| Insecticidal activity of Bt is directly affected by the activation of Bt after insect fed on Bt protoxin.The toxicity of Bt will be reduced by incomplete hydrolysis or excessive hydrolysis.Serpins,serine proteases inhibitors,are functionally diverse proteins found in many insects,involve in digestion,growth and development,immune responses and other physiological processes.As the most effective inhibitory factors of trypsin and chymotrypsin,serpins may be take part in the insecticidal process of Bt toxin through regulating the activity of midgut proteases.In this study,four serpin genes were cloned from Helicoverpa armigera,their structure were predicted and phylogenesis evolution were analysed by bioinformatics software.The expression levels of serpins in different development stages and different tissues were compared using RT-q PCR,and the changes of the expression quantity of serpins after larvae fed Cry1 Ac were also compared.The effect of serpin-e on protease activity in midgut and the effect on Cry1 Ac toxicity against H.armigera were determined by prokaryotic expression protein.The amino acid sequence,expression level of serpin-e and the effects of serpin-e on protease activity in midgut between Cry1Ac-resistant and-susceptible H.armigera were compared.And the function of serpin-e in Cry1 Ac insecticidal mechanism was further analyzed by using RNAi interference techniques.The main results are as follows:1.The full-length c DNAs of four serpins from H.armigera were about 1000 bp,named serpin-a,serpin-b,serpin-cand serpin-e,respectively.Their Gen Bank accession numbers were MT066041,MT066042,MT066043 and MT066044.Bioinformatics analysis showed that serpins encoded about 400 amino acids with molecular weight of 50 KDa and isoelectric point of 4-7.The serpin-a,serpin-b and serpin-c had signal peptides sequences in N-terminals compased of about 21 amino acids,and all four serpins had potential exposed reactive center loop(RCL)in the conserved domains.The phylogenetic-tree analysis showed that serpins of H.armigera had high homology with Spodoptera litura,Trichoplusia ni,Spodoptera frugiperda and Bombyx mori.2.Four serpins were expressed in all developmental stages and different tissues.The expression levels of serpin-a and serpin-e were the highest in the 4th instar larvae,the expression of serpin-b was high from the 4th instar and reached the highest in the adult stage,and the highest expression of serpin-c was in the pupal stage.In different tissues,the highest expression of serpin-a was found in midgut and malpighian tube serpin-b and serpin-c in head,and serpin-e in hemolymph.The expression levels of serpin-b and serpin-e in midgut of H.armigera were significantly up-regulated after larvae fed on low concentrations of Cry1 Ac.These results indicated that serpin-b and serpin-e could be induced by Cry1 Ac toxin,and they maybe involved in the insectical toxicity process of Cry1 Ac.3.After prokaryotic expressions,serpin-a and serpin-b were produced as insoluble proteins,serpin-e in both soluble and insoluble proteins,and serpin-c failed to induce protein.After serpin proteins were purified by Ni-Sepharose column,we tested their protease inhibitor activities.Without the inhibitors,the activation was inhibited by only serpin-e protein,but not serpin-a and serpin-bproteins.The inhibitory activities of serpin-e lasted for 48 hours and the activities were lost after that.In vivo,the toxicity of Cry1 Ac to H.armigera was obvious reduced after larvae fed Cry1 Ac protoxin and serpin-e protein mixtures compared the larvae treated with Cry1 Ac alone.Therefore,serpin-e could inhibite the activity of midgut proteases,further inhibit the activation of Cry1 Ac and reduced the toxicity of Cry1 Ac.4.The expression levels of serpins in different resistant strains were compared.It was found that the relative expression of four serpins were significantly increased in LF30 resistant strain.The expression level of serpin-b,serpin-c and serpin-e were increased in LF120.Although serpin-e was expressed in both resistant and susceptible strains,the expression levels in all resistant strains were significantly higher than those in the susceptible strain.The difference of serpin-e between Cry1Ac-susceptible strain and LF30 resistant strain was further compared.Compared with the susceptible strain,the serpin-e gene in resistant cotton bollworm had five mutation sites,resulting in the mutation of two amino caids(D128?Eand I353?L).The expression levels of serpin-e in the resistant strain were significantly higher than that in the sensitive strain at each developmental stage.The expression levels of serpin-e in midgut,hindgut and malpighian tube in resistant cotton bollworm were significantly higher than that of sensitive strain.We then compared the digestive activity of midgut juices to activate the Cry1 Ac protoxin from resistant and susceptible strains.The results showed that within the first 20 minutes,the digestive ability of midgut juices from the resistant strain were lower than that from susceptible strain.We speculated that the increased expression of serpin was related with the resistance of H.armigera to Cry1 Ac.Compared with the control,the interference efficiency of serpin-e gene in resistant and sensitive strains was 77.95% and 82.65%,respectively.Compared with the control group,susceptible and resistant larvae fed on prototoxin Cry1 Ac had significantly lower body weight and slower growth,with weight inhibition rates of 77.78% and 77.72%,respectively.In this study,four serpins of H.armigera were cloned,it is confirmed that serpin could be induced by Cry1 Ac,the toxicity of Cry1 Ac against H.armigera reduced by inhibiting the activity of midgut juice.In the resistant strain,the expression level of serpin-e was significantly increased and the activation ability of midgut juice in resistant strain decreased.It was suggested that the increased expression of serpin-e might be related to the resistance of H.armigera to Cry1 Ac.The results will provide a foundation for further analysis the role of serpin gene in Bt toxin against cotton bollworm,and further study the insecticidal mechanism and insect resistance mechanism of Bt. |
