Cloning And Functional Identification Of The Serpin Genes Involved In Melanization In Plutella Xylostella

Serine protease inhibitors(serpins),a kind of ancient inhibitors,are widely distributed in animals,plants and prokaryotes.Serpins participate in a broad spectrum of physiological processes including complement activation,inflammatory reactions,cell death,growth and development,cell differentiation,apoptosis and protein folding by inhibiting their cognate enzymes.In the innate immune response of insects,serpins play a crucial role in the melanization reaction and the Toll and Imd pathways.To study the function of Plutella xylostella serpins,we identified two serpin genes,serpin-3 and serpin-6,through sequence comparison with Drosophila melanogaster serpin-27A and Manduca sexta serpin-3 and serpin-6.We cloned the open reading frames(ORF)of serpin-3 and serpin-6 and expressed them in E.coli BL21(DE3).We analyzed the function of serpin-3 and serpin-6 using inhibition activity detection and RNA interference.The results of our study are outlined below.1.We cloned the serpin-3 and serpin-6 ORF from the DBM genome.Our results show that the ORF of serpin-3 and serpin-6 encode 451 and 414 amino acid residues,respectively.Sequence alignment shows that the binding sites of P.xylostella serpin-3 are same as of Ostrina furnacalis serpin-3 and Manduca sexta serpin-3.The binding sites of P.xylostella serpin-3 and M.sexta serpin-3 are also the same.Phylogenetic tree analysis shows that P.xylostella serpin-3,O.furnacalis serpin-3,and M.sexta serpin-3 have a close relationship.P.xylostella serpin-6 has a close relationship to M.sexta serpin-5.2.We analyzed expression profiles of serpin-3 and serpin-6 genes at developmental stages by qRT-PCR.Our results demonstrate that serpin-3 has the highest expression at the adult stage and serpin-6 is highly expressed at the egg and pupal stages.We injected 4th instar larvae of P.xylostella with E.coli and S.aureus.We measured the relative expression levels of the two serpins by qRT-PCR at 0 h,3 h,6 h,9 h,and 12 h after injection.The results show that the mRNA levels of serpin-3 have the highest expression after injection at 3 h and the highest expression levels of serpin-6 are observed at 12 h after injection.Serpin-3 has a strong response to gram-negative bacteria,and the response of serpin-6 to gram-positive bacteria is faster than for gram-negative bacteria.3.We carried out RNA interference experiments by injecting dsRNAs that target specific regions of serpin-3 and serpin-6 genes.We collected P.xylostella larvae at 6 h,12 h,24 h,and 36 h and then measured transcript levels using qRT-PCR.These data show that the transcript levels of serpin-3 are significantly decreased 6 h after injection,and serpin-6 transcript levels are significantly decreased 24 h after injection.RNAi-mediated knockdown of serpin-3 and serpin-6 influences the expression of some genes involved in melanization,and antimicrobial peptides,as well as in the Toll and Imd pathways.After 6 h of reduced serpin-3 expression,the expression of PAP-1,PAP-3a,PAP-3b,and PPO-2 are not significantly changed,but theexpression of PPO-1 is decreased.The expression of some antibacterial peptide genes such as Gloverin-1,Gloverin-2 and Cecropinincreases with serpin interference.The expression of and Spaetzle-4 increases and the expression of FADD decreases with serpin interference.After 24 h of reduced serpin-6 expression,the expression of PAP-1 and PPO-2 decreases andthe expression of some antibacterial peptide genes such as Glover in-1,Gloverin-2 and Cecropin increases.The expression of Toll-9 and Dorsal decreases,but the expression of Spaetzle-4 increases with reduced serpin-6 expression.The expression of FADD and IKK-r increase with reduced serpin-6 expression.(4)We further ligated the ORP of serpin-3 and serpin-6 into pET28b and expressed it in E.coli BL21.Both recombinant serpin proteinsare approximately 50 kDa in size.We purified the recombinant proteinsby nickel chelated agarose gel affinity chromate graphy and obtained antibodies against serpin-3 and serpin-6.The molecular weights of the purified proteins are the same as those identified by Western Blot.The far UV CD spectrum shows that serpin is composed of a mixture ofα-helices,β-sheets,and(3-turns.Serpin-3 has a minimum peak at 200 nm and contains 39.1%a-helices,14.6%β-sheets,and 16.1%β-turns.Serpin-6 has a minimum peak at 222 nm preceded by a shoulder at 208 nm and contains 99%a-helices,0.3%p-sheets,and 2.7%β-turns.To identify the functions of these serpins,we measured the inhibition activity of the purified recombinant proteins.PO activity is blocked after incubation of recombinant proteins with the larval plasma of P.and E.coli.Our results show that the recombinant proteins of serpin-3 and serpin-6 can inhibit melanization in plasma,highlighting their important roles in the regulation of prophenoloxidase activationin P.xylostella.Our findings indicate that serpin-3 and serpin-6 play significant roles in the innate immunity of P.xylostella.