Studies On The Cloning And Functional Analysis Of Pdp1and Slc35b1in Ostrinia Furnacalis (Lepidoptera:Crambidae)

The Ostrinia furnacalis(Asian corn borer, ACB) is a major pest in agriculture which feeds on corn or sorghum crops, causing serious economic losses.The defects of the traditional control methods in controlling ACB makes it a inevitable trend to seeking environmentally friendly strategys,meanwhile transgenic technology offers possibility for genetic control of ACB. The key to build conditional gene lethalgenetic system of ACB is to seek for target genes under functional verification of individual level, which can play a conditional lethal function in ACB finally. Meanwhile it is significant in genetic control of ACB to seek for key genes which take important parts in egg stage and larva stage,then to prevent the growth and development of ACB before they feed and harm. It’s still blank currently to study the target genes which can play a lethal effect. In this study, cDNA sequences of Ofpdpl [PAR(Protease-activated receptor)-Domain Protein1], Ofslc35b1[homolog of human solute carrier family35member B1(UDP-galactose transporter-related protein1)] and Ofhsc70(heat shock cognate70) was successfully cloned from eggs of ACB, with subsequent research of mRNA expression characteristics and possible physiological function in the process of growth and development. The results are as follows.1. the cDNA sequence (960bp) of Ofpdpl was obtained using RT-PCR and PCR methods, including a complete open reading frame (ORF) of804bp, which encoded267amino acids. A search of the protein database showed that OfPDP1was a transcription factor of PAR bZIP (basic leucine zipper) subfamily which included several conserved regions in the carboxy terminal region of the protein. The result of phylogenetic analysis was consistent with the classification of the species in biology. The qPCR results showed a high level expression at the76h of eggs’development process, which suggested that Ofpdpl could take an important part in the changing process of embryo physiological structure during the larva’s forming period. It could be declined by71%in the transcript levels of Ofpdpl in the eggs injected with Ofpdp1-specific dsRNA in48h compared to those in the control eggs.2.(1) the cDNA sequence (1472bp) of Ofslc35b1was also obtained using RT-PCR and PCR methods, with an ORF encoded258amino acids. A search of the protein database showed that OfSLC35B1with conserved evolution relationship was a member of human UDP-galactose transporter-related protein SLC35B1.The structure prediction of hypothetic amino acid sequence shows that: OfSLC35B1was located in the cytoplasmic membrane mostprobably;amino acids locaded in NO.10-258was a typical UAA domain in nucleotide-sugar transporter; there are seven hydrophobic transmembrane region in existing OfSLC35B1sequence, figureing out eight in whole OfSLC35Bl.(2) the result of qPCR indicated that:the mRNA expression profile of Ofslc35bl in the early stage of eggs was higher than late stage, and expression levels would rised up in1st larva stage and5th larva stage,together it showed that Ofslc35bl may be required for the maintenance of homeostasis in insect;expression of Ofslc35bl and Ofhsc70were increased,which were seemed undergoing ER stress conditions caused by tunicamycin at16h after injection into eggs.(3) in addition,Injecting dsSLC35B1-1into eggs at blastocyst stage sucessfully inhibited the mRNA expression level of Ofslc35b1(62%down), meanwhile the hatching rate decreased by about10%;dsSLC35B1-1feeding in the larval stage hasn’t caused growth abnormalities,but the mRNA expression level of Ofslc35bl was still interfered significantly(55%down).However, after RNAi of Ofslc35bl at egg stage or larvae stage,the mRNA expression level of Ofhsc70hasn’t showed an up trend, which may attributed to that dsSLC35B1-1treatment hasn’t cause endoplasmic reticulum stress in Asian corn borer.The results above shows that:the expression of target gene could be suppressed significant after dsRNA injection (dsPDP1or dsSLC35B1) in egg stage or dsRNA feeding (dsSLC35B1) in larva stage; however, the hatching rate of eggs could be declined only for Ofslc35bl(about10%) after dsSLC35Bl injection in egg stage. It’s most likely becaused that RNAi would not completely eliminate the expression of genes, and could only caurse the partly reduction of the genes, while the remnants of gene expression activity was sufficient to maintain normal growth and development of ACB. Therefore, we need to try other methods to eliminate the expression of Ofpdpl or Ofslc35bl completely, and to observe the growth and development of the Asian corn borer.