Cloning And Expression Analysis Of A Matrix Metalloproteinase Gene From Bombyx Mori

Matrix metalloproteinases(MMPs),a family of proteolytic enzymes,are involved in the degradation of extracellular matrix and/or basement membrane protein component.Studying on MMPs is not only significant in researching the physiological process of the metamorphosis in Bombyx mori,but also provides a basis for the further study on MMPs family's function and mechanism through the study of pattern insect, Bombyx mori.To study the essential function of MMPs in metabolism of Bombyx mori, in the thesis,the work we did are showing:1.Cloning and sequences analysis of a matrix metalloproteinases from Bombyx mori:A full-length cDNA of Bm-MMP gene was first cloned from the silkworm pupae with RACE and RT-PCR,and named Bm-MMP.The sequence analysis shows that there are two alternative splice variants in the mRNA of Bm-MMP (Bm-MMP-V1 and Bm-MMP-V2)(GenBank no:EU496376,EU496377).The full-length cDNA of Bm-MMP-V1 is 2 257 bp,contains a 1 686 bp ORF,encodes 561 amino acids,and the protein molecular weight is about 62.3 kD.The full-length cDNA of Bm-MMP-V2 is 2 188 bp,contains a 1 617 bp ORF,encodes 538 amino acids,and the protein molecular weight is about 59.9 kD.The gene structural analysis shows that there are 13 exons in Bm-MMP,and the alternative splicing site locates on the exon 12. The ORF of Bm-MMP-V2 is 69 bp less than that of Bm-MMP-V1,and the similarity of the rest nucleotide sequences is 100%.The amino acid analysis indicates that Bm-MMP has all the typical structural features of MMP family,such as signal peptide sequence, propeptide,catalytic domain,hinge region and hemopexin-like domain,and there is a conserved sequence,the center of the catalytic activity in the catalytic domain.The analysis of amino acid sequence homology represents that the Bm-MMP-V1 and Bm-MMP-V2 share the highest similarity(88.8%) with sequences of Gm1-MMP in Galleria mellonella,and their identities with Drosophila melanogaster Dm1-MMP are 61.2%and 64.3%,respectively.2.Prokaryotic expression:The Bm-MMP-V1 was then cloned into pET28a⁽⁺⁾ for prokaryotic expression.The results of SDS-PAGE and Western blot indicate that a fusion protein--62 kD protein with 6×His-tag was expressed in Escherichia coli BL21.3.The expression profiles of Bm-MMP at the different developmental stages and post immune stimulitated:The RT-PCR result shows that the Bm-MMP-V1 and Bm-MMP-V2 are higher expressed in 4th-instar moulting larvae,mature larvae,36 h and 48 h of mounted silkworms,prepupae.It is supposed that those genes are associated with the molting and metamorphosis.In addition,we detected a higher expression of Bm-MMP in hemocytes of 5th-3 d larvae induced by LPS.It is deduced that Bm-MMP may be associated with immunity.We have cloned and characterized a full-length cDNA encoding Bm-MMP,the first matrix metalloproteinase(MMP) identified in Bombyx mori.The prokaryotic expression deduces that the Bm-MMP may have the proteolytic enzyme activity.This gene may be associated with the metamorphosis and immune stimulation of Bombyx mori under the study of expression profiles in different development stages and immune stimulation.This research is not only significant in researching the mechanism of metamorphosis and development in Bombyx mori,but also provides a reference for the further study on physiological process of the metamorphosis and development in insects.