Cloning Analysis And The Functional Investigation Of Metallocarboxypeptidase Gene In Bombyx Mori

Metallocarboxypeptidases are the key enzymes participating in forage digestionand absorption within the digestive tube in insects like Bombyx mori mCp, adigestive-tube specific metallocarboxypeptidase gene, was excavated and firstly clonedin this study. The influence of protein content and starvation to mCp expression wereinvestigated, as well as that of high temperature and insect hormones treatments, hopingto figure out the effects of mCp and its translation products on silkworm development.The results are as follows:1Complete cDNA cloning of the novel metallocarboxypeptidase gene mCpThe specific expression tag (AAATAAAAACTCTATTC) in our constructed SAGElibrary of silkworm digestive tube was utilized to excavate the Bombyx mori ESTsequence BY926338, which was supposed to be the seed sequence. PCR and RACEwere employed to clone and verify the elongated1462bp cDNA sequence. mCp waslocalized to nscaf2818of the25th linkage group with6extrons and5introns. And thesequence of the deduced protein was458amino acid residues. Protein sequence analysisfound that there were binding sites for zinc metalloprotein on the secondary structure ofmCP protein, supporting that it was a metallocarboxypeptidase-like protein. Analyzingthe constructed cladogram of insect metallocarboxypeptidase system, we found that theprotein of the newly cloned Bombyx metallocarboxypeptidase gene was closest toCarboxypeptidase A in Danaus plexippus. Homology comparison between activesequences of carboxypeptidases suggested that the amino residues in carboxypeptidasesequences were conservative between species, but a certain amount of amino residuesites resembled in vertebrates or insects. In all, integral analysis from cloning andbioinformatics preliminarily suggested that the newly cloned gene was ametallocarboxypeptidase gene. 2mCp gene was specifically and highly expressed in silkworm digestive tubeThe chip expression profile of total genes on day3of the5th instar larvae showedthat the expression levels of mCp gene in9tissues of Dazao strain were remarkablydifferent, among which high expression level was only detected in midgut, while traceamounts or no mounts of the transcripts were detected in the other tissues. The result ofRT-PCR indicated that midgut was the only one with high expression of mCp geneamong the11tissues investigated on day3of the5th Ysh larvae, which approved theresult of gene chip expression profile. No mCP protein was observed in silkgland by theresult of western blot but only in midgut. Expression profile of development stages byRT-PCR showed that mCp was significantly expressed in dormant stage and food-intakeperiod, while the level rapidly decreased from spinning time, indicating that mCpexpression had sort of relationship with food intake.3Functional analysis of mCP in silkwormTo elucidate the relationship between mCp translation products and digestion andabsorption of Bombyx proteins, the influence of starvation on digestive tube mCp genewas firstly investigated. The result indicated that starvation for6to72hoursdown-regulated the mRNA level of mCp gene within the digestive tube, and starvationover72hours also significantly cut down the mCP protein content. However, thesedecreases were restored after re-feeding to the experimental larvae, and the expressionlevel of mCp was even tremendously up-regulated. All the above indicated that theexpression of mCp was induced by food intake. Interestingly, the fact that digestiveenzyme genes like mCp were restrained under starvation but restored withcompensatory amount coordinated with the compensatory growth in animals.The great influence of protein content on nutrition indexes such as feed efficiency,which would directly affect the growth and development of silkworm, has been reportedpreviously. In this study, casein was added to the basic artificial diet, to imitate themodel of silkworm growth and development. In the observations, the increase of proteincontent between13.3%and33.3%would up-regulate the expression of mCp gene remarkably, as well as the content of mCP protein to a certain amount. Among thevalues, the most efficient amount of protein was23.3%, while higher percentages wouldactually impair both the mCp expression and translation products. Notably, the mCpgene in males was more efficiently induced by the amount of protein, which may relateto the fact that the feed efficiency of males was higher than that of females.The effects of high temperature, which would influence food intake, digestion andabsorption, were further investigated. The result indicated that the mCp expression wasup-regulated for a certain period and then down-regulated when the larvae were placedunder34°C from day3of the5th instar. And also the alimentation and development inlarvae were impaired. Metallocarboxypeptidase inhibitor gene within the digestive tubewas significantly up-regulated, which restrained the expression of mCp, when the larvaewere placed under38°C from molting of the5th instar. And Ysh larvae even graduallydied out. The result indicated that high temperature did affect the mCp expression insilkworm, and further influenced the alimentation absorption and development.The endogenous molting hormone titer was low on day3of the5th instar.Supplement of exogenous20E at this period induced aging and ecdysis, and alsodown-regulated the mCp expression level within the digestive tube. However, juvenilehormone would temporarily increase the mCp expression within the digestive tube.Knock out by50.4%to55.3%of mCp expression on day3of the5th instar bysiRNA would pull off the pupation by6days, and the5th instar was1fold longer with a45%decrease in pupation rate, indicating that mCp knock-out probably influence theprotein absorption and transportation of digestive tube, to impair the metamorphosisfrom larvae to pupae.