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作物学报 ›› 2010, Vol. 36 ›› Issue (09): 1484-1489.doi: 10.3724/SP.J.1006.2010.01484

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

紫花苜蓿LEA蛋白基因ihpRNA表达载体构建及烟草转化

白永琴1,康俊梅1,孙彦2,杨青川1,*,李燕1   

  1. 1 中国农业科学院北京畜牧兽医研究所，北京 100193；2 中国农业大学动物科技学院草地研究所，北京 100094
  • 收稿日期:2010-01-08 修回日期:2010-04-20 出版日期:2010-09-12 网络出版日期:2010-05-20
  • 通讯作者: 杨青川, E-mail: qchyang66@yahoo.com.cn
  • 基金资助:
    本研究由国家科技支撑计划(2008BADB3B05)和国家科技支撑计划子课题(2008BADB3B04-1)资助。

Construction of ihpRNA Expression Vectorof MsLEA3-1 Gene from Medicago sativa L. and Genetic Transformation in Tobacco

BAI Yong-Qin1,KANG Jun-Mei1,SUN Yan2,YANG Qing-Chuan1,*,LI Yan1   

  1. 1 Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2 Institute of Grassland Science, China Agricultural University, Beijing 100094, China
  • Received:2010-01-08 Revised:2010-04-20 Published:2010-09-12 Published online:2010-05-20
  • Contact: YANG Qing-Chuan, E-mail: qchyang66@yahoo.com.cn

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1564

被引次数

9

摘要： 根据紫花苜蓿LEA蛋白MsLEA3-1基因(GenBank登录号： EU665182)序列，设计两对含有酶切位点的特异性引物LEAf1/ LEAf2和LEAr1/ LEAr2，以构建好的PMD-LEA质粒为模板，分别合成用于构建干扰载体的正反义片段pMD-F和pMD-R，将正反义片段分别插入表达载体pART27的相应位置，构建成含有发夹结构的RNAi载体pART-F-R，经过Not I酶切鉴定，证明载体构建成功。通过农杆菌介导的方法，以干扰表达载体pART-F-R转化烟草，经过PCR检测，得到16株阳性转基因植株，为MsLEA3-1基因的功能研究奠定基础。

关键词:  紫花苜蓿, MsLEA3-1基因, RNA干扰, ihpRNA表达载体, 烟草转化

Abstract: During long-term evolution, plant has developed various physiological functions and bio-chemical mechanisms to respond the diverse stresses in different environments. Plant cell accumulates a series of proteins to reduce cell dehydration in the period of water shortage, of all proteins, late embriogenesis abundant LEA protein has been paid attention, which is one of the hot topics in plant stress physiology. In the paper, an RNAi expression vector harboring MsLEA3-1 gene fragment from Medicago sativa L. was constructed. On the basis of the sequence of Medicago sativa LEA protein (MsLEA3-1) gene (GenBank accession number: EU665182), two pairs of specific primers containing different enzyme sites were designed. With the template of PMD-LEA plasmid constructed, positive-sense strand and antisense strand were obtained, which were separately inserted into the expression vector pART27. The RNAi vector pART-F-R containing a hairpin structure was confirmed by the digestion of restriction enzymes. pART-F-R was transformed into tobacco by Agrobacterium mediated transformation system. PCR testing showed that 16 transgenic plants were obtained.

Key words: Medicago sativa L., MsLEA3-1 gene, RNA interference, ihpRNA expression vector, Tobacco transformation

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