The Cloning And Functional Analysis Of Stress-related Gene In Medicago Varia Xinmu No.1

Medicago varia Xinmu No.1 is of moderate salt tolerance and planted widely in the slightly-alkaline land with excellent economic and social benefits. The salt tolerance of Medicago varia Xinmu-1 is still to be improved further for its wide application in alkaline land due to its limited salt tolerance. This study offers references for researching salt-tolerance molecular mechanism and future molecular breeding of Medicago varia Xinmu No.l through analysis of gene functions and regulation and control of genetic expression with different types of genes related to adversity tolerance.(1) MvNHX1, MvP5CS and MvDREB1 of Medicago varia Xinmu No.l were cloned with RT-PCR and homology cloning. The RT-PCR and a Real-time PCR assay showed that the level of MvNHX1 and MvP5CS transcriptions both were up-regulated obviously in the seedlings while MvDREB1 transcription showed no remarkable variation after high salinity treatment, which indicated that MvNHX1 and MvP5CS are related to salt tolerance of the plant.(2) The promoters of MvNHX1 and MvP5CS were cloned respectively with Genome walking technology. Promoter analysis showed that both promoters have a common cis-acting element and core promoter element, and MvP5CS promoter also has cis-acting element conferring high-level transcription. With Bioinformatics method, the structure and function as well as location of MvNHX1 and MvP5CS were analyzed. Homology analysis showed that MvNHX1 belongs to Na+/H+ antiportor family, and MvP5CS shared an identity more than 70% with the known P5CS in other plants, in addition to six conservative domains of P5CS enzyme. Further analysis showed that MvNHX1 was a transmembrane protein in plasmalemma with signal peptide. MvP5CS is a hydrophilic protein in cytoplasm, which has the same domain as P5CS in other plants.(3) The MvNHX1 and MvP5CS genes of Medicago varia Xinmu No.l were transferred successfully into tobacco via agrobacterium mediation. RT-PCR analysis showed that the expression level of MvNHX1 and MvP5CS transcription in T1-generation genetic-modified tobacco was steady during salinity treatment. The germination percentage of transgenic tobacco were higher than non-transgenic tobacco under drought and salt stress. MvNHXl transgenic tobacco showed better effect on salt tolerance and MvP5CS on drought resistance. The increment of proline content of transgenic tobacco exceeded non-transgenic tobacco under salt stress, and MvP5CS transgenic tobacco had an increment of proline content higher than MvNHXl transgenic tobacco. While MDA content in non-transgenic tobacco increased sharply under salt stress, MvP5CS genetic-modified tobacco had an accumulation rate of MDA slower than MvNHX1 genetic-modified tobacco. Comprehensive analysis indicated that MvNHX1 and MvP5CS genes both could improve salt tolerance and drought resistance of transgenic tobacco, and MvP5CS had a better effect on osmotic adjustment and protection of cell membrane.