Influence Of CBF₃ And COR15a On Cold Resistance Of Tobacco

Tobacco (Nicotiana tabacum L.) ,which belongs to Nicotiana,Solanaceae and derives from America Oceania and south Pacific islands, was introduced into China in the Ming Dynasty Wanli and now contains about 66 species. It is a warm-season crop, ceasing growth at 10¹3℃ and perishing at 2℃. The optimum temperature for the growth of seedlings is 24℃C, and the fast growing temperature is at 25²8℃. While temperature above 35℃ will do harm to the seedlings. Average tempetature should be better at 24²5℃ in maturation phase. Tempetature below 17℃ will degrade the leaf quality. Therefore, chilling injury is the main limited factor in the growth of tobacco.GUS gene controlled by chilling-induced promoter RD29a of Arabidopsis thaliana was introduced into tobacco to clarify expression characteristics. And CBF3 cold response transcription activators of Arabidopsis thaliana controlled by RD29a, was introduced in order to rapidly and highly activate expression of cold-induced gene of CRT/DRE element. And cold-induced gene COR 15a was also introduced for making use of its mighty ability of defending the intracellular membrane system such as photosynthetic membrane. Cold tolerance and photosynthesis of tobacco at low temperature are expected to be improved as a reference to the improvement of cold tolerance in other crops. And the results showed as follows.1. PCR results showed that positive amplified band of tobacco K326 was obtained after RD29a-GUS and bivalent gene RD29a-CBF₃ and RD29a-COR15a were introduced, which proved that exogenous genes were successfully transformed into tobacco.2. Histochemical analysis of GUS of the RD29a-GUS transgenic plantlet showed that: Promoter RD29a which is a chilling-induced promoter doesn't have any special tissue expression and efficiently and stably expressed at 4℃. Its critical temperature of expression is at 10°C and without expression at room temperature 25℃.3.Cold tolerance test showed that transgenic plantlet with RD29a-CBF₃ and RD29a-COR15ab bivalent gene didn't wilt during 48h at 1℃, but non-transgenic plantlet wilt in 12h at 1℃. Their ice point are -2℃ and -1℃, respectively. Electric conductivity was measured and described with logistic equation, and the results showed that LT₅₀ of transgenic plantlet was -1.01℃, but non-transgenic plantlet was 0.86℃ . Transgenic plantlet is lower 20% and 1.87℃ than non-transgenic plantlet in cell membrane injury percentage and the semilethal low temperature,...