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Acta Agron Sin ›› 2010, Vol. 36 ›› Issue (4): 596-601.doi: 10.3724/SP.J.1006.2010.00596

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2010-04-12 Published: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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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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