Expression Profiles And Activity Characterization Of Lipase-related Proteinin Malpighian Tubules Of The Chinese Oak Silkworm,Antheraea Pernyi

Lipases are ubiquitous enzymes in nature,play a crucial role in fat metabolism by catalyzing the hydrolysis of triacylglycerol to free fatty acids and glycerol.But rare reports about insect lipase are available.Lipases are serine hydrolases with a conserved catalytictriad: serine,aspartate or glutamate,and histidine.In this study,we studied the expression profiles and activity of ApLSP.In this study,the specific primes were designed to clone the cDNA of 1032 bp open reading frame?ORF?.We constructed pET-28a-ApLSP prokaryotic expression vector,then recombinant vector was transferred into Escherichia coli and the transferred E.coli was induced by different concentrations of IPTG,SDS-PAGE and Western Blot analysis showed that the molecular mass of the mature protein was 40.2 kDa,which was consistent with prediction,indicating that the ApLSP protein was successfully expressed.The recombinant proteins were purified under native conditions using an Ni-NTA column,and the interest protein with high purity was obtained.The acquired protein was employed as antigen to immunize a New Zealand white rabbit and ultimately the polyclonal antibody serum against ApLSP was achieved.the polyclonal antibody serum against ApLSP was used for subsequent functional study.To determine the expression of ApLSP transcripts,integument,silk gland,fat body,midgut,malpighian tubules,and hemocytes were isolated from larvae at the third day of the fifth instar stage.QRT-PCR results showed that ApLSP was widely expressed in all tissues analyzed,and showed particularly high expression in the midgut.Western blotting results showed that the ApLSP protein could be detected in malpighian tubules.Larvae from the third day of the fifth instar stage were respectivily injected the heat-inactivated Escherichia coli,Micrococcus luteus,Beauveria bassiana,nuclear polyhedrosis virus and PBS as the negative control.The transcript and protein expression analysis of ApLSP were performed by qRT-PCR or Westerrn Blot,the results suggested that the other four pathogenic microorganisms immune-challenged all can significantly upregulate or downregulate the expressions of ApLSP mRNA and protein,but the up-regulated or down-regulated expression degree and time of ApLSP were different.The purified ApLSP protein activity was determined in vitro.The optimum temperature and pH for enzyme activity were 35�C and 8.0,respectively.ApLSP activity was stimulated in the presence of Mg²⁺,Na+,Ca²⁺ and b-mercaptoethanol,while Zn²⁺,Cu²⁺,and Fe3+ inhibited its activity.Detergents such as SDS,glycerol and Tween-20 were able to increase lipase activity by 20 % to 30 %.In conclusion,our results elucidated the ApLSP activity characterization,and indicate that ApLSP might be play an important role in the innate immunity of insect of A.pernyi,further analyticed the relationship between ApLSP and immunity of A.pernyi at the molecular level,and also have an important referential meaning for studing innate immunity regulation mechanism of A.pernyi and other lepidoptera insects.