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作物学报 ›› 2010, Vol. 36 ›› Issue (4): 596-601.doi: 10.3724/SP.J.1006.2010.00596

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

紫花苜蓿锌指蛋白基因RNAi表达载体的构建及在苜蓿的转化

秦智慧1,2，晁跃辉1，杨青川1,*，康俊梅1，孙彦3，王凭青2,*，龙瑞才1   

  1. 1中国农业科学院北京畜牧兽医研究所，北京100193；2重庆大学生物工程学院，重庆400030；3中国农业大学100193
  • 收稿日期:2009-11-10 修回日期:2010-01-07 出版日期:2010-04-12 网络出版日期:2010-02-05
  • 通讯作者: 杨青川, E-mail: qcyang66@yahoo.com.cn, 王凭青, E-mail: wang_pq@21cn.com
  • 基金资助:

    本研究由国际科技支撑计划项目（2008BADB3B05）和现代农业产业技术体系建设专项资金资助。

Construction and Transformation of RNAi Vector of MsZFN Gene from Alfalfa (Medicago sativa L.)

QIN Zhi-Hui1,2,CHAO Yue-Hui1,YANG Qing-Chuan1,*,KANG Jun-Mei1,SUN Yan3,WANG Ping-Qing2*，LONG Rui-Cai1   

  1. 1Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing100193,China;2Bioentineering College of Chongqing University,Chongqing 400030,China;3College of Animal Science and Technology,China Agriculture University,Beijing 100191,China
  • Received:2009-11-10 Revised:2010-01-07 Published:2010-04-12 Published online:2010-02-05
  • Contact: YANG Qing-Chuan, E-mail: qcyang66@yahoo.com.cn, WANG Ping-Qing, E-mail: wang_pq@21cn.com

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2205

被引次数

15

摘要：

根据紫花苜蓿锌指蛋白MsZFN基因(GenBank登录号为EU624138)序列，设计两对含有酶切位点的特异性引物，以紫花苜蓿cDNA为模板，分别合成用于构建干扰载体的正反义片段，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。利用农杆菌介导方法，将pART-F-R转化到紫花苜蓿中，经过PCR检测，获得了3株转基因植株。经过RT-PCR检测，证明转基因植株中MsZFN基因表达量与未转基因的植株相比，明显降低。结果表明，构建成功具有发夹结构的RNAi载体pART-F-R，它可有效的抑制紫花苜蓿MsZFN基因。

关键词: 紫花苜蓿, 锌指蛋白, RNAi, 转基因

Abstract:

Based on the sequence of Medicago sativa Zinc Finger Protein (MsZFN) gene (GenBank accession No. EU624138), two pairs of specific primers containing different enzyme sites were designed. With the template of full-length cDNA , 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 constructed. Mediated by Agrobacterium tumefaciens, pART-F-R was transformed into alfalfas. PCR testing showed that three transgenic plants were obtained. Result of RT-PCR showed that transgenic alfalfas had lower expression level of MsZFN gene than wild alfalfas. Those results indicated that the RNAi vector pART-F-R containing a hairpin structure was constructed successfully and highly efficient for the simultaneous silence of MsZFN gene.

Key words:   Alfalfa(Medicago sativa L.), Zinc finger protein, RNAi, Transgene

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