Chorispora Bungeana CbCBFx1 And CbCBFx4 Cloning And Function Analysis

Low temperature is one of the most important abiotic environment that affects the plant grow and development, restricts distribution and yield of the crop. In recent years, with the changes of the global climate,low temperature due to the decrease in agricultural production is severe. The yield reduction caused by the low temperature solution is imminent. It is very important to increase food production. Therefore, reinforce the research of plant freezing resistance mechanism, through the genetic and molecular techniques to improve the freezing resistance of plants is One of the important problems in the study.Many plants increase in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. Large research show that cold acclimation involves expression the C-repeat-binding factors or dehydration response elments(CBF/DREB1) core transcriptional activators, it will cause large number of physiological and biochemical changes by stimulate downsteam genes, and improve the freezing resistance of plants. Chorispora bungeanais a perennial subnival alpine plant that can survivefreezing temperature. Despite the close relationship with arabidopsis in the genus, but freezing resistance is much higher than that of arabidopsis thaliana. Both from the aspects of morphology and transcriptome analysis results, the freezing tolerance mechanism of Chorispora bungeana and Arabidopsis is very different. So to understand and make clear the freezing tolerance mechanism of Chorispora bungeana is very important.In this study, two Arabidopsis CBF homology genes, named KJ447117.1 and KJ447118.1 was isolated from Chorispora bungeana by RACE techology, Sequence analysis showed that they and arabidopsis CBF1 and CBF2 has higher homology, named CbCBFx1 and CbCBFx4 respectively. To understand the regulating of CbCBFx1, we cloned 1kb size CbCBFx1 promoter by Tail-PCR. Sequence analysis showed that the cis-acting element of CbCBFx1 promoter is similar to CBF2 promoter, it also contains ICEr1 and ICEr2 which is cold crucial regulatory regions in CBF2 promoter. RT-PCR analysis of gene expression show that those two genes can be induced by cold, in addition to, CbCBFxl can also be induced by drought, abscisic acid and NaCl. We get the overexpression CbCBFxl and CbCBFx4 transgenic Arabidopsis respectively by floral-dip method of agribacterium-mediated plant transformation. Ultimately determine the appropriate transgenic Arabidopsis by testing purpose gene expression used RT-PCR. We study CbCBFxl and CbCBFx4 function by use those two transgenic Arabidopsis. We pass the test plant half lethal temperature(LT50) to get to know its freezing resistance. In the non cold acclimation, the LT50 of Col-0 and overexpression CbCBFxl transgenic Arabidopsis are-4.05 ℃ and-7.5 ℃, and the LT50 of overexpression CbCBFx4 transgenic Arabidopsis are-6 ℃, so the freezing resistance of these two kinds of transgenic arabidopsis thaliana higher than the Col-0 significantly, surprisingly the freezing resistance is increase with expression quantity. In the cold acclimation, the LT50 of Col-0 and overexpression CbCBFxl transgenic Arabidopsis are-8℃ and-11℃, the transgenic arabidopsis freezing resistance is higher than the Col-0, but overexpression CbCBFx4 transgenic Arabidopsis LT50 is-8℃, so the freezing resistance are little difference between overexpression CbCBFx4 transgenic Arabidopsis and Col-0. In addition to this, the salt resistance of those two transgenic arabidopsis thaliana are increasing. And the Soluble sugar and proline in those two transgenic arabidopsis thaliana compare with the Col-0 are also increasing. The phenotype of the overexpression CbCBFxl transgenic Arabidopsis show that it inhibited the growth and development of plant Significantly, dwarf, leaf blade stack, leaf hairs thick, bolting later. But the phenotype of overexpression CbCBFx4 transgenic Arabidopsis have no obvious influence on the growth of arabidopsis thaliana, it show that leaf edges curl and Yellowish leaf.In brief, this paper by studying the CbCBFxland CbCBFx4 function in Arabidopsis indicated that those two genes play very important function to freezing tolerance in Chorispora bungeana.