CN-113846100-B - Gene editing technology for improving resistance of rice plant to black-streaked dwarf virus

Abstract

The invention provides a method for improving the resistance of rice plants to rice black-streaked dwarf virus by using a gene editing technology. The method can be used for rapidly cultivating rice varieties with high resistance to rice black streaked dwarf, and reduces the loss caused by diseases.

Inventors

• SUN FENG
• WANG WEI
• MA SHUHUI
• JI YINGHUA

Assignees

• 江苏省农业科学院

Dates

Publication Date

20260508

Application Date

20210924

Claims (1)

1. 1. A method for improving the resistance of rice plants to rice black-streaked dwarf virus by utilizing a gene editing technology is characterized in that a gene editing vector is constructed by utilizing a sgRNA sequence of specific targeted rice eIF4G, the rice eIF4G gene editing plant is obtained through agrobacterium-mediated rice transformation, the resistance of the rice plants to the rice black-streaked dwarf virus is improved, and the sgRNA sequence of the specific targeted rice eIF4G refers to a sequence of GAGGGATTTATGTCCCAGCG.

Description

Gene editing technology for improving resistance of rice plant to black-streaked dwarf virus Technical field: the invention relates to a method for improving resistance of rice plants to rice black-streaked dwarf virus by utilizing a gene editing technology, belonging to the technical field of agricultural science. The background technology is as follows: Rice black-streaked dwarf virus (RBSDV) belongs to the family of reoviruses (Reoviridae), genus Fijivirus (Fijivirus), and is transmitted in a persistent manner by the insect mediator Laodelphax striatellus. In China, RBSDV causes black streaked dwarf disease of rice, and the symptoms of infected plants are dark green dwarf, and no heading or small heading is caused. The yield of the disease field is generally reduced by 10% -40%, and the serious disease field and even the particles are not recovered. Although many studies on virus particle morphology, genome sequence, physicochemical properties of encoded proteins and the like of RBSDV exist at present, the antiviral mechanism of rice is rarely reported. Because of lack of disease-resistant varieties in production, the prevention and control of the diseases mainly depend on chemical pesticides to prevent and control insect with poison transfer mediators. Therefore, the CRISPR-cas9 gene editing technology is utilized to create disease-resistant rice materials and cultivate disease-resistant varieties, and has important significance on current rice production. CRISPR-cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated genes) gene editing system is widely available in bacteria and archaea, and is an adaptive immune mechanism of RNA-mediated degradation of viral or phage DNA formed during long-term evolution of organisms (Makarova et al., nat. Rev. Microbiol.,2015, 13:722-736). Cas9 is a nuclease consisting of 1490 amino acids, comprising RuvC-like and HNH nuclease domains (domiiguez et al, nat. Rev. Mol. Cell biol.,2016, 17:5-15). Cas9 can cleave double-stranded DNA complementary to crRNA, with a cleavage site 3nt upstream of PAM (protospacer adjacent motif, NGG) creating a DNA double-strand break (double strand breaks, DSBs) (Gaj et al, trends biotechnol, 2013, 31:397-405). DSBs generated by DNA damage activate two different repair mechanisms, non-homologous ending-joining (NHEJ) or Homologous Recombination (HR), to repair damaged DNA, thereby enabling site-directed editing of the genome (WYMAN AND KANAAR, ANNU.REV.GENET.,2006, 40:363-383). The CRISPR-cas9 technology has been widely used in bacteria, yeast, animals, plants. Since the acting target of the CRISPR-cas9 gene editing system is DNA, in the plant antiviral study, CRISPR-cas9 gene editing is firstly applied to the resistance study of the DNA virus Geminiviridae (GEMINIVIRIDAE). Tobacco transient expression of sgRNA-cas9 constructs targeting BSCTV (beet severe curly top virus), beYDV (bean yellow dwarf virus) genomic DNA, inhibition of viral accumulation and induction of mutations at the target sequence, overexpression of sgRNA-cas9 transgenic tobacco plants showed immunization against virus (Ji et al, nat. Plants,2015,1:15144;Baltes et al, nat. Plants,2015, 1:15145). Studies of the target sequence of sgRNA showed that expression of sgRNA against the IR (intergenic region) region of the TYLCV (tomato yellow leaf curl virus) genomic replication point was most effective in slowing or reducing viral DNA accumulation in tobacco, significantly alleviating disease symptoms (Ali et al, genome biol.,2015, 16:238). Whereas CRISPR-cas9 gene editing systems targeting the TYLCV CP (coat protein) coding sequence induce viral production mutants that overcome CRISPR-cas9 gene editing resistance, replicate and expand in plant cells (Ali et al, sci.rep.,2016, 6:26912). Recent studies have shown that the CRISPR-cas9 gene editing system can successfully enhance the resistance of plants to RNA viruses. Potato virus Y (potyviruses) recruits the host plant eIF4E (eukaryotic translation initiation factors) or its isoform (isosporm) eIF (iso) 4E with the viral VPg (viral genome-linked protein) protein, the interaction of which plays a critical role in viral replication (Robaglia AND CARANTA, TRENDS PLANT sci.,2006, 11:40-45). Cucumber eIF4E gene mutant plants were constructed using the CRISPR-cas9 gene editing system, exhibiting resistance to Cucumber vein yellowing virus, zucchini yellow mosaic virus, PAPAYA RING spot viruses (CHANDRASEKARAN ET al., mol. Plant Pathol.,2016, 17:1140-1153). Similarly, construction of Arabidopsis eIF (iso) 4E gene point mutated non-transgenic plants using CRISPR-cas9 gene editing significantly enhanced resistance to TuMV (Turnip mosaic virus) (Pyott et al., mol. Plant Pathol.,2016, 17:1276-1288). The above studies indicate that constructing host factor gene editing plants involved in viral replication, motility and pathogenesis by CRISPR-cas9 is an effective way to enhance viral resistance. The invention compris