Protokaryotic Expression And Genetic Transformation Of 2 Osvdac Genes In Rice

Voltage-dependent anion channel (VDAC) is a kind of ion channel protein, which is encoded by a small multi-gene family, They primarily depend on voltage to regulate the selectivity of negative and positive ions. VDACs locate mainly in the mitochondrial membrane, It is not only plays a decisive role in energy metabolism and mitochondrial metabolism, but also are in relation to induced apoptosis.Previous studies in our laboratory has mapped eight vdac candidate genes on rice genome by whole Nipponbare and 9311 genome scanning with different bioinformatics tools. Compared with three vdac genes identified, why there are eight vdac genes existed in rice genome is worthy to probe for their struction, location and biologic function.In this article, two ORFs for vdac3 and vdac5 genes are cloned, and their efficient prokaryotic expression and protein purification conditions are optimized in order to prepare for their antibodies. The two plant expression vectors with fused ORFs-GFP, pET-30a-osvdac3 and pET-30a-osvdac5 were constructed and then genetic transformation were carried out. The main results are as follows.1. Two ORFs for vdac3 and vdac5 genes were obtained from cDNA of YTB by PCR with the specific PCR primers. The recombinant prokaryotic expression vector pET-30a-osvdac3 and pET-30a-osvdac5 were constructed by inserting the vdac genes into a prokaryotic expression vector pET-30a with a His tag, and transformed into competent cell E.coli BL21. The optimal cultivting conditions for inducing the expression of two fused recombination proteins were at 15℃for 17 hours with 0.7 mmol/L IPTG (isopropy-β-D-thiogalactoside). The target proteins were further isolated and purified through affinity chromatography, and dected as a single band by SDS-PAGE. The N side sequence analysis by the mass spectrometry coincided with the expected expression products of the two genes, and teir antibodies were then prepared. Western Blotting indicated that these two antibodies had better specificity.2. CaMV35S∷osvdac3∷GFP and CaMV35S∷osvdac5∷GFP expression vectors were also constructed and transformed into rice line YTA. Some positive transgenic plants of YTA were obtained, which can be further used for cellular and tissues location of VDAC3 and VDAC5 proteins.