Prokaryotic Expression Of DsRNA Of V-ATPaseA,AK2 And HR3 And Its Effects On Growth And Development Of Lymantria Dispar

Lymantria dispar is an international defoliator pest,which has the characteristics of plant hosts,strong vitality and extensive damage.It seriously threatens the growth of plants and causes huge economic and ecological losses in China.Vacuolar-type proton ATPase（V-ATPase）is a cationic ATPasethat transports eukaryotic cells,existing widely in the inner membrane system of protoplasmic membrane and organelles,which affects the physiological activities such as molting,reproduction,growth and development of insects.Adenylate Kinase 2（AK2）plays a key role in adenine nucleotide metabolism in mitochondrial intermembrane space and affects insect growth and development.Hormone Receptor 3（HR3）,as a molting transcription factor,regulates insect ecdysis,reproduction,growth and development.In this study,Lymantria dispar was used as target insect to clone the full length of c DNA of V-ATPaseA,AK2 and HR3genes.The spatio-temporal expression patterns of V-ATPaseA,AK2 and HR3 genes were analyzed by quantitative RT-PCR.The prokaryotic expression vectors were successfully constructed and transformed into HT115（DE3）strain of Escherichia coli,and the expression of dsRNA was successfully induced by IPTG.The effects of gene silencing on the growth and metagenesis of L.dispar larvae were investigated by feeding diets containing Escherichia coli expressing dsRNA of V-ATPaseA,AK2 and HR3 genes.The results are as follows:1.LdV-ATPaseA,LdAK2 and LdHR3 gene sequences were obtained from Lymantria dispar transcriptome.The open reading frame（ORF）of LdV-ATPaseA,LdAK2 and LdHR3gene was 1851 bp,1473 bp and 729 bp,encoding was 616,490 and 242 amino acids,respectively.Their protein molecular weights were 68.04 kDa,26.91 kDa and 55.23 kDa with5.24,8.64 and 6.98 of theoretical isoelectric points（p I）,respectively.LdV-ATPaseA and LdHR3 were alkaline proteins,and LdAK2 was acidic protein.There were no signal peptides and transmembrane domains in the three proteins.2.The expressions of LdV-ATPaseA,LdAK2 and LdHR3 gene in different developmental stages and tissues were quantitatively analyzed by quantitative RT-PCR.The results showed that LdV-ATPaseA,LdAK2 and LdHR3 genes were expressed in all developmental stages,and the expression levels in female pupae were 1.81-,6.63-and 19.25-fold of that in control egg stage,respectively.It is speculated that LdV-ATPaseA,LdAK2 and LdHR3 genes maybe participate in the reproductive function of L.dispar.The expression levels of LdV-ATPaseA,LdAK2 and LdHR3 genes were specific in different tissues.The expression levels of LdV-ATPaseA and LdAK2 in testis were 30.80-and 15.49-fold of that in fat body,respectively.The expression levels of LdHR3 in epidermis were 22.98-fold of that in the control fat body.The expression levels of the three genes in the intestinal tract and epidermis were relatively high,which might affect the feeding and molting of L.dispar.3.The prokaryotic expression vectors were successfully constructed and transformed into HT115（DE3）strain of Escherichia coli,and expressed dsRNA corresponding three genes was induced by IPTG.The 2^nd and 3^rd instar L.dispar larvae were fed with dsRNA of three genes respectively,and the silencing efficiency of the three genes was analyzed.The results showed that the silencing efficiency of LdV-ATPaseA,LdAK2 and LdHR3 gene was 96.24%～98.35%,27.06%～85.73%and 62.15%～95.64%in the 2^nd instar larval stage.In the 3^rd instar larval stage,the silencing efficiency of LdV-ATPaseA,LdAK2 and LdHR3 gene was 39.71%～63.97%,33.63%～91.46%,and 61.81%～95.63%.These results indicated that the m RNA expression levels of target genes in the 2^nd and 3^rd instar larvae were significantly lower than those in the control,suggesting that feeding prokaryotic dsRNA diets could effectively silence LdV-ATPaseA,LdAK2 and LdHR3 genes.4.Silencing LdV-ATPaseA,LdAK2 and LdHR3 genes affects the growth and development of L.dispar.Consumption rate,body weight and the cumulative growth rate of body weight were significantly reduced by feeding dsRNA of LdV-ATPaseA,LdAK2 and LdHR3.Feeding dsRNA from the 2^nd and 3^rd instar larval stages could effectively reduce the weight of female L.dispar pupae and feeding dsLdAK2 and dsLdHR3 could significantly reduce the weight of male pupae.The mortality of L.dispar larvae was significantly increased through feeding dsLdV-ATPaseA,dsLdAK2 and dsLdHR3 from the 2^nd and 3^rd instar larval stage.LdV-ATPaseA,LdAK2 and LdHR3 genes were silenced from the 2^nd instar larval stage,and the malformation rate was significantly increased.The malformation rate of feeding dsLdHR3 was significantly increased from the 3^rd instar larval stage,and the molting defect emerged.The pupation rate of L.dispar was significantly reduced through feeding dsLdV-ATPaseA and dsLdHR3 from the 2^ndinstar stage,and LdAK2 gene silencing from the 3^rd instar stage significantly decreased pupation rate.Gene silencing from the 2^nd and 3^rd instar larval stages significantly reduced the number of eggs of female adults.The development time of the 6^th instar larvae,total developmental period from 2^nd to 6^th instars and pupae was significantly prolonged by feeding with dsLdV-ATPaseA.Feeding dsLdAK2 and dsLdHR3 significantly prolonged the development time of the 6^th instar larvae,and total developmental period from 2^nd to 6^th instars.Feeding dsLdAK2 significantly prolonged the development time of the 4^th instar larvae and pupae at the 3^rd instar larval treaments,and feeding dsLdHR3 significantly prolonged the development time of the pupae.In conclusion,dsRNA of LdV-ATPaseA,LdAK2 and LdHR3 were initially fed at the 2^ndand 3^rd instar L.dispar larvae,which confirmed that LdV-ATPaseA,LdAK2 and LdHR3 genes affected the growth and development of L.dispar.This study provides a theoretical basis for the development of molecular control targeting LdV-ATPaseA,LdAK2 and LdHR3.