Study On Cloning And Functional Analysis Of Pupscs Gene In Puccinellia Chinampoensis And Alfalfa Transformation

In this study, a new P5CS homologous genes was separated by RT-PCR method in Puccinelliachinampoensis, named: PuP5CS (from Genebank: HQ637435). And introduced it into Medicago sativaL.cv.Gongnong No.2by Agrobacterium-mediated genetic transformation, in order to gain the newbreeding of alfalfa.The main result were as following:Semi-quantitative RT-PCR results show that in the roots and leaves, the gene PuP5CS is expressed,but the expression level in the root of the PuP5CS gene is lower than that in the leaves. In the leaves,PuP5CS gene was expressed higher under salt stress, alkaline stress and salinization combined stress,compares to the drought stress. In the leaves, the four kinds of stress can induce PuP5CS geneexpression with different pattern.The subcellular localization result shows that PuP5CS genes are mainly expressed in the cellmembrane and nucleus.When testing the salt tolerance and cold resistance of transgenic tobacco, the overexprssiontransgenic tobacco plants under low high salt stress and temperature stress can significantly improve itsproline accumulation, chlorophyll content and soluble sugar content compares to the control, whilethese in antisense transgenic tobacco plants under abiotic stress is relatively lower than the control.According to the above, it can be concluded that PuP5CS gene affects not only the tobacco prolinesynthesis process, but also participates in the soluble sugar, MDA, chlorophyll a and chlorophyll b.From the increased osmotic adjustment substances alleviate membranous oxidation, enhancedphotosynthesis and other aspects, PuP5CS gene increases the overall stress resistance of tobacco.An efficient regeneration method of Medicago sativa L. cv. Gongnong No.2has been estanblished.The cotyledon was selected as explant for induction. Select the UM medium+2,4-D2mg/L+KT0.25mg/L as the callus induction medium,UM medium containing1.2mg/L KT for regeneration and1/2MS medium as the rooting medium. After co-cultured for3days, the cotyledon was then transferred onthe UM medium containing2,4-D, KT20days, then transferred on the embruogenesis inductionmedium, which contains1.2mg/L KT,2mg/L glufosinate,500mg/L cefotaxime. After20-40days,shoots grew up from the surface of the calli, then transferred on the1/2MS medium which contains1mg/L glufosinate,500mg/L cefotaxime, after rooting transplant the shoots to vermiculite: soil1:1mixture soil. By using PCR and RT-PCR validation, the result shows that the PuP5CS gene has beenintegrated into the genome of the regenerated plants.