Amplification, Expression Analysis And RNA Interfernce Of Bt Receptors Genes In Chilo Suppressalis

Bacillus thuringiensis (Bt) is a member of the Bacillus cereus group of bacteria. One important characteristic of Bt strains is that they produce insecticidal d-endotoxin proteins which are highly specific to their target insects. Nowadys, Bt toxin has been widely used in pest control.Little has been known about the mechanism the Bt toxins. There are two thypothesis: One is pore formation, the other is signal transduction. The receptors genes of Bt toxin in C. suppressalis were sledom reported. In this work, we searched the trasncripts of four kinds of receptors genes1. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in C. suppressalisHousing keeping genes (HKG) are often empirically chosen as the reference genes in quantitative real-time polymerase chain reaction (qRT-PCR). However, the suitability of HKGs used as reference genes has been seldom validated. We evaluated the expression stability of five HKGs (actin Al, GAPDH, G3PDH, E2F, rp49) in C. suppressalis. The algorithms of geNorm, Normfinder, stability index and△Ct analysis were used to estimate the expression stability of selected HKGs. In all tested samples and different tissues, the expression of actin Al was the most stable C. suppressalis. In different developmental stage, E2F and G3PDH are the best choice.2. Gene clone and transcriptions analysis of Cry protein receptor in C. suppressalisThere are four classes of Bt recepotor genes. The cDNA fragments of all genes in C. suppressalis were amplified.Ten genes include one cadherin gene; two aminopeptidase N genes, three alkaline phosphatase genes and four Glycosyltransferase genes were obtained. The molcular trees were constructed for all four kinds of Bt receptors genes. The phylogentic analysis indicated that CAD1is similar with the cadherin of Lepidoptera whereas CAD2is similar with those of Diptera. 3. Expression analysis of Bt receptors genes in C. suppressalisThe genes expressions levels of four kinds of Bt receptors genes were detected in the midgut of the1st,2nd,3rd,4th,5th,6th larver and adult. The resutls showed that CAD1, CAD2, ALP1and ALP2had the silmilar expression changes in difference development stages. Their mRNA levels increased from the2nd instar but decreased in the adult. The expression of APN genes had little changes in the larvae stages. The expression of GTl and GT2are low in adult stage. The expression of GT3is little change in all stages.4. RNA interference of Cadherin (CAD) in C. suppressalisRNAi technology was used to silence two CAD genes in the rice stem borer. The gene functions of two CAD genes in C suppressalis were analyzed. Real time-PCR shows that the mRNA abundance of treated insects was reduced by40%at two days after feeding on siRNA. The mortalities of siRNA-treated insects significantly decreased, suggesting that these downregultion of CAD genes confers the Bt resistnace...