Developing Striped Stemborer Resistance Genetically Modified Rice By Using RNA Interference Technology

Stripped stem borer(Chilo Suppressalis)is one of the most destructive pests of rice.Yield loss caused by C.Suppressalis and expense due to pest prevention lead to huge economic loss annunally.Although pesticides and Bt rice can be adopted to manage C.Suppressalis,but C.Suppressalis may evolve the resistance in a short period.To address this issue,finding new ways against this insect is a long-term that have to be faced in rice production.With the development of RNA interference(RNAi)technology,RNAi strategy have been widely used to develop insect-resistant transgenic plant and become the most promising new method for insect managment technique.So far,many reports have confirmed that RNAi can be induced in some insect species by dsRNAs or miRNAs delived by transgenic plants.In this study,five C.Suppressalis target genes have been determined by in vitro dsRNA feeding assay in our previous studies.Fifteen dsRNA/miRNA expression vectors were constructed according to the cDNA sequences of these five target genes.All the 15 RNAi vectors were introduced into rice,and the insect resistance of acquired transgnenic rice plants were identified.We found that only RNAi transgenic plants targeting VATP enzyme exhibited siginifcant insect resistance.Several independent experiments confirmed that insect resistance of VATP RNAi rice plants is stable.After feeding on VATP RNAi rice plants,C.Suppressalis larvae exhibited less average body weight and lower average growth rates compared with that feeding on non-transgenic rice control.The average worm weight can be reduced 50.8% ~ 29.4% compared with the control,and the growth rate can be reduced 17.2% ~ 18.1% compared to the control.Finally,the average growth period of C.Suppressalis feeding on VATP RNAi rice was extended 2.3 to 3.3 days compared with the control feeding on non-transgenic rice.Moreover,this RNAi effects seemed to be passed from the parents to the next generation.Our feeding assay showed that the offspring derived form C.Suppressalis that feeding on RNAi rice plants still showed slower body weight incress,even they fed on non-transgenic wildtype rice as soon as they were hatched.Small RNA samples of C.Suppressalis collected form six different dvelopmental stages were sequenced by using high-throughput sequencing technology to identify C.Suppressalis small RNAs.By bioinformatic analysis,many conserved small RNAs were found in C.Suppressalis.Moreover,104 C.Suppressalis-specific unique novel miRNAs were identified,of which 57 novel mirRNAs seem to expresse in midgut tissue.All of these 104 were validated by stem loop RT-PCR,qPCR and sequencing.Five C.Suppressalis novel miRNAs that may express in the midgut were selected to construct artificial miRNA interference vectors.These amiRNA vectots were then introduced into rice,and the insect resistance of the acquired transgenic rice plants was identified.The test results of insect resistnace showed that there are two of five amiRNA interference vectors had RNAi effects,and transgenic rice plants contained these two vectors affected the growth and development of C.Suppressalis larvae.Similarly,C.Suppressalis larvae feeding on transgenic rice plant exhibited less average body weight,lower growth rate,and extened gowth period.This strategy has not been reported in previous studies,but our research demonstrated that this might be a new effective way to constrcut RNAi vector for developing insect resistant transgenic plants.In this study,two strategies weres used to designed RNAi vectors: targeting C.Suppressalis endogenous important genes or using C.Suppressalis endogenous small RNAs.Although in plant cells,RNAi mediated by miRNAs seems not as effective as that mediated by dsRNAs,but transgenic rice expressing miRNAs showed a overall better insect-resistance compared with that expressing dsRNAs.