In Vivo And In Vitro Expression And Activity Analysis Of β-FFase In Two Non-mulberry Feeding Silkworms—Samia Cynthia Ricini And Antheraea Pernyi

Samia cynthia ricini and Antheraea pernyi, for which mulberry trees are not the host plant in natural conditions, are the relative species of the Bombyx mori.Mulberry latex contains extremely high concentrations of toxic component--- alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1)and 1-deoxynojirimycin (DNJ).These sugar mimic alkaloids have been demonstrated to be highly toxic to the eri-silkworm S. c. ricini. However, these compounds are not toxic to larval of the silkwormB. mori,which feed only on mulberry leaves. The findings that B. mori encodes a functional β-fructofuranosidase (EC 3.2.1.26,β—Fase) BmSUC1 and its activity was not inhibited bya a-glucosidase (EC 3.2.1.20) inhibitor DNJ might give a clue to unlocking the unique regulatory system in B. mori vs. mulberry defense system. In our previous studies, we have isolated two P-FFase genes from S. c. ricini (ScSucl and ScSuc2), and three from A. pernyi (ApSuc1a, ApSuc1b and ApSuc2). These genes are almost not transcribed in the fifth-instar larvae of both S. c. ricini and A. pernyi, suggesting that the expression and regulatory mechanism of β—FFase genes take probably part in the alternative feeding adaptation of mulberry or non-mulberry feeding insects.In this study, the featureofsucrase activity in A. pernyi, S. c. ricini and B. moriin vivowas firstly investigated. We found that the sucrase activity in the midguts of S. c. ricini and A. pernyi was markedly inhibited by the addition of DNJ because of a-glucosidase in these two insects was main type of sucrase, while that of B. mori was nearly not inhibited by DNJ and the sucrose activity mainly came from β-FFase. Furthermore, the activity of β-FFase in the midgut extracts was detected by using two substrates, sucrose plus DNJ (to inhibit the activity of a-glucosidase) and raffinose.The results showed that neither S. c. ricini nor A. pernyi canhydrolyze the fructose residue as compared to B. mori, indicating that there was no β-FFase activity in the midguts of S. c. ricini and A. pernyi.Meanwhile, in order to perform prokaryotic expression and analysize enzymatic activity in vitro, we spliced the upstream, middle and downstream fragments of each β-FFase genes of S. c. ricini and A. pernyi based on our previous studies. As a result, complete ORFs of all five genes were successfully obtained by splicing overlap extension PCR.They were ligated to pET-24b vectortogether with BmSucl, respectively, and transformed into E. coli BL21 (DE3) strain. The condition for expressing soluble proteins was optimized. Western blot analyses showed that the recombinant proteins of ScSUCl, ApSUC1a and BmSUC1 have been expressed successfully. Then we expressed the three recombinant proteins in large scale and purified them using Ni-NTA affinity chromatography.Enzymatic assay of pH-activity relationship showed that neither ScSUCl nor ApSUC1a had β—FFase activity when compared to BmSUC1. The result suggested that either ScSUC1 and ApSUC1a themselves do not possess theβ—FFase properties or their functions are dependent on the post-translational modification and procession.This study assayed the β—FFase activity of two non-mulberry feeding silkworms S. c. ricini and A. pernyiin vivo and in vitro. our findings proved that there were no functional β — FFase exsiting in the midguts of S. c. ricini and A. pernyi, which help to understand the role of lepidopteran β — FFase in the molecular mechanismfor mulberry feeding insects to resist toxic alkaloids.