| Abstract: | Rice stripe virus(RSV) often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs(si RNAs) with transgenic virus resistance of plants using RNA interference(RNAi) is known for decades, no systematical research has been done on the profiling of si RNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein(NCP) gene. In this paper, three independent RSV-retsistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of si RNAs were confirmed by Southern blotting and Northern blotting analyses, and the majority of si RNAs were in lengths of 21, 22, and 24 nucleotides(nt), which have validated a connection between the presence of the RSV NCP homologous si RNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines(T4-B1), the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confirmed the large population of NCP-derived si RNAs in transgenic plants, and the RSV-infected library(T4-B1-V) possessed more si RNAs than its mock inoculated libraries(T4-B1-VF), these results indicating the inverted repeat construct and RSV could introduce abundance of si RNAs in transgenic rice. Moreover, a varied expression level of specific si RNAs was found among different segments of the NCP gene template, about 47% of NCP-derived si RNAs reads aligned with the fragment from 594 to 832 nt(239 nt in length) in NCP gene(969 nt in length) in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the first study to address the si RNA profile in RSV-resistant transgenic plant using next generation sequencing(NGS) technique, we confirmed that the massive abundance of si RNA derived from the inverted repeat of NCP is the major reason for RSV-resistance. |
