Field Investigation And Molecular Characterization Of Transgenic Rice With The Resistant To Rice Black Streaked Dwarf Virus(RBSDV)

Rice black streaked dwarf virus, short for RBSDV, is mainly spreaded in China rice-growing region carried by Laodelphgax striatellus. Because the disease is difficult to be effectively controlled with conventional method, the degree of harm accumulates annually, which results heavy economic losses to agriculture. RNA-mediated virus resistance to cultivate GM crops antiviral is by far the most effective and efficient way which provides plants with high resistance to disease, high bio-safety features and great value. In this study, laboratory provided transgenic rice KRBSDV with the resisitance to rice black streaked dwarf disease as experimental material and the receptor Zhendao 88 as control. Agronomic traits, herbicide resistance, virus resistance nutrional ingredients of the transgenic rice were detected. The differences expression between anti-viral transgenic rice and wild-type were also analzed at RNA and protein levels. Meanwhile, the food safety of transgenic rice was assessed. The main results and conclusions are as follows:(1) Analysis on agronomic traits, resisitance and nutrional ingredients of genetically modified riceThe results of the agronomic traits and virus resistance showed that there were no significant differences from the appearance between Transgenic rice KRBSDV and Zhendao 88 at each period of plant phenotype. The KRBSDV showed strong resistance to PPT and RBSDV. These results of nutrional ingredients analysis indicated that the GM rice had no significant differences with wild-type on water content, ash content, Vitamin content, mineral content, protein content, plant acid content and Trypsin inhibitor content. The result showed that the transferred RBSDV-CPM hairpin RNA gene(hpRNA) provided the transgenic rice KRBSDV significant resistance to RBSDV without changing its biological charactors.(2) Southern blot hybridization demonstrated that transgenic rice exogenous gene is a single copy. The hiTAIL-PCR method was used to amplify insertion sites on both sides of the wings of the genomic sequence with the database comparison and found that the insertion point is on the number of rice chromosome 2, the insertion site region expresses no protein.(3) Extracted mRNA rice leaf of tillering stage and analyzed with RNA-seq sequencing technique. According to statistics, there were 6869 significantly mRNA expression differences(P<0.05) between KRBSDV and Zhendao 88. Transgenic rice mRNA expression differed from wild-type on all 12 chromosomes, not focus on chromosome 2 around the insertion region. The transgenic rice Bar had 10,215 counts of relative expression, and the antiviral RBSDV-CPM gene had 6531 counts of relative expression. There were no Bar and antiviral gene expression detected in wild-type rice. Analysis of the significant differential expression of mRNA, we found they were mainly involved in transport, regulation of transcription and translation, transcriptional regulation, protein binding transcription, binding protein, structural molecular activity and other variety of different types of protein, such as ribosomes, ATP synthesis associated transport proteins, chaperones, heat-sensitive protein, Histone. Further screening of the mRNA data showed there were no new allergen proteins and toxic protein.(4) Protein level differences between transgenic rice KRBSDV and receptor Zhendao 88The SWATH analysis detected 1887 different type of proteins in seed of KRBSDV and Zhendao 88, in which 346 proteins indicated significant difference on expression. The PPT N-acetyl transferase(Phosphinothricin N-acetyl transferase, PAT), expressed by maker gene bar, could be detected but the relative expression was low and the expression protein of aimed gene RBSDV-CPM was not detected. The analysis of the significant differential protein indicated that the proportion of functional proteins was similar to the proportion of whole functional proteins. There were significant amount differences on the 20 s, 40 s, 60 s calibration protein, ATP transfer and synthesis relative proteins, chaperone proteins, glutenin and histone and other pathogen induced response proteins and heat-shock protein between transgenetic rice and wild-type rice. The significant differential protein sequences were blased on NCBI in data base and screened in The Allergen Online Database, and there were no new toxic or allergen proteins in transgenetic rice seed.Overall, the current data suggested that the transgenic rice, with reliable PPT and RBSDV viral resistance, has no significant differences with its wild-type rice in growth patterns, agronomic traits and nutritional ingredients. Although the transferred gene has brought expression differences on RNA and protein level, there are no new toxic or allergen protein expressed, which indicates that the transgenetic rice has good food safety.