Host-mediated RNAi To Control Aphids

Aphid, one of the major harmful homopterous insects in agricultural forestry,having strong fertility and wide range of distribution, can directly cause serious damageto economic plants like crops, fruit trees etc., and spread a variety of plant viruses. Sofar, methods of control the aphids mainly depend on chemical pesticide, which mayeasily result in serious contamination of environment and agricultural by-products, andendow the aphid with ability of drug resistance. With the enhancement of people’scognition of environmental protection and quality of life, more safe, durable andefficient ways to control aphids have been obtained much more attention.Gene PCOO2is a very important protein gene secreted by the pea aphid salivarygland, which is essential in the process of aphids feeding on the host plant. PCOO2protein has a secretion signal in its N-terminal, which has not any homology in otherspecies. Navdeep S. Mutti injected siRNA which can cover most of ORFs of PCOO2gene into pea aphids. Level of PCOO2transcription decreased significantly in the thirdday of injection, the deaths by half of the pea aphids demonstrated that Gene PCOO2isessential to pea aphid.This study found out the gene sequence of pea aphid via GenBank.The APCOO2was cloned from Myzus persicae by Homology-based cloning method, which contained525bp, and then constructed the silenced vector pBIN19：：APCOO2i.Through planttransgenic technology, cultivated transgenetic plants that can produce siRNA of targetgene, and then fed the Myzus persicae with transgenetic tomato plant, observed theexpression of target gene in aphid. Attempts to use transgenetic plants to mediate RNAito achieve the goal of controlling the aphid safely, durably and efficiently have greatcreativity in science, and practical value in agricultural production. Main methods andresults are as following:①Cloning of Myzus persicae APCOO2geneUse the total RNA of Myzus persicae as templates to syntheses cDNA via reversetranscription in vitro. Use cDNA as the templates, through PCR amplification to get asequence of525bp, named as APCOO2. Biological information analysis through NCBIBLAST manifested that this gene sequence has an85-percent similarity with pea aphidPCOO2.②Construction of silenced vector pBIN19：：APCOO2i Utilized Myzus persicae life essential gene APCOO2to.construct a silence vectorof pBIN19：：APCOO2i. Firstly, respectively introduced two pairs of restriction sites（Hind III&Xba I, Kpn I&Xho I）to the gene fragment, then constructed anintermediate vector pHANNIBAL-APCOO2i, and then the fragment including a35Spromoter, APCOO2gene, and a35S terminator was cut and. ligated to the expressionvector pBIN19to construct of Myzus persicae silenced vector pBIN19：：APCOO2i.③The cultivation of transgenic tomato plantsCotyledons of tomato AC++were used as explants and to be infected byAgrobacterium tumefaciens, which transformed the expression vector pBIN19：：APCOO2i regulated by CaMV35S promoter into tomato. Through the resistancemedium As, we selected the screening induced calluses, and then we put these callus onbuds inducing culture medium to induce buds, some regenerated tomatoes have beenobtained after screened by50mg/L Kan and250mg/L Carb.④Expression levels of APCOO2Total RNA was isolated from2th and4th instar nymphs，then it was used forpolymerase chain reaction，the2th instar nymphs is twice the levels of expression of the4th instar nymphs.