Identification For Cold Resistance And CBF₂Gene Cloning And Expression Analysis In Winter Wheat Cultivar Of Dongnongdongmai1

The cold resistant level of winter wheat varieties that could be grown in the alpine region is one of the most adapted traits. The accurate identification for the cold resistant level of breeding materials is one of the most key steps in the winter wheat breeding for the strong cold resistance. The identification results for cold resistance often appeared to show larger diversity because of the instability of the weather factors in different years based on the overwinter survival rate as the evaluation standards for the breeding materials in the field. At present, measuring the level of cold winter wheat general method is done by cooling stages after material tested the organization relative electric conductivity, simulation that the determination and a half lethal temperature (LT50).But there is also a larger difference between this approach and the common cold resistance level. To research and develop the evaluating technology for the cold resistant level of winter wheat breeding materials is the major problem which have to solve in winter wheat breeding for cold tolerance. And the genetic basis research for the cold resistance is another major aspect for cold resistance breeding, and provides the foundation of cold resistant level of genetic improvement in winter wheat.The evaluation of hardiness of winter wheat was using simulation frozen processing in this study. In the laboratory, Dongnongdongmai1, the cultivar with strong cold tolerance which registered and approval in2007, and JIMAI22which is sensitive to cold were used as the experimental material. The program for evaluating the largest freezing ability of winter wheat cultivar was determined by checking the cold hardiness level of variety in different cold conditions. The cold hardiness of Dongnongdongmai1was measured through the method of the investigation for overwinter survival rate and determination of conductivity, and the comparison of the two varieties in cold hardiness was conducted, A large number of experimental research showed that CBF transcription factors play a certain role in the cold adaptation of the plant cold resistance and enable the plant stronger cold resistance.The CBFs famliy gene and related gene were cloned with high cold resistance variety, Dongnongdongmai1as material in this study, which were CBF2(AY951945), CBF4(AY951945), CBF5(AY785902.1), CBF9(AY785905.1), CBF14(AY785901.1), ICE41(EU562183.1) and ICE87(EU562184.1) and verificated the sequence of cloned, and then furthermore, the real-time fluorescence quantitative PCR technology was used to analyze the verified genes quantitatively and to learn the CBF gene expression in Dongnongdongmai1, the high cold resistance varieties. The research results are:1) The were reviving rate was above70%at low-temperature of-12℃for24h in Dongnongdongmai1.2) In measuring the electricity conductivity in the leaf and sheath of wheat, it was found that the conductivity in leaf was always higher than that in leaf sheath after chilling, or the injury in leaf was heavier than that in sheath; The extent of the damage in leaf did not related with the reviving rate in wheat, but the leaf sheath is crucial part in deciding the reviving rate level in wheat. Through the fitting Logistic equation the LT50of sheath Dongnongdongmai1was19.55℃.3) By using the simulation freezing condition designed in the trial the cold resistance potential of winter wheat seedlings can be accurately identified.The plant age in identification of the cold resistance should be up to3～5leaves, freezing test should use program G. Dongnongdongmai1by the program G after treatment, and the half a lethal temperature (LT50) is-33℃; The LT50JIMAI22is-16℃.4) The sequences of cloning in Dongnongdongmai1are named C2、C4、C5、C9、C14、141and187.The results showed that the sequence of C2was the member of CBF2in the CBFs family; C4,141and187sequence determined respectively the common wheat3B on the chromosome part of the sequence. The similarity of Emmer wheat with C5sequence was97%. The similarity of C14and Triticum aestivum clone BAC1031P08was up to100%.5) After the frozen processing, the multiple sequences were obtained from cloning of Dongnongdongmai1, one of them was CBF2gene fragments, and its length was482bp, and there were AP2/EREBP structure domain and2sagments of conservative sequence. The similarity of this segment with Triticum monococcum of COR/DRE bindg fartor2(AY951945.1), Secale cereale of CBFIVa-2B (ABY59787.1) and the barley CBF2(ABE02653.1) were100%,99%and100%.6) CBF2in Dongnongdongmai1leaves at4℃for4h marked the beginning of expression, the peak at6h, and the minimum at12h, and the expression in other treatments changed little, and increased slightly in the treatment of3℃2h and then up to the nomal temperature, the amount of CBF2in Dongnongdongmai1in leaf sheath at4℃10min was the largest, with the extension of time expression decreased.7) The corresponding protein sequence of N end40-60regional amino acid translated by CBF2sequence showed a strong hydrophobic in Dongnongdongmai1.Other cloning sequence and corresponding target sequences showed a greater difference, but mostly was on chromosome3B of wheat on one sequence of instructions, indicating the wheat chromosome3B was activated in the early cold acclimation.