Enzyme analysis,gene CDs cloning and expression in E.coli of Mulberry Borer(Apriona germari hope) endogen β-1,4-endoglucanase
XU Wei-jia1,HAN Wei-jie1,LI Wang1,2,CHEN Yu-lin1(1 College of Animal Science and Technology,Northwest A&F University,Yangling,Shaanxi 712100,China;2 College of Animal Science and Technology,Henan Science and Technology University,Luoyang,Henan 471003,China)
【Objective】 This objective focused on endogen β-1,4-endoglucanase gene of mulberrying borer,with the purpose of supplying the theory basis for constructing engineering bacteria which can decompose cellulose effectively.【Method】 The activities of endogen β-1,4-endoglucanase were measured by 1% of CMC-Na,total RNA was isolated from Mucberry Borer gut by trizol,the first cDNA chian was used as template,a pair of primers was designed based on the gene whose accession number is AY771358.1,for PCR endogen β-1,4-endoglucanase gene of Mulberry Borer,DNASTAR and BLAST of NCBI were used for sequence analysis;another pair of primers was designed based on the sequence of this study,which was to add restriction enzyme cutting site BamHⅠfor up site and SacⅠfor down site,PCR endogen β-1,4-endoglucanase gene of Mulberry Borer,and structure pET-APEG for prokaryotic expression in Escherichia coli BL21(DE3).【Result】 The most suitable pH range is from 4.4 to 5.6,with peak at 5.0;the most suitable range temperature is from 30 to 50 ℃,with peak at 50 ℃.If solution of endoglucanase is treated in water bath of 37 ℃ for 30 min,the activities hardly change;However,after treated in water bath of 60 ℃ for 30 min,the activities plummet.The length of CDs of β-1 and 4-endoglucanase is 978 bp,ecoding 325 amino acid residues;The result of BLAST showed that,the sequence identity of this gene is 85% compared with the cellulase gene of yellow-spotted longicorn beetle,Psacothea hilaris which belongs to the GHF5,and the sequence identity of protein reaches 90%,the sequence identity of this gene is 98% compared with the sequence of Ag-EaseⅢ which has been reported in mulberry borer,the sequence identity of protein reaches 99%.Structed pET-APEG for expression in E.coil BL21(DE3) and SDS-PAGE result showed,pET-APEG vector was expressed as about 55 ku polypeptide in E.coli BL21(DE3).【Conclusion】 The mulberry borer of Shannan area belongs to GHF5,and has multi-domains of endogen β-1,4-endoglucanase,and it's gene can be expressed in E.coil BL21(DE3).
【CateGory Index】： Q786
【CateGory Index】： Q786