Cloning And Expression Analysis Of V-atpase Subunit A、B、B And H Gene From Helicoverpa Armigera（hübner）

V-ATPase hydrolyzes ATP to generate energy, transporting H+in lumento cells, which would maintain of alkaline environment in lumen. Then the V-ATPaseplays an important role in growth, development and reproduction of insects. Forexample, maize root worm (Diabrotica virgifera virgifera) feed dsRNA of V-ATPasesubunit A gene, the mortality of pests increased. Silencing V-ATPase subunit H geneof Locusta migratoria could lead to the death of individuals and various moltingdefects. Those practical researches attract us more attention on molecular structure andfunction of V-ATPase subunits. In this experiment, selecting cotton bollworm(Helicoverpa armigera) as the target pest, we cloned the V-ATPase subunit A、 B、 Cand H gene in cotton bollworm, and preliminarily analysed the relative expressionlevel of V-ATPase subunit A、B、C and H gene in different instars of cotton bollworm.The main results were as follows:（1）The full-length cDNAs-encoding V-ATPase subunit A、B、C and H werecloned by RT-PCR technique from cotton bollworm. The length of subunit A openreading frame was1863bp which encoded a protein of621amino acid residues, thepredicted molecular weight and isoelectric point were68kD and4.92. Respectively bythe phylogenetic tree analysis and sequence homology analysis, cotton bollwormV-ATPase subunit A gene has a close phylogenetic relationship with silkworm’s(Bombyx mori), both amino acid sequences had the highest homology, the homologywas about96%. The length of subunit B open reading frame was1485bp whichencoded a protein of495amino acid residues, the predicted molecular weight andisoelectric point were55kD and4.98. Respectively by the phylogenetic tree analysisand sequence homology analysis, cotton bollworm V-ATPase subunit B gene has aclose phylogenetic relationship with Heliothis virescens’s, both amino acid sequenceshad the highest homology, the homology was about99%. The length of subunit Copen reading frame was1155bp which encoded a protein of385amino acid residues,the predicted molecular weight and isoelectric point were44kD and8.66.Respectively by the phylogenetic tree analysis and sequence homology analysis,cotton bollworm V-ATPase subunit C gene has a close phylogenetic relationship withtobacco hornworm’s (Manduca sexta), both amino acid sequences had the highest homology, the homology was about87%. The length of subunit H open reading framewas1428bp which encoded a protein of476amino acid residues, the predictedmolecular weight and isoelectric point were55kD and6.80. Respectively by thephylogenetic tree analysis and sequence homology analysis, cotton bollwormV-ATPase subunit H gene has a close phylogenetic relationship with tobaccohornworm’s and silkworm’s, those amino acid sequences had the highest homology,the homology was about93%. It is indicated that each gene of subunit A、B、C and Hfrom cotton bollworm has much closer phylogenetic relationships with other insects’,both amino acid sequence homology are also higher.（2）The result of real-time PCR show that the expression level of V-ATPasesubunit A、B、C and H gene are different in every cotton bollworm developmentstages, and the relative expression level of each subunit in different instars existssignificant difference. The expression level of subunit A in1instar larvae is thehighest, and then is in adult stage, in pupal the expression level is the lowest. Theexpression level of subunit B in adult stage is the highest, and then is in1instar larvae,in male pupal the expression level is the lowest. The relative expression trends ofV-ATPase subunit A、B gene are roughly same in every cotton bollworm developmentstages, showing that structure and function of A、B subunits are closely linked。 Theexpression level of subunit C from high to low is6instar、male adult、5instar、femaleadult、1instar、4instar、egg、2instar、female pupal、3instar、male pupal. Theexpression level of subunit H from high to low is male adult、6instar、female adult、1instar、4instar、5instar、2instar、female pupal、3instar、egg、male pupal. Therelative expression trends of V-ATPase subunit C、H gene are roughly same in everycotton bollworm development stages, showing that structure and function of C、Hsubunits are closely linked.The above results will provide reference for subsequent interference research. Ifit would silent target genes of V-ATPase, and the death or deformity rate of cottonbollworm would rise, the RNAi-based insect management strategies would be a goodmeasure, beneficial to delay the pest resistance.