Cultivating Striped Stemborer Resistance Transgenic Rice Using RNA Interference Technology And Developing Full-length CDNA PET Library

As the staple food for over half of the world population,rice is one of the most important food crops.Stablitiy of rice production maintains the food safety and social peace.During almost the whole growth period of rice,the yield is affected by various pests,and the striped stemborer(SSB,Chilo suppressalis)is one of the most important insect pests.In last decades,the application of BT transgenic crops reduces the economic losses and protects the ecological environment by effectively managing some major pests.It is worthwhile to note that,the BT-resistance pest populations have appeared under the laboratory conditions.Therefore,it is important to explore new strategies for pest controlling.In the last two decades,RNAi technology has gradually become the most promising strategy for pest controlling,that is potentially comparable with BT technology.In this study,we attempted to apply the RNAi strategy for rice pest management.The main results of this study are as follows:1.A comphresive transcriptome database(RPTDB,http://rptdb.hzau.edu.cn)consisting of the transcriptome data of eight main rice pests was organized and constructed.RPTDB aims at providing well-organized information for worldwide researchers who require genetic materials for insect functional research,pest management or others,and thus filled the genetic information lacks.And it has average 25 unique visitors per day in last 90 days.2.Artificial dsRNAs of 22 candidate targets were designed and synthesized,and then tested by the diet-based feeding test.Six dsRNAs(dsCSE,dsGLC,dsACD,dsTAS,dsVAS and xse7)showed application potential in pest management based on the feeding test.A total of 31 dsRNAs expression vectors were constructed and introduced into Japonica rice,ZH11,however none of the transgenic rice showed stable insect resistance.3.We assessed the SSB resistance ability of miRNA mediated transgenic rice line csu-15.Both short-term and consecutive feeding tests indicated that csu-15 exerted influences on growth of SSB.Larvae fed on stemcutting of csu-15 line for 7d,and their total RNAs of midgut issue were isolated for differential expression analysis by RNA-seq.We found 33 significant differential expression unigenes,and one of them was predicted to have a target side of miRNA csu-novel-15.4.A full-length cDNA PET library construction and data analysis pepline was developed for rapid gene annotation of a newly sequenced species and further insects' genome annotations.Total RNA from the leaf of the rice variety Nipponbare was used for the library construction and the evaluation of the full-length cDNA PET library construction and data analysis pepline.Rice full-length cDNA database KOME and wide-used annotation database RARG 7 were used to evaluate the efficiency of our approach.At least 75.1%non-redundant reads could map to KOME clones,for those concordance mappings,the distances of 73.3%TSSs' 0-bases with corresponding KOME clones' 0-base below 6.The results reveal that our method shows high authenticity and accuracy.