Cloning And Genetic Transformation Of A. Annua EβF Synthetase Gene And Ecdysone Receptor Genes Of USP And EcR Of Grain Aphid (Sitobion Avenae F.)

As a major pest of wheat, aphid makes heavy damage to production and quality ofwheat every year as well as acting as the major transmitter of virus in wheat. In this study,the EβF ((E)-β-farnesene) synthetase gene from Artemisia annua referred to HHH wascloned and subjected to expression vector generation and wheat transformation viaagrobacterium tumafeciens. In addition, two hormone receptor genes in grain aphiddenoted as EcR and USP were cloned. The synthesized double strand RNA ofaforementioned genes (EcR and USP) were used to feed the aphid.Their roles in the aphidthrough RNAi interference as well as the feasibility in controlling the wheat aphid byregulating aforementioned genes were evaluated. The main results are as follows.1. The EβF synthetase gene (HHH) was cloned from Artemisia annua by RT-PCR.Based DNA recombinant technology, the expression vector referred to185HHH-167Barthat fused this synthetase gene was constructed.2. Using the hexaploid wheat KN199as the material,7transgenic wheat linesoverexpression HHH were generated based on the transgenic approach via agrobacteriumtumafeciens. The7lines referred to K01-1-1, K01-3-5, K01-1-6, K02-1-4, K02-5-4,KHH-1-5and KH-5, all of these lines had Bar resistance. There were another5linesdenoted as K01-3-6, K02-2-5, K02-3-4, K03-1-1and K06-3-4which expressed Bar genebut without identification of HHH expression. The functions and the expression patterns ofHHH in the transgenic wheat lines were investigated. These lines provide a basis forfurther understanding the roles of HHH in regulation of aphid growth and the effects onaphid’s predators.3. Accoding to the conserved sequences, the specific primers for cloning of thehormone receptor genes in peach aphid and pea aphid referred to USP and EcR weredesigned. Using the cDNA as the template, the double strand RNA (dsRNA) of USP andEcR was synthesized using the corresponding kit.4. The dsRNA of USP and EcR was separately used to feed aphids, in which thesalivary gland expressing gene designated COO2that largely interfere aphid growth and development as well as induce aphid’ death was used positive control, and the greenfluorescence gene (GFP) was used as negative control. The results indicated that thedsRNA fed with7.5ng/μL behaved dramatic roles in controlling aphids, with similareffects to COO2in inducing aphid’ death. Therefore, the results in this study confirm thatUSP and EcR could act as important genes in regulating aphid’s growth and development.5. After feedings of2d,4d,6d and8d by the dsRNA of USP and EcR, theexpression levels of USP, EcR and COO2in aphids were investgated by q-RT PCR. Theresults showed that the expression levels of aforementioned genes after feeding treatmentwere significantly down-regulated. These expression results of tested genes withdown-regulated patterns were in consistent with the dramatically elevation of aphids’death.