麻牛膝TONSOKU基因克隆、生信分析及其抑菌作用

Cloning,bioassay,and antibacterial activity of TONSOKU gene from Cyathula capitata(Wall.)Moq.

目的克隆编码麻牛膝(Cyathula capitata(Wall.)Moq.)CcTSK蛋白的基因全长,通过基因重组技术制备表达质粒,对表达产物进行生物学预测和抑菌分析。方法以麻牛膝嫩叶为材料,完成CcTSK基因cDNA的克隆。采用生信软件分析该基因及编码蛋白的生物学特征。构建CcTSK基因原核表达载体,并转入大肠埃希菌BL21(DE3)感受态中,IPTG诱导表达,考察CcTSK-GST融合蛋白的表达菌株对低浓度抗生素的敏感性、自身生长变化及在较高浓度抗生素下的存活率。结果CcTSK基因的gDNA为12613 bp,CDS为3855 bp,共编码1284个氨基酸。CcTSK蛋白为亲水性不稳定蛋白,没有跨膜结构域,属于没有信号识别功能的非分泌蛋白,亚细胞定位预测该蛋白可能位于细胞核,并且具有核定位信号,含有TPR重复序列与LRR重复序列结构域,二级结构主要有α-螺旋(占53.89%)、无规卷曲(占32.79%)、延伸链(占8.88%)和β-转角(占4.44%)。经IPTG诱导后,CcTSK-GST融合蛋白的表达抑制大肠埃希菌生长。低浓度抗生素测试结果显示,CcTSK-GST表达菌株对氯霉素(chloramphenicol,Cam)最敏感;高浓度抗生素处理下,CcTSK-GST的表达使得抑菌现象显著增强。结论成功获得编码CcTSK蛋白的基因全长及原核表达质粒,并对其生物学特征进行了分析比较,该蛋白与拟南芥AtTSK蛋白有较高相似性。此外,CcTSK-GST融合蛋白的表达对大肠埃希菌增殖具有抑制作用,并且显著增强抗生素所产生的抑菌现象。

Objective In order to predict the biological and antimicrobial analysis of the expressed product,the full length of the gene encoding CcTSK protein of Cyathula capitata(Wall.)Moq.was cloned and the expression plasmid was prepared by gene recombination.Methods The cDNA of CcTSK gene was cloned from tender leaves of Cyathula capitata(Wall.)Moq.Bioinformatics software was used to analyze the biological characteristics of the gene and its encoding protein.The prokaryotic expression vector of CcTSK gene was constructed and transferred into E.coli BL21(DE3)competent strain,and induced by isopropylβ-D-thiogalactopyranoside(IPTG),to investigate the sensitivity of strains expressing CcTSK-GST fusion protein to low concentration of antibiotics,and their own growth and cell survival rate under high concentration of antibiotics.Results The gDNA and CDS of CcTSK gene were 12613 bp and 3,855 bp,encoding 1,284 amino acids.CcTSK protein is a hydrophilic unstable protein with no transmem­brane domain and belongs to a non-secretory protein lacking the ability to recognize signals.Subcellular localization predicts that it may be located in the nucleus and has nuclear localization signal,as well as two domains containing TPR repeats and LRR repeats.The main secondary structures wereα-helix(53.89%),random coil(32.79%),extend­ed chain(8.88%),andβ-rotation(4.44%).Induced by IPTG,expression of CcTSK-GST fusion protein inhibited the growth of E.coli.The results of low concentration antibiotic test showed that CcTSK-GST expressing strain was the most sensitive to chloramphenicol(Cam),and the antibacterial effect was significantly enhanced at higher concen­tration of antibiotic.Conclusions The full length of the coding gene and prokaryotic expression plasmid of CcTSK protein were successfully obtained,and the biological characteristics of the protein were analyzed and compared.The protein had high similarity with the TSK protein of Arabidopsis(AtTSK).In addition,the expression of CcTSK-GST fusion protein could inhibit the proliferation of E.coli,and significantly enhanced the bacteriostatic phenomenon pro­duced by antibiotics.