gene-edited plants (A, design of sgRNA specifically targeting rice eIF4G; B, detection of eIF4G gene-edited plants by PCR-enzyme digestion method; C, detection of eIF4G gene-edited plants by cloning and sequencing method; D, detection of eIF4G transcription level in gene-edited plants by qRT-PCR).

[0019] Figure 2Genetic analysis of T1 generation rice plants with eif4g gene editing (A, RT-PCR detection of transgenic components in T1 generation eif4g gene-edited plants; B, RT-PCR detection of genetic analysis of eif4g large deletion mutants).

[0020] Figure 3 : Identification of rice black-streaked dwarf virus resistance in rice eif4g gene-edited plants (A, B, symptoms of rice black-streaked dwarf virus inoculation in rice eif4g gene-edited plants; C, disease incidence of rice black-streaked dwarf virus inoculation in rice eif4g gene-edited plants; D, qRT-PCR detection of RBSDV P9-1 transcription level in rice eif4g gene-edited plants; E, protein hybridization detection of RBSDV P6 expression level in rice eif4g gene-edited plants).

[0021] Figure 4 Detection of agronomic traits in rice eif4g gene-edited plants (A, growth phenotype of rice eif4g gene-edited plants; B, plant height statistics of rice eif4g gene-edited plants; C, thousand-grain weight statistics of rice eif4g gene-edited plants; D, number of tillers per plant of rice eif4g gene-edited plants; E, panicle length of rice eif4g gene-edited plants). Detailed implementation method:

[0022] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0023] Example 1: Gene editing technology enhances the resistance of rice plants to rice black-streaked dwarf virus.

[0024] (1) Design sgRNA specifically targeting the eIF4G gene

[0025] Based on the nucleotide sequence of the rice eIF4G gene (LOC_Os07g36940), a targeting RNA sequence gaggatttatgtcccagcg was designed using CRISPR PLANT tools (http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html) to target eIF4G. Figure 1 A).

[0026] (2) Constructing rice eif4g gene-edited plants

[0027] The gRNA sequence targeting the eIF4G gene was synthesized by Nanjing Genscript Biotech Co., Ltd. and cloned into pRGEB32 (Yinong Yang, Pennsylvania State University, Addgene plasmid #63142). After sequencing verification, it was transformed into Agrobacterium (GV3101) and then into rice (Nipponbare) using Agrobacterium-mediated rice callus transformation. The T0 generation transgenic rice plants with the eif4g gene edited were identified using PCR-enzyme digestion and cloning sequencing methods. Figure 1 B, C). Genomic DNA was extracted from 10 eif4g T0 generation transgenic rice plants. PCR amplification and BsgI restriction electrophoresis showed that the restriction patterns of 5 plants (#2, #3, #4, #5, #7) differed from those of the wild type, indicating that these plants underwent gene editing at the eif4g target site, with a gene editing rate of 50%. Figure 1 B). Sequencing of the eif4g PCR product from the eif4g#2 gene-edited plant showed that the eif4g#2 plant lacked three bases at the eIF4G target site, resulting in a protein-level deletion of one amino acid (Pro, proline). Figure 1 C).

[0028] Gene-edited rice plants of generation eif4g#2 and eif4g#4 (T1 generation) were planted. Genomic DNA was extracted from individual plants. Cas9 PCR analysis showed that the eif4g#2 gene-edited plants (plants 4, 7, and 8) did not contain transgenic components. Figure 2 A). Different editing types were observed in the eIF4g#4 T1 generation rice plant population, including large deletions and deletions of three amino acids as well as wild-type, but no homozygous large deletion mutants were found. This suggests that the eIF4G gene is crucial for rice growth and development, and loss-of-function homozygous mutants are lethal. Figure 2 B).

[0029] (3) Identification of rice black-streaked dwarf virus resistance in rice eif4g gene-edited plants

[0030] Rice black-streaked dwarf virus (RBSDV) was artificially inoculated into homozygous gene-edited rice plants of generation T2 (eif4g#2 and eif4g#5). RBSDV resistance identification showed that the eif4g gene-edited plants significantly enhanced resistance to the virus; the disease incidence rate in wild-type rice was 90%, while that in eif4g#2 and eif4g#5 gene-edited rice plants was 38% and 41%, respectively, less than half that of the wild type. Figure 3 A, B, C). qRT-PCR experiments showed that the RBSDV P9-1 transcription level in eif4g gene-edited plants was significantly lower than that in wild-type rice. Figure 3D). Protein hybridization results also showed that RBSDV P6 protein expression was significantly lower in eif4g gene-edited plants compared to wild-type rice. Figure 3 E). The data above indicate that rice plants with the eif4g gene edited significantly enhance their resistance to RBSDV.

[0031] (4) Agronomic traits analysis of rice plants with eif4g gene editing

[0032] To analyze the effects of eif4g gene-edited plants (eif4g#2, eif4g#5) on rice growth and development, we examined multiple rice agronomic traits ( Figure 4 A). The test results showed that the eif4g gene-edited plants did not have an adverse effect on the growth and development of rice, and their plant height ( Figure 4 B), 1000-grain weight ( Figure 4 C), number of tillers ( Figure 4 D) No difference from wild-type rice; the panicle length of eif4g gene-edited plants was significantly increased compared to wild-type. Figure 4 E). The above results indicate that eif4g gene editing in rice plants does not affect normal growth and development, and has important application value in rice breeding for resistance to RBSDV.

[0033] Applications of gene editing to enhance rice plant resistance to rice black-streaked dwarf virus include: using gene editing to breed rice black-streaked dwarf virus resistant varieties.

[0034] The above embodiments do not limit the invention in any way.

Claims

1. A method for improving the resistance of rice plants to rice black-streaked dwarf virus using gene editing technology, characterized in that: Gene editing vectors were constructed using sgRNA sequences that specifically target rice eIF4G. Rice plants with eif4g gene editing were obtained through Agrobacterium-mediated transformation, thereby improving the resistance of rice plants to rice black-streaked dwarf virus. The "sgRNA sequence that specifically targets rice eIF4G" refers to the following sequence: gaggggatttatgtcccagcg.

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