Cloning And Procaryotic Expression Of GroEL Genes Of Endosymbiotic Bacterium From Schizaphis Graminum And Rhopalosiphum Maize Yangling Biotype

Aphid is the most important insect vector transmitting plant virus.Many virusdiseases of crop, vegetable and economic crop transmitted by aphid cause severe economicloss. Control on aphid-borne virus diseases may be wishful and have new methods if wefully understand the molecular mechanism of aphid transmission. Previous work about aphid transmission have identified and analyzed a lot of molecular determinants from plant virus. However, the aphid vector factors involved in aphid transmission only have a few reports. Buchnera GroEL of aphid is a protein involved in virus circulative transmission and paid much attention to.Our study designed two pairs of specific primers and cloned two molecular weight of 63ku from Schizaphis graminum, and Rhopalosiphum maize utilizing PCR technique and have done the prokaryotic expression, the main results are as follows:(1)Amplified the Buchnera groEL of Schizaphis graminum, and Rhopalosiphum maize Yangling Biotype at first time all over the world ,and sequenced the full lenghth gene.Furthermore,we compared the related nucleotide of ten homogenous genes.The two Buchnera groEL gene of Schizaphis graminum and Rhopalosiphum maize are all 1647bp.Their GenBank accession numbers are AF3 87863 and AF434719.The deduced amino acid sequences indicated the Buchnera groEL encode 548 amino acids.By compared the nucleotides and the deduced amino acid we protract their evolution tree and found the relative of Buchnera spp is related not only to the relative of the vector,but also to the vectors'biotype.(2) The cloned Buchnera groEL genes of Schizaphis graminum and Rhopalosiphum maize were ligated into pBV221,pET30a and pTrcHisA expression vector for construction of pBVRM,pBVSG, pETRM,pETSGPR,pTrcRM and pTrcSG. They were transformated into E. coli BL21(DE3),DH5a and JM109 for studying their expression. The SDS-PAGE electrophoresis results suggested that pBVRM and pBVSG containing aim gene can express 63 ku aim proteins, but have lower expression quantities;pETRM and pETSGfusion expression vectors can promote aim gene expression in high levels when induced by IPTG.The fusion proteins expressed have a molecular weight of 69 ku, expression quantities were higher and highest when induced by IPTG for 4h. Vector pTrcHisA could express aim fusion proteins whose MW is 69ku,too,but the product of pTrcSG and pTrcRM is the lowest in the three prokaryotic expression vectors by different receptor cell,sometimes even they can't be detected by SDS-PAGE.(3)Purified the fusion protein pETSG GroEL and immuned the rabbit to prepare for the antibody of the GroEL.(4)The Western blot indicated the prokaryotic expression is successful and the fusionand non-fusion protein are homogenous.We gained the Buchnera groEL gene of Schizaphis graminum, and Rhopalosiphummaize biotype through this study , and have done a primary study of expression. Based on this study, we can further probe the role during aphid transmission of Buchnera GroEL and utilize them through the ways of genetic engineering.