The Cloning Of Cold Tolerance Gene ICE1 Among Invasive Plant Croftonweed

Croftonweed (Eupatorium adenophorum Spreng.) originated from tropical and subtropical regions in central America. During its invasion process to China, however, this specie had naturally involved cold-tolerant populations through physiological adaptability differentiation, which may be genetically determined.Thus it could adapt to invade northeastern regions with lower temperature further. By comparing tolerance abilities to the low-temperature, the former researchers showed that Baise Guangxi(BSG) population was identified as the cold-susceptible population with the highest semi-lethal low-temperature and freezing injury index, while, Huangguoshu Guizhou(HGG) population was regarded as the cold-tolerance population which had the lowest indexes of the two parameters. The key low-temperature responsive transcription factor CBF (C-repeat binding factor) and its downstream COR(cold responsive) gene were cloned, and the expression of the two genes were also compared between BSG and HGG populations under cold stress. The results showed that HGG population had a higher expression lever of the two genes than BSG population.However, this has not clarified what causes these differences in gene expression.ICE1(inducer of CBF expression) is an upstream factor that positively regulates the expression of CBF, which regulates the expression of a series of cold induced genes by associating with CRT/DRE DNA regulation element,and it can improve the ability of plants to cold tolerance. Through cloning and expression analysis of EaICE1, the role of EaICE1 is studied in regulation of downstream genes EaCBF and EaCOR in order to clarify the cause of differences in gene expression of those two genes,then explain the reasons for the high cold-tolerance of HGG population.1. Screening the differentially expressed genes between the cold-susceptible and cold-tolerance populations of Croftonweed by SSH.In this study, a subtractive cDNA library was constructed using suppression subtractive hybridization(SSH) with the HGG population as the tester and the BSG population as the driver under cold stress. Part of clones were randomly selected for DNA sequencing, and the results may provide some information for the further study of Croftonweed adaptability differentiation.2. The cloning and structural analysis of gene EaICE1, an upstream transcription factor of CBF, isolated from Croftonweed.EaICE1 was cloned from Croftonweed(HGG) by RACE(rapid amplification of cDNA ends). The full-length cDNA of EalCEl is 1652bp long, with a 1503bp ORF(open reading frame) encoding a Myc-like bHLH transcription factor of 501 amino acids. The amino acid sequence coded by EaICE1 possessed a characteristic structure domain(bHLH), which plays an important role in DNA binding and the activation of downstream CBF. Comparing the EaICE1 amino acid sequences isolated from cold-tolerance population(HGG), cold-susceptible population(BSG) and two intermediate populations of Yunnan Jinghong (JHY) and Dali (DLY), it showed that two histidines was truncated in the HGG poputation compared to the others at the point of 45th aa. The truncation of the amino acid sequences is speculated to be the result of the directional selection in low temperature. The differences in protein function, and its relationship with cold-resistance need to be verified further.3. EaICE1 expressions of dfferent Croftonweed populations under low-temperature stress.Semi-quantitative RT-PCR, Northern blot and real-time PCR were used to study EaICE1 gene expression of four Croftonweed populations under 4℃. The results shows that EaICE1 was expressed constitutively, and the expression differences were significantly found between HGG and the other populations. The expressional level is higher in BSG population than HGG population before treatment, but EaICE1 is not activated at this time because ICE1 could activate CBF expression only at low temperatures. After 4℃treatment for 0.5h, this gene expression of the cold-tolerance population(HGG) rapidly rose, then slowly decreaced. However, the expression of the cold-susceptible population(BSG) reached the peak after 4℃treatment for 4h. This difference may cause the different expressions of the downstream genes, which leads to different low-temperature tolerance abilities between the two populations.In summary, we speculate that,the truncation of the amino aeid sequences and different expressions of EaICE1 in cold-tolerance population (HGG) maybe caused by genetic adaptability differentiation under low-tempreture.