Exploration On The Control Of Nilaparvata Lugens By DsRNA-transgenic Rice And Evaluation Of Bt Transgenic Rice Line S21

The brown planthopper (BPH), Nilaparvata lugens (Stal) is a major rice pest in many Asia countries. BPH feeds on plants by sucking phloem sap through its stylet mouthparts and causes substantial physiological damage to rice. N. lugens outbreaks frenquently and it is difficult to be controled effectively. BPH has become a serious threat to rice production because as it causes severe yield lossess. Novel methods for controlling BPH are highly desirable.RNAi (RNA interference) has been developed into a powerful tool for functional genomics study, and to date it has been widely used in insect genetic research. Gene knockdown via dsRNA has been successfully demonstrated in several insect orders. Moreover, it has been demonstrated that transgenic plants producing dsRNA of insect target genes could successfully control lepidopteran and coleopteran insect pests.We found dsRNAs were stable for48hours when they were incorprated into artificial diet. We chose two N. lugens EST sequences as target genes. One encodes the vacuolar ATPase subnite D (named173), and the other was ATPase subnite H (named322), respectively. The specific dsRNAs were synthesized in vitro. We studied the RNAi effects on N. lugens after dsRNAs were delivered into3rd instar larva of N. lugens by artificial feeding. We found that transcript levels of the two target genes in N. lugens decreased significantly by qRT-PCR analysis. The transcript levels of the173gene was reduced by47%after continuous ingestion of0.5μg/μl dsRNA for8days, and the transcript levels of the322gene was reduced by42%after continuous ingestion of0.5μg/μl dsRNA for8days. Additionally, the development of N. lugens larvae that were fed with the0.5μg/μl173gene dsRNA was interfered and resulted to lethality. This suggested that173gene may plays an essential role in the development of N. lugens. Therefore,173gene may be a good target gene to control N. lugens by plant-mediated RNAi.Previous studies demonstrated that EcR(Ecdysone receptor) and TPS (Trehalose phosphate synthase) play important role in the development of N. lugens. In addition to the gene173(encoding vacuolar ATPase subnite D), we also chose EcR and TPS as our target genes to construct the RNAi vectors for production of hpRN As of the target genes. The vectors were introduced into rice by Agrobacterium-mediated transformation method. The transgenic rice plants were found to produce dsRNAs and siRNAs of the target genes. When nymphs were fed on transgenic rice plants expressing the dsRNA, the transcript levels of the target genes were found to be reduced, and the nymph dutation and period of generation cycle were significantly prolonged. However, feeding on the dsRNA transgenic rice did not result to any lethal phenotype, nor the weight and survival rate were not affected. We further studied the RNAi effects on3rd generation N. lugens feeding on transgenic rice plants. The1st instar nymph duration,5instar nymph duration and the whole nymph dutation were significantly prolonged. However, the transcript levels of the target genes in insects were still suppresed. The reproduction ability of brachyptery female adult, weight and survival rate were not affected by transgenic rice plants. It is still not efficient to use the plant-mediated RNAi strategy for controlling N. lugens.Rice is a critical food crop of the world, and lepidopteran pests, such as striped stem borers Chilo suppressalis (Walker), Tryporyza incertulas (walker) and rice leaf folders Cnaphalocrocis medinalis (Guenee), were it major pests. Bt transgenic rice events have been successfully developed to control pest. However, commercial planting of Bt rice has been delayed by the concern over food safety. We have obtained the transgenic insect resistant rice S21to address the concerns of food safety and the development of insect resistance. The line S21was constructed with green tissue-specific promoter to direct the Cry1Ac/Cry1I-like fusion gene. In this study, we further evaluated the insecticidal activity of line S21.Laboratory and field bioassay demonstrated that the S21were highly active against the striped stem borer C. suppressalis and the rice leaf folder C. medinalis. While the Xiushui-134plants were infested by C. suppressalis at8.8%and by C. medinalis40.5%, the S21plants were completely undamaged by both C. suppressalis and C. medinalis under field conditions in2013. ELISA results indicated that the fusion Bt protein was specifically expressed in green tissues but not in seeds. The Bt fusion protein was at670-1160ng per gram of fresh stems and at1050-1510ng per gram fresh leaves, but less than10ng per gram in seeds. Our study suggested that S21has great potential for commercial planting.