Cloning, Characterization And Functional Analysis Of Hsp27.6from Apis Cerana Cerana

The function of small heat shock proteins （sHSPs） is one of the hot topics in the field ofmolecular biology. sHSPs play an important role in the cellular defense of prokaryotic andeukaryotic organisms against a variety of internal and external stressors. And the immunefunction of sHSP has been found gradually. The Chinese honeybee, Apis cerana cerana, is animportant indigenous species. It is cold and disease resistance and good at collecting scatterednectar, and have important economic value and social benefits for the maintenance of theecological balance as the pollinator of flowering plants. In this research, we selected theChinese honeybee as the experiment material, and a series of research have been managed onthe isolation, sequence and expression profile analysis, and functional identification ofAccHsp27.6, which can greatly help to study the function and mechanism of sHSP. The mainresults are as follows:1. The Hsp27.6gene was cloned from Apis cerana cerana by RT-PCR and RACE-PCR, andnamed as AccHsp27.6（GenBank accession number GQ254650）. The full-length AccHsp27.6cDNA was1,014bp, with a76-bp5’ untranslated region （UTR）, a223-bp3’ UTR and a708-bp ORF encoding a protein of236amino acids with a calculated molecular weight of27.6kDa and an isoelectric point of7.53. The sequence analysis revealed AccHsp27.6has theα-crystallin domain （ACD） of metazoan α-crystallin-type sHSPs and the predicted amino acidsequence of AccHsp27.6exhibited43.88%and44.07%similarity to the sHSP fromMacrocentrus cingulum （GenBank accession number EU624206） and Hsp21.7from Nasoniavitripennis （GenBank accession number XP001604512）, respectively. The clone of genomicDNA indicated that there was no intron in this gene and7HSEs and3NF-κB binding siteswere present in the5′-flanking region, suggesting a possible function in immunity.2. A semi-quantitative RT-PCR analysis indicated that AccHsp27.6was expressed in all testedtissues and at different developmental stages. Furthermore, expression of the AccHsp27.6 transcript was induced by exposure to heat shock, H₂O₂, a number of different chemicals（including SO₂, formaldehyde, alcohol, acetone, chloroform, and the pesticides phoxime andacetamiprid）, and the microbes Staphylococcus aureus and Micrococcus luteus. In contrast,the mRNA expression could be repressed by CO₂, the pesticides pyriproxyfen and cyhalothrin,and the microbes Bacillus subtilis and Pseudomonas aeruginosa.3. AccHsp27.6-pET-30a（+） was constructed using pMD18-T simple and pET-30a（+） ascloning vector and expression vector, respectively. And then it was induced by IPTG toexpress in E. coli strain BL21（DE3）. The fusion protein with the approximately molecularweight at36kDa was then purified. Notably, the recombinant AccHsp27.6protein exhibitedsignificant in vitro molecular chaperone activity and antimicrobial activity.Taken together, these results suggest that AccHsp27.6might play an important role in theresponse to abiotic and biotic stresses and in immune reactions.