Spatiotemporal Expression And Analysis Of The Subunits Of V-ATPase V₁ Domain In Helicoverpa Armigera ?H???bner?

Vacuolar-type proton ATPase plays an important role in the growth and development of insects.V-ATPase mainly exists in the plasma membranes of epithelial cells such as martensitic duct,midgut and salivary gland in insects.It transports K+or Na+ outside the cells by coupling K+/2H+reverse transport vectors to promote the absorption of nutrients and provides energy for various life activities.The V-ATPase on the apical membrane of midgut goblet cells of lepidoptera insects creates an alkaline environment for midgut intestinal cavity through the transport of H?,so that the alkaline hydrolyzed protein of midgut can function normally.In this experiment,we cloned the V-ATPase E and G subunits gene in Helicoverpa armigera(Hubner).Real-time PCR was used to determine the expression levels of subunits in the VI domain of V-ATPase at different development stages and in different tissues of H.armigera,and the effect of sublethal dose of chlorarnphenicol on its expression level.The main results were as follows:(1)The open reading frame(ORF)region encoding the V-ATPase E subunit and the G subunit of H.armigera was cloned by PCR amplification with specific primer.The length of E subunit ORF is 681bp which encod a protein of 226 amino acid residues.The predicted molecular weight and theoretical pI are 26.1KD and 8.30,respectively.According to the homology analysis of amino acid sequences and phylogenetic tree based on amino acid sequences,V-ATPase E subunit is a conservative subunit.The first 70 amino acid residues of V-ATPase E subunit are completely conserved.And the homology with Spocloptera litura and Trichoplusia ni are as high as 92%and 91%,respectively.The length of G subunit ORF is 354 bp which encod a protein of 117 amino acid residues.The predicted molecular weight and theoretical pI are 13.7 KD and 9.4,respectively.According to the homology analysis of amino acid sequences and phylogenetic tree based on amino acid sequences,V-ATPase E subunit is a conservative subunit,and the amino acid sequence mainly has few amino acid differences at the C-terminus.And the homology with Spodoptera litura and Trichoplusia ni are as high as 96%and 95%,respectively.(2)The results of gene expression analysis of subunits of V-ATPase V1 domain in different developmental stages of H.armigera showed that 8 subunits of V-ATPase V1 domain are expressed in the whole development stage of H.armigera,and the expression levels are different.A subunit has the highest expression in the female moth,and the male pupae and female pupae are the lowest.B subunit has the highest expression in the 5 instar larvae,and the male pupae and female pupae are the lowest.C subunit has the highest expression in the 5 instar larvae and 3 instar larvae,and the male pupae and female pupae are the lowest.D subunit has the highest expression in the 5 instar larvae,and the male pupae and female pupae are the lowest.E subunit has the highest expression in the 5 instar larvae,and the male pupae and female pupae are the lowest.F subunit has the highest expression in the 4 instar larvae,and the male pupae and eggs are the lowest.G subunit has the highest expression in the 2 instar larvae,and the eggs,male pupae and female pupae are the lowest.H subunit has the highest expression in the 2 instar larvae,and the eggs,male pupae and female pupae are the lowest.(3)The results of gene expression analysis of subunits of V-ATPase V1 domain in different tissues of H.armigera showed that 8 subunits of V-ATPase Vi domain are expressed in the different tissues of H.armigera,and the expression levels are different.A subunit has the highest expression in the heads of female moths and male moths,and the expression in the wing of female moth is the lowest.B subunit has the highest expression level in the antennae of the male moth,and the expression in the abdomen of female moth is the lowest.C.subunit has the highest expression level in the midgut,and the expression in the abdomen of female ruoth is the lowest.D subunit has the highest expression level in the midgut,and the expression in the abdomen of female moth is the lowest.E subunit has the highest expression level in the midgut,and the expression in the abdomen of the female moth is the lowest.F subunit has the highest expression level in the antennae of female moth,and the expression in the abdomen of the female mothis the lowest.G subunit has the highest expression level in the midgut,and the expression in the abdomen of the female moth is the lowest.H subunit has the highest expression level in the chest of female moth,and the expression in the abdomen of the female moth is the lowest.(4)After LD5o treated with chlorantraniliprole,the expression levels of V-ATPase V1 domain subunits were as follows:the expression of A,B and G subunits are increased;the expression of C,D,E,F and H subunits are decreased.There are significant differences in subunits A,B and E between control and treatruent.The above results indicate that V-ATPase does play an important role in the growth,development and reproduction process of H.armigera.If a certain subunit gene of V-ATPase in H.armigera can be knocked out to cause the increase of mortality and abnormality rate or the decrease of fecundity.Then it is a good method to use genetic editing technology to find new target genes to control lepidoptera pests such as H.armigera.