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Acta Agron Sin ›› 2010, Vol. 36 ›› Issue (09): 1484-1489.doi: 10.3724/SP.J.1006.2010.01484

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

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 Online:2010-09-12 Published:2010-05-20
  • Contact: YANG Qing-Chuan, E-mail: qchyang66@yahoo.com.cn

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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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