内含子介导的TBSV病毒表达载体的构建和功能分析  

Construction and Function Analysis of an Intron-containing TBSV-based Vector System

作　　者：吴乐乐 徐丽[1] 丁向真[2] 李志英[1,2] 王盛[1,2,3] WU Le-Le1 XU Li1 DING Xiang-Zhen2 LI Zhi-Ying1,2 WANG Sheng1,2,3(1 Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in the Western China, Yinchuan 750021, China; 2 School of Life Science, Ning Xia University, Yinchuan 750021, China; 3 Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia, Yinchuan 750021, Chin)

机构地区：[1]西部特色生物资源保护与利用教育部重点实验室,银川750021 [2]宁夏大学生命科学学院,银川750021 [3]宁夏优势特色作物现代分子育种重点实验室,银川750021

出　　处：《农业生物技术学报》2018年第4期719-728,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31660037)

摘　　要：重组植物RNA病毒对寄主的侵染力下降是影响植物RNA病毒表达载体效率的一个限制因素。为了探索提高病毒载体表达效率的途径,在番茄丛矮病毒(Tomato bushy stunt virus,TBSV)表达载体p TBSV-G的基础上,于病毒编码的两个关键基因编码区内插入不同数量拟南芥(Arabidopsis thaliana)基因组内含子序列,构建了内含子数目和位置不同的系列病毒表达载体。接种寄主植物Nicotiana excelsiana后,通过比较分析病毒携带的绿色荧光蛋白基因(green fluorescent protein,gfp)在寄主植物叶片中的表达情况,对在病毒基因组编码区内引入内含子的有效性和规律性进行了探索。结果显示,Net Plant Gene软件可以准确地识别出插入病毒序列中的全部内含子序列,表明利用该软件对内含子剪接情况进行提前预判的方法具有一定的实用价值。反转录PCR(reverse transcription PCR,RT-PCR)检测结果显示,对照组的扩增产物片段均大于实验组且条带大小与理论预期一致;Western blots实验能够检测到完整的P19、P33和P92目的蛋白表达产物。RT-PCR和Western blots实验结果表明,病毒载体中内含子在寄主植物细胞核中确实能够被正确的识别和剪接。GFP在接种叶片上的表型和蛋白质水平定性与定量实验结果显示,插入2个内含子和11个内含子可以使病毒表达载体的表达效率提高约2倍,而插入9个内含子对病毒表达载体效率无明显的增效作用。由此推测,增效作用与内含子数量无关,内含子的插入位置可能发挥着关键的作用。研究结果将为内含子在植物病毒RNA表达载体中的应用奠定一定的理论和实践基础。The low efficiency for recombined RNA viruses is the limiting factor in the study of plant RNA virus-based expression vectors. To enhance the accumulation levels of recombinant proteins in plants, we explored the application potential and effect of addition of multiple introns into the viral vector in plant virus RNA-based expression system. According to the characteristics of introns in higher plant, 11 introns were selected from the Arabidopsis thaliana genome. The intron sequences were synthesized in vitro and the introncontaining viral vectors were constructed based p TBSV-G, a Tomato bushy stunt virus（TBSV）-based vector.The Net Plant Gene server was employed to predict coding and non-coding sequence features in synthetic viral vectors in vitro. And then reverse transcription PCR（RT-PCR） and Western blots were used to verify whether the introns in the viral vector could be correctly identified and processed by the nuclear pre-m RNA processing machinery in vivo. The expression levels of green fluorescent protein gene（gfp） in inoculated Nicotiana excelsiana leaves were examined by Western blots and enzyme linked immunosorbent assay（ELISA）respectively. Those data were gathered to evaluate the validity and regularity of introducing introns in the coding region of the viral genome. The Net Plant Gene software could accurately identify all introns inserted into the viral coding region. The result indicated that it is a useful tool for prediction of coding and non-coding sequence features in designed viral vector before experimentally inoculated to plant. The results of RT-PCR showed that the sizes of amplification products in control group were larger than those in the experimental group and it was consistent with the theoretical prediction. Western blots analysis showed that the tagged proteins（P19, P33 and P92） could be detected by using the respective antibody. The results of RT-PCR and Western blots indicated the artificial introns in the viral vector can be correctly processed in the nucleu

关 键 词：植物RNA病毒 番茄丛矮病毒(TBSV) 表达载体 内含子 烟草 

分 类 号：S-3[农业科学]