Transformation And Analysis Of Temporal-spatial Expression Of Ptcbf Trifoliate Orange (poncirus Trifoliata (l.) Raf.) And Ponkan (citrus Teticulata Blanc)

Citrus suffer environmental stress frequently.The stress,such as cold,drought, saline-alkali and so on,causes the citrus to serious quality and quantity drop and become a serious obstacle to citrus industry.Therefore,researching citrus response to environmental stress mechanism and using genetic engineering to improve its resistance have become the key work for further citrus development.Being a key factor in regulating the transcription of a series of genes related to cold acclimation,CBF(C-repeat binding factor) is induced by low temperature rapidly and improve transcriptional level of related genes by binding to CRT/DRE (C-repeat/dehydration responsive element) element specially,further lead to the synthesis of sugar,proline,proteins contributed to the stabilization of biomembrane,and some other substances,thus enhance the cold tolerance of plant.Further studies on gene transformation testify that the cold tolerance of plant containing high expressed foreign CBF gene is improved greatly.This suggests a new way of improving cold tolerance of plant by genetic engineering technique.The formers in our laboratory have constructed fusion gene expression vector CG2301,with the fusion gene of CBF-GFP.The main purpose of the present research is to transform trifoliate orange and ponkan by the way of hypocotyl via agrobacteriummediated. Treat the transgenic seedlings under low temperature,and observe the fluorescence by fluorescence microscopy.Main results of this study are as follows:1) Localization of CG2301.Transformed CG2301 to onion epidermal cell,then observed the fluorescence by fluorescence microscopy.It was found that the fluorescence focused on nuclear.It proved that PtCBF gene localized to nuclear.2) Fusion gene expression vector CG2301 was transformed to trifoliate orange and ponkan by the way of hypocotyl via Agrobacterium-mediated,and received resistance seedlings.Some of the seedlings had been potted and regenerated buds.3) Temporal expression of PtCBF.Treated the resistance trifoliate orange under 4℃at different times.We found that the root was more sensitivity,which fluoresce strongly after exposured 4℃15 min,and the stem and the back of leaf fluoresced weakly.With the longer processing time,we detected that it fluoresced more strongly at the spots which were just the joint of complex leaf in the face of leaf,as well as the the little leaf,the joint of leavestalk and stem.The spots that flashed greenish fluorescence in resistance trifoliate orange were uneven.We didn't find any leaves or seedlings full of greenish fluorescence. The greenish fluorescence tended to focuse on the thin,such as leaves and roots, compared with the stems.The greenish fluorescence of ponkan,whose cold resistance was weaker than trifoliate orange,when exposured 4℃,exhibited slightly.We found stronger greenish fluorescence at the main nervation and stem.4) Spatial expression of PtCBF.In this work we showed that the greenish fluorescence had dramatically changes after 4℃exposured in resistance trifoliate orange. It followed the law "strong-weak-strong".The greenish fluorescence generated at 15min under 4℃,it showed stronger and maintained a high level in a matter of hours(such as 2h, 4h,8h),but it became slighter in 16h,and re-brightened in 20h.The greenish fluorescence was found much brighter at 24h and 48h.However,the greenish fluorescence of ponkan, whose cold resistance was weaker than trifoliate orange,was not related to the treated time.