Cloning And Transfomation Of Stress Related Mitogen-Activated Protein Kinase Gene CDNA From Medicago Sativa L.

In this article, total RNA of Medicago sativa L.(Apollo Supreme) s seedling leaves was isolated using the acid guanidium thiocynnate-phenol-chloroform extraction methed. A full length cDNA sequence of MAPK(mitogen-activated protein kinase) was cloned by RT-PCR. Sequnce analysis showed that the cloned full length cDNA sequence of MsMMK4 gene have 1116 base pairs, encod 371 amino acid. Comparing with the reported cDNA sequence of MsMMK4(X82270)gene in GeneBank, the cDNA sequence of MsMMK4 gene had 14 base pairs exchange, but the deduced amino acid sequence had 100% homology. The obtainnned gene fused with expression vecter pRDN that involved inducible promoter rd29A and pPZP221 involved constitutive promoter CaMV35S, and the constructed expression vecters pRMN and pPZM were obtained, respectively.Tobacco plants were transformed with the two recombined expression vecters by Agrobacterium-mediated transformation. Then, the gentamylin-resistant tobacco transfomants were obtained. PCR detection demonstrdted that the target gene has been integrated into tobacco genome. Six and ten Transgenic tobacco plants transformed with the pRMN and pPZM were obtained, respectively. And the pRMN transgenic tobacco plants could nomally grow under the theatment of drought, while the control tobacco was faded. This results demonstrated that the pRMN transgenic plants showed drought tolerance under the theatment of drought. Some obvious morphological changes including dwarf and growen slowly have been observed in a few constitutive promoter CaMV35S transgenic tobacco plants compared to inducible promoter rd29A.