Construction and transformation of an over-expression plasmid of the MsZIP gene from Medicago sativa

Abstract

Abstract: Based on the MsZIP gene sequence (GenBank No. HQ911778), a cDNA fragment was cloned and connected to the PMD18-T vector to construct PMD-MsZIP. The target fragment and linear plasmid were obtained from the cloning vector PMD-MsZIP and from the plant expression vector PBI121 using dual digestion with XbaI and BamHI. The plant over-expression vector PBI-MsZIP was built through directional connections using T₄ DNA ligase. The plasmid was transferred to Agrobacterium LBA4404 by the CaCl₂ freeze-thaw method and was then transferred into alfalfa by an Agrobacterium-mediated transformation system. Eleven kanamycin-resistant plants were obtained. Four of them were sampled to detect target fragments and PCR identification showed that the recombinant plasmid had been transferred into alfalfa. The MsZIP gene was successfully over-expressed in transgenic Medicago sativa CV. Zhongmu No.1. To test the function of this gene, the transgenic alfalfa was treated with 200 mmol/L NaCl and 25 μmol/L PEG-6000 for three days, and some physiological parameters were measured. The contents of soluble sugar, soluble protein and proline significantly increased, while the MDA content declined. Over-expressed MsZIP gene enhanced the salt and drought tolerance of alfalfa.

CLC Number:

LI Yan, SUN Yan, YANG Qing-chuan, KANG Jun-mei, ZHANG Tie-jun, FANG Feng. Construction and transformation of an over-expression plasmid of the MsZIP gene from Medicago sativa[J]. Acta Prataculturae Sinica, 2012, 21(6): 182-189.

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