摘要
以盐藻总RNA为模板,利用RT-PCR技术扩增了盐藻DsCDPK基因的cDNA序列,将其克隆到pMD^(TM)19-T simple载体上,经测序获得的克隆片段全长1650 bp,与已发表的盐藻DsCDPK(GenBank:JQ964113)的编码区序列同源性达100%。将DsCDPK基因的开放阅读框与质粒pET-32a(+)连接,构建原核表达载体pET-32a-DsCDPK。将该重组质粒转化到大肠杆菌BL21中,经IPTG诱导融合,蛋白在大肠杆菌BL21中得到成功表达。通过SDS-PAGE检测发现,融合蛋白为部分可溶性表达,将上清蛋白经过His柱纯化后获得了纯度较高的可溶性融合蛋白,Western杂交检测显示,融合蛋白能被抗His单克隆抗体特异性识别,初步证明该融合蛋白就是带有His标签的DsCDPK蛋白。采用实时荧光定量PCR方法分析了高盐胁迫下DsCDPK基因的表达模式。试验结果表明,盐藻DsCDPK基因为盐胁迫上调基因,在高盐(3.0 mol/L NaCl)胁迫下,DsCDPK基因表达量显著增加,盐胁迫1 h时表达量达到最高,为正常生长状况(1.0 mol/L NaCl)下的3倍,差异达到极显著水平(P<0.01)。该研究成果为进一步阐明盐藻钙依赖蛋白激酶基因的功能及作用机制奠定了基础。
In this study,cDNA corresponding to Ca^2+-dependent protein kinase (CDPK) gene (DsCDPK) was isolated from green alga Dunaliella salina using the total RNA of the green alga s as a template by reverse transcription PCR (RT-PCR),and the DsCDPK gene was cloned into pMDTM19-T simple vector. The DNA sequence of DsCDPK gene was shown to contain a 1650 bp open reading frame. Alignment analysis showed that the amino acid sequence of the gene was 100% identical to DsCDPK gene (GenBank No. JQ964113) summited in previous study. Then the DsCDPK gene was cloned into pET-32a (+) expression vector,and introduced into Escherichia coli BL21 (DE3) for expression. The fusion protein was induced with isopropyl β-D-1 thiogalactopyranoside (IPTG),and the soluble recombinant protein was expressed successfully and purified using a His-SefinoseTM Kit. The purified DsCDPK protein was identified further by SDS-PAGE and western blotting analysis. Real time quantitative RT-PCR demonstrated that DsCDPK was up-regulated genes with salt stress. The expression level of DsCDPK was induced by 3.0 mol/L NaCl,with the maximum in 1 h. The relative expression level of DsCDPK was 3-fold higher than that by 1.0 mol/L NaCl ( P <0.01). These findings provide the bases for research of biological function and mechanism of DsCDPK gene.
作者
丛玉婷
邢震宇
岳金荣
高相楠
张晓琳
柴晓杰
CONG Yuting;XING Zhenyu;YUE Jinrong;GAO Xiangnan;ZHANG Xiaolin;CHAI Xiaojie(Key Laboratory of Hydrobiology in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian 116023,China)
出处
《水产科学》
CAS
CSCD
北大核心
2019年第2期248-253,共6页
Fisheries Science
基金
国家自然科学基金资助项目(31472260
30972240)
