Identification Of Resistant Candidate Gene Of Rice Black-streaked Dwarf Disease In Rice And Excavating New Materials

The pathogen of Rice black streaked dwarf disease is Rice black-streaked dwarf virus(RBSDV),and it is a viral disease transmitted by vector,small brown planthoppers(SBPH;Laodelphax sreiate sriatellus Fallen),in a persistent and propagative manner.Rice show typical stunting,less tiller,dark leaves compared with healthy plants and small enations on stem and leaf backs after infected,causing great losses to rice.At present,explore resistant resources and breeding resistant varieties are the most economical and effective methods for the prevention and treatment of the disease.However,so far,there has been no reports of the resistance genes related to RBSDV have not been cloned.Since Southern rice black-streaked dwarf virus(SRBSDV)produces similar symptoms compared with RBSDV,in order to eliminate the interference of the source of poison,in this study,RT-LAMP assay was used to rapid detecte RBSDV.And in this study,we identified 18 rice black dwarf disease resistance candidate genes in resistant QTL by early identification of parents and F2 generation and functional prediction.After the transgenic plants were obtained,the positive plants were screened with hygromycin specific primers and target gene primers.The transcription level of transgenic To generation was determined by real-time fluorescence quantitative PCR,and the variation range was between 0.15 and 150 times.According to the level of transcription of each candidate gene,the Ti generation with high expression and low expression were selected for artificial inoculation resistance identification.We identified Ti generation of eight candidate genes,in which Os06t0351500-01,Os06t0486400-01,Os06t0486400-01,Os06t0557100-01,Os06t0480000-01,Os06t0529800-01 and Os06t0556300-01 were found to have no significant difference from the transgenic receptor,while the incidence of 1 line of Os06t0348800-03 was significantly different from that of susceptible cultivar Huaiai No.5,and the resistance was significantly improved.At the same time,the gene transcription level and virus content of the resistant plants in this line were measured.The results showed that the variety of candidate gene expression level was related to the variety of virus content.Secondly,a rice black dwarf dwarf disease resistant material-15HP0187 was screened out.In order to clarify the resistance characteristics of 15HP0187 to rice black-streaked dwarf virus and SBPH,antibacterial and non-tonal experiments were carried out on 15HP0187.The results showed that the resistance of 15HP0187 to rice black-streaked dwarf disease include resistance to SBPH.At the same time,we utilized the susceptible cultivar Nipponbare to hybridize,and has been gotten F2 generation.This work laid the foundation for the follow-up study.At the end of the study,in order to clarify the regulatory pathway of rice black streaked dwarf disease resistance genes,a yeast two-hybrid cDNA library was constructed using Nipponbare(Oryza.Sativa L.spp.japonica,var Nipponbare),which was infected by RBSDV.The samples used in the construction of the library were collected at different time points after Nipponbare were infected by RBSDV.The time of collection was determined by the interval at which the virus could detecte by RT-PCR on the plant until the typical symptoms were observed.Therefore,the final time points were Od,7d,14d,21 d,31d.A decoy vector was constructed by using protein encoded by rice black streaked dwarf disease resistance gene,and the interaction proteins in virus and in the regulatory pathway were screened to provide a basis for studying the mechanism of action of disease resistance genes.