The Identification On The Function Of EaCBF1 Gene In Eupatorium Adenophorum

Croftonweed (Eupatorium adenophorum Spreng), originated from Mexico to Costarica in Central America, is a poisonous and invasive weed. In China, Croftonweed has invaded Yunnan, Sichuan, Guangxi, Guizhou, and Xizang Province. It still continues to spread northward from the south to the north in China. In this process, the temperature is a key environmental factor that will impact significantly on the range and speed of its invasion. Thus, it’s meaningful to discover its invading mechanism, forecast its potential invading ranges, and prevent its occurrence in other places by researching the mechanism on the cold tolerance of croftonweed. The physiological parameters and expression of EaCBF1 of cold-susceptible BS, cold-tolerant HGS, and transgenic plant TG1 transferred into BS, with HGS-CBF1 were compared to reveal the difference in physiological metabolism and gene expression level so that it could enucidate the effect of EaCBF1 on cold-tolerant capability of the croftonweed. That will provide theraticl basis with adaptation mechanism to cold stress.The low Semi-lethal temperature (LT50) and the change of electrolyte leakage of transgenic BS plant TG1 with over expression of HGS-CBF1 was compared with BS and HGS populations. It was found that TG1 had -6.53℃LT50, which was similar to -7.20℃of HGS and much diiferent from BS..After treatment at -8.0℃, TG1 exhibited similar level of electrolyte leakage with HGS, whereas BS had much higher up to 73.8%. At -10.0℃or below, all three plants maintained the same level in electrolyte leakage. The result showed that over expressing of EaCBF1 could improve the resistance of croftonweed to the cold stress.The comparison of Chlorophyll fluorescence parametre Fv/Fm changes of BS, HGS and TG1 showed the value of Fv/Fm of TG1 began to decrease at -8℃, being 2℃higher than that of BS at -6℃. Fv/Fm of HGS did not reduce upto at -10℃. That indicated that over-expression of EaCNF1 provented freezing from injury that could maintain the normal photosynthesis of chloroplasts.The content of MDA, soluble sugar, soluble protein,SOD, chlorophyll of BS, HGS and TGI have been bioassayed after treated at -2℃for four days.The results showed that the content of MDA in BS and HGS increased 3.02 and 2.74 times higher respectively than that of the control, while the content in TGI was just 1.19 times higher. The contents of soluble sugar in BS, HGS and TG1 were 1.24,5.98 and 2.79 times higher than those of the controls respectively. There was just 48.7% soluble protein left in BS after the treatment, but in TG1 there was still 71.9% left while in HGS it was 1.31 times higher than the control. After treated for 4 days, SOD content in BS, HGS and TG1 were 1.16,1.21 and 1.24 times higher than the controls respectively after treated for 2 days. The contents of chlorophyll of BS and TG1 were significantly different with 59.4% and 90.6% after treated for 2 days, while 85.8% content in HGS. Hence, overexpression of EaCBFl could reduce the peroxidate injury caused by freezing to the lipid of croftonweed leaves, and positively adjust the substance content dissolved in the cell to maitain osmotic balance of the cell that could improve cold-resistant capacity of croftonweed.The expressions of EaCBF1 in BS,HGS and TGI after treated at 4℃for different time have been detected by qPCR and semi-quantitative RT-PCR.The results showed that the over expression of EaCBF1 in TG1 was much higher than that in BS and HGS, it increased gradually by treating the plant in 4℃for longer, and it didn’t reach the maximum until treating the plant for 24h;the expressions of EaCBF1 in BS and HGS increased firstly and then go down during the treatment,but the maximums occur at different time, BS get to the maximum in 1h when being treated while HGS reached the maximum in 4h.This difference in gene expression level and time might be the main reason contributing to differences in their cold-resistance.Using the stem of croftonweed which were cultred in virtro as the explant,we Transferred the HGS-EaCBFl into HGS by Agrobacterium-mediated transferation,so that the transgenic can express EaCBF1 in a higher level,then observe and test how cold the transgenic can endure.We have got a few seedlings that are resistant to the kanamycin by now, and a further step is being needed to identify the positive plant by molecular test.