Molecular Cloning And Functional Analysis Of β-fructofuranosidase Genes In Diaphania Pyloalis Walker

Plant latex and other exudates at the position of damage caused by insect herbivory are defensive system against insect. Mulberry latex contains extremely high concentrations of alkaloidal sugar mimics, such as 1-deoxynojirimycin (1-DNJ), which are effective glycosidase inhibitors and highly toxic to the insects that do not normally feed on mulberry leaves. Although these compounds do not harm the silkworm, Bombyx mori, the defense mechanisms have not been well documented. BmSucl, a sucrose hydrolase gene in B. mori, is the first cloned β-fructofuranosidase(β-FFase) gene from animal intestine. BmSucl may play an important role in the process of silkworm resisting mulberry alkaloids. Diaphania pyloalis Walker, a common mulberry pest, is a distantly related species but has consistent diets withB. mori. Larvae of D. pyloalis do very serious harm to the production of mulberry fields. We previously found that there are five β-FFase homologous genes in the genome of D. pyloalis. The expression patterns and functional features of these genes in vitro and vivo remain unknown.In this study, we firstly investigated the transcriptional profiles of the β-FFase homologous genes in different tissues of D. pyloalis larvae. The results of RT-PCR showed DpSuc1a mRNA was expressed abundantly in the tissues of foregut, midgut and silk gland and DpSuc2a mRNA was highly expressed in the foregut, midgut and fat body, while the transcribes of DpSuc1b, DpSuc2a and DpSuc2b were nearly not detected. Next, we assayed the β-FFase activity in the total extracts of midgut and silk gland, respectively. The results indicated that the midgut extracts of D. pyloalis displayed strong β-FFase activity and the activity was not inhibited by the addition of DNJ, similar to that of B. mori..For further studying the function of D. pyloalis β-FFase genes, we cloned the ORFs of 5 genes with specific primers and constructed recombinant vectors respectively for prokaryotic expression in vitro. SDS-PAGE and western blot analyses showed that the recombinant proteins could be expressed except DpSUC1b. After purifying the expressed proteins, we performed enzyme assay to detect the β-FFase activity. Interestedly, the recombinant proteins of D. pyloalis showed no β-FFase activity as compared to that of BmSUC1. Therefore, we prepared polyclonal antibody of DpSUC1a in rabbit and detected its expression profile in vivo. The result of western blot indicated that DpSUC1a was highly expressed in the midgut, which is consistent with that of RT-PCR. The expression of DpSUC1a in the midgut cells was also captured by immunohistochemical analysis. In conclusion, these results suggested that the mechanism regulating expression of DpSucla might be different from BmSucl, in which action of DpSUC1a is mostly depended on post-translational modifications and processing.As one of the major mulberry pests, D. pyloalis is an important object for pesticides. The study of D. pyloalis Walker β-FFase contributes to explaining how the mulberry feeding insects can circumvent the mulberry’s defense system at the molecular level. Also, this study is a necessary and foundational research for revealing the evolutionary interaction mechanism between mulberry trees and the silkworm.