Low Temperature Response Analysis And Transformating Into Lemon Of Cold-induced Gene From Citrus

A cold acclimation related gene from the P.trifoliata named Ptcor8, had been cloned by our group. There exist highly homologous genes in Fortunella and C.limon, the expression of the homologous gene has the same tendency as the ability against cold in cirtus, tthe P.trifoliata could survive from -26?, while C.limon could not Besides,25-fold in Poncirus than that in lemon. The existing study by our group revealed that the homology of the pPtcor8 and pClcor8 were 88.17%, and three special regulatory elements related to stress responsiveness in pPtcor8 were found. In order to uncover the function of cold-induced gene and its promoter in citrus cold hardiness, the plant expression vector containing cold-induced gene and their promoter should be constructed. The agrobacterium-mediated transient expression was used to inject agrobacterium which containing different vectors to C.aurantifolia leaves, and transformed into C.limon, Concluding that the promoter of P.trifoliate is more sensitive to low temperature. On this occasion, the plant expression vector of pPtcor8::Ptcor8::YFP, pClcor8::Ptcor8::YFP, pPtcor8::Clcor8::YFP, containing cold induced gene and their promoter had been constructed to transform into C.limon. Finally, we got transgenic C.limon trees.The main result of this study were as follows:1.Plant expression vectors(35S::Ptcor8::YFP, pPtcor8::Ptcor8::YFP, pClcor8:: Ptcor8::YFP, pPtcor8::Clcor8::YFP) were transient expressed in C.aurantifolia leaves mediated by agrobacterium. The result showed that the fused gene was successfully expr-essed in C.aurantifolia, comparing with the leaves in room temperature, concluding that the promoter of P.trifoliate is more sensitive to low temperature.2. The C.limon transformation system was explored and optimized, finally the best formula of regeneration medium was MT+0.75 mg/L 6-BA, while adding 75 mg/L kan and lOmg/LBasta.3.The 3 expression vectors pPtcor8::Ptcor8::YFP,pPtcor8::Clcor8::YFP, pClcor8:: Ptcor8::YFP were transformed into C.limon which was not resistanted to cold, finally we got 34 resistance buds which contained pPtcor8::Ptcor8::YFP,48 resistance buds which contained pPtcor8::Clcor8::YFP,29 resistance buds which contained pClcor8::Ptcor 8::YFP. In the final,3 resistance buds which contained pPtcor8::Ptcor8::YFP,4 resistance buds which contained pPtcor8::Clcor8::YFP and 2 resistance buds which contained pClcor8::Ptcor8::YFP were successfully transformed by testing the reporter gene.