Study On Small Heat Shock Proteins Genes In The Silkworm, Bombyx Mori

Silkworm is one of the first domesticated insects,it is not only one of the most important economic insects in agriculture but also one of the crucial experimental insects.Small heat shock proteins have been found in all organismsso far examined. Small heat shock proteins play an important role in the cellular defence of prokaryotic and eukaryotic organisms against a variety of internal and external stressors.Small heat shock proteins of various origins exhibit chaperone-like activities in vitro and in vivo,sHSPs appear to be also involved in various biological processes including cell proliferation and differentiation,embryogenesis and aging. While different sHSPs are highly heterogeneous in sequence andsize, the common trait is the presence of a conservedα-crystallin domain. In order to research the distributing character.gene structure, expressive speciality and the function of sHSPs systematically, we cloned BmsHSPs and carried through expression of recombinant protein BmHSP24.3 in vitro and study on the function based on the silkworm genome database and chip database. All the results are as below:1 Bioinformatic analysis of BmsHSPsA blastp research was performed by using thesilkworm genome database. We had found 14 BmsHSPs in total. Thereinto, 6 genes were identical with the known sHSPs in sequence. The other 8 genes were newly discovered of which no identical sequences had been found in NCBI. There are only three BmsHSPs having introns and the intron number of different BmsHSPs ranges from 2 to 4. All the BmsHSPs hadα-crystallin domain, but there were still differences in sequence and size according to the scan to conservative domain of the BmsHSPs. The BmsHSPs were highly heterogeneous in the sequence of N-terminal domain and C-terminal domain.the peptide length of conserved anα-crystallin functional domains ranges from 60 residues to 90 residues.2 Expression profile of BmsHSPsBased on the silkworm genome database and chip database.we found multiple sHSPs expressed in processing of normal development of silkworm. In different tissues, the transcriptive products of sHSPs are different. Generally speaking, the transcriptive products of BmsHSPs are tissue-and temporary-specific expressed. Nearly all the BmsHSPs had strong expression in germen and make remarkably difference between male and female. Express level analysis of using RT-PCR revealed that BmsHSPs expressed in all the tissues under normal condition, the expression involved along with cell proliferation and differentiation, and after heat stimulation, the expression of BmsHSPs increased remarkably, which showed that organisms response to heat-stress.3 Cloning and the systematic evolution analysis of BmsHSPsWe designed primers on the base of the silkworm genome CDS sequence and cloned five BmsHSPs. These five clones were identical with the silkworm genome CDS sequence without any base change.10 sHSPs of H.sapiens, 11sHSPs from D.melanogaster, 8 sHSPs from Mus and the 6 known sHSPs of B.mori were found in GenBank, and evolution of 8 sHSPs was analyzed together.The NJ tree of sHSPs was constructed by using MEGA3 software. From the systematic evolutionary tree of sHSPs, all the sHSPs are classfied into two different groups.sHSPs of D.melanogaster and B.mori made up the first group,sHSPs of H.sapiens and Mus made up the second group, at the same time we found that different species shared very high homolog in sequence. The deduced amino acid sequence of Bmhsp21.4 was similar to that of D.melanogaster CG14207-PA, The deduced amino acid sequence of Bmhsp26.4 was similar to that of D.melanogaster CG4533-PA.4 Cloning and expression of recombinant plasmid pET-50b-Bmhsp24.3To gain recombinant plasmid pET-50b-Bmhsp24.3 in E.coli, the complete ORF of Bmhsp24.3 wassubcloned into the pET-50b(+) expression vector, the recombinant plasmid was transformed into Escherichia coli BL21 and then induced to express by IPTG. We demonstrated that recombinant protein BmHSP24.3 expressed in inclusion bodies under normal conditions (37℃with IPTG to the final concentration of 1mM).Then we changed the expressional conditions constantly in order to acquire the dissolve form, at last we acquired the dissolvable and insoluble forms under 18℃with IPTG to the final concentration of 0.25mM and constantly shake overnight.5 Immunoblotting and function of recombinant protein BmHSP24.3Western blot analysis indicated that the mature protein has a mass of about 80kDa by SDS-PAGE consistent with the deduced size. Western blots were probed with antiserum to a T7·Tag (MetAlaSerMetThrGlyGlyGlnGlnMetGly).The expressed protein reacted strongly and specifically toT7. Tag by western blotting.To investigate the chaperone properties of recombinant protein BmHSP24.3, we analyzed the interactions of recombinant protein BmHSP24.3 with Rhodanese understress conditions.The results demonstrated that recombinant protein BmHSP24.3 can formstable complexes with Rhodanese, Native-PAGE showed that a complex was detected, also free Rhodanese and recombinant protein BmHSP24.3 could be observed. SDS-PAGE of the collected complexshowed both proteins were detected, indicating the formation of a complex。the interactions of recombinant protein BmHSP24.3 with Rhodanese is a reversible interaction .Taken together, our experiments proved that the recombinant protein BmHSP24.3 was fully functional as a chaperone protein. It represented protection of proteins from irreversible aggregation.