杨树微管功能研究植物表达载体构建及验证

[目的]分别构建84K杨的微管蛋白TUA5和TUB16与红色荧光蛋白mCherry融合的植物过表达载体,瞬时表达验证载体在植物体内表达后的荧光信号,为研究杨树微管功能奠定基础。[方法]以毛果杨微管蛋白TUA5和TUB16的基因序列为模板,设计84K杨的TUA5和TUB16基因的引物,提取野生型84K杨的RNA并反转录成c DNA,同源克隆得到84KTUA5和84KTUB16基因,分别连接在pCAMBIA 1300载体mCherry荧光标签的N’端和C’端,转化到大肠杆菌TOP10感受态细胞中,通过菌落PCR和测序鉴定获得阳性单克隆,并通过电击法将重组质粒转化到农杆菌GV3101中,瞬时转化烟草后进行荧光观察。[结果]克隆得到了84KTUA5和84KTUB16基因,成功与pCAMBIA 1300-mCherry载体连接,烟草瞬时表达荧光观察结果显示:仅目的基因与mCherry标签C’端相连的融合蛋白能够成功表达,且荧光明显。[结论]成功构建了84K杨TUA5和TUB16基因与pCAMBIA 1300-mCherry的融合表达载体,为进一步研究杨树微管功能提供了背景材料。

Construction and Verification of Plant Expression Vectors for Poplar Microtubule Function Research

[Objective]To lay the foundation for studying the microtubule function of poplar,plant overexpression vectors of 84K poplar by constructing the tubulin TUA5 and TUB 16 fused with red fluorescent protein mCherry,and using transient expression to verify the fluorescent signal after vectors expressed in plant.[Method]Primers for TUA5 and TUB16 genes of 84K poplar were designed using TUA5 and TUB16 homologous gene sequences from Populus trichocarpa,RNA from wild type 84K poplar was extracted and reversely transcribed into cDNA,homologous cloning yield 84KTUA5 and 84KTUB16 genes,which were respectively linked to N'and C'terminal of the mCherry fluorescent tag from pCAMBIA 1300 vector,and then transformed into E.coli TOP 10 competent cells.Positive monoclonal were conducted by means of colony PCR,and after sequencing identification,the recombinant plasmid was transformed into agrobacterium GV3101 by electrochemical reaction to transiently transformed into tobacco leaf,and then observed fluorescence excitated from mCherry.[Result]The 84K TUA5 and 84K TUB16 genes were cloned and successfully linked to pCAMBIA 1300-mCherry vector,fluorescence observation of tobacco transient expression showed that only the fusion protein of the target gene linked to the C″terminal can be successfiilly expressed,and the fluorescence was obvious.[Conclusion]The fusion expression vectors of TUA5 and TUB16 genes of 84K poplar and pCAMBIA 1300-mCherry were successfully constructed respectively,which provids background materials for further study of poplar microtubule function.