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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (33): 162-167.doi: 10.11924/j.issn.1000-6850.2014-2440

所属专题： 园艺

• 生物技术科学 • 上一篇    下一篇

miR172超表达载体的构建及其在micro-Tom番茄中的遗传转化

许慧珍,鞠正,杨永芳,田慧琴,朱本忠   

  1. 中国农业大学,中国农业大学食品科学与营养工程学院,中国农业大学食品学院,中国农业大学食品科学与营养工程学院,中国农业大学食品科学与营养工程学院
  • 收稿日期:2014-09-09 修回日期:2014-11-13 接受日期:2014-09-29 出版日期:2015-01-08 发布日期:2015-01-08
  • 通讯作者: 许慧珍
  • 基金资助:
    国家自然科学基因 “番茄果实成熟相关 smallRNAs的全基因组分析与功能鉴定” (31271959)。

Construction of miR172 Over-expression Vector and Its Transformation into micro-Tom Tomato

  • Received:2014-09-09 Revised:2014-11-13 Accepted:2014-09-29 Online:2015-01-08 Published:2015-01-08

摘要： 为了进一步阐明miRNA在番茄不同生长发育阶段尤其是在果实成熟阶段的调控途径，利用聚合酶链式反应(PCR)从番茄(Solanum lycopersicum)的cDNA克隆出miR172基因核心片段，将该基因片段，以及pCAMBIA1300-221载体通过特异性的限制性内切酶双酶切，然后将miR172片段正向连接到载体上，转化大肠杆菌Trans5α，鉴定重组质粒正确后，利用冻融法将重组载体转入农杆菌EHA105中。再次回转大肠杆菌验证获得的重组载体，通过PCR以及双酶切鉴定是否符合预期结果，将测序结果与NCBI上的基因序列相比较，同源性高达100%。结果成功构建了适用于番茄农杆菌遗传转化的植物表达载体，接下里进行转基因植株的培育和鉴定。为通过miR172基因进一步研究果实成熟衰老机理奠定了物质基础。

关键词: 肉质性状, 肉质性状

Abstract: To reveal the pathway of miRNA regulating tomato different growth and development stage, especially in fruit ripening, the research obtained tomato (Solanum lycopersicum) miR172 gene by the polymerse chain reaction technique (PCR), then we used the restriction enzymes to digest especially gene segments and pCAMBIA1300-221 vector, and connected the gene to the plasmid pCAMBIA1300-221, then transformed into Agrobacterium by Freeze-thaw method when the recombinant plasmid was accurate. The recombinant plasmid was transformed again into Escherichia coli in order to obtain the exact plasmid by PCR and double enzyme digestion in the research, and the sequence result was homology to NCBI database up to 100%. The results indicated that tomato expression vector pCAMBIA1300-221-miR172 was constructed successfully and then we cultivated and identificate the transgenic plants. It would provide base for genetic research in fruit ripening and aging through miR172 transgenic tomatoes.