Isolation Of CBF Family Genes From Chrysanthe-Mum Lavandulifolium And Transformation Studies Of ClCBF In Arabidopsis Thaliana

Chrysanthemum(Chrysanthemum×morifolium Ramat) is traditional famous flowers of China, whose flowers are gorgeous, flowering is long and variety is wide. But the abiotic stresses limit their growth and ornamental value, such as low temperature, drought and saline. Therefore, it is an important significance to cultivate the Chrysanthemum cultivars of comprehensive resistance for broadening the scope of application, extending the viewing time, reducing the production cost. Chrysanthemum lavandulifolium is wild diploid species near the source of the cultivated-chrysanthemum, whose genetic background is simple, distribution is wide and salt tolerance is strong. The study is that the homologous genes of CBF genes family will be separated from Chrysanthemum lavandulifolium, which is important to improve plant stress tolerance in adversity conditions. Analysis of genes expression pattern, bioinformatics and transformed into Arabidopsis thaliana in order to explore the expression pattern and function of CBF genes so that understanding CBF pathways and providing the basis for cultivating chrysanthemum of new strains of strong resistance.The main results were summarized as follows:1. Searching and comparing nucleotide sequences of3genes which are in chrysanthemum transcriptome database which is built by our research group by NCBI website BlastX program. It shows that the3genes are members of AP2gene family and they all have conserved structure domain of AP2. Analysis of the expression pattern of CICBF under different abiotic stresses conditions, we processed the CICBF in different developmental stages such as salt, cold, heat, and ABA induced treatments. The results show that CICBF1is induced by stress in cold, heat and salt, it is more sensitive to salt stress. ClCBF2is only induced by salt and its expression is inhibited by high temperature. CICBF4is induced by cold, salt and ABA, and the expression is significant by cold induce. In the same stress condition, the expression is different, which suggests expression pattern of the3genes are different.2. The complete cDNA sequence of CICBF1and CICBF4were cloned by3’RACE. Its full-length sequence were1149bp and823bp, and contained an open reading frame633bp and823bp. Through analyzing CICBF promoter element, we found that CICBF1and CICBF4promoter sequence contained MYC, W-box, TATC, etc response element. While we detected the ABA response element in CICBF4promoter and which were not found in the CICBF1, this information is consistent with CICBF4gene expression.3. Through comparing multiple sequences of different species, we found that CICBF1and ClCBF4have characteristic sequence of CBF protein and they are members of CBF gene family then CICBF2is not a member of gene family. Analysis of3genes use MEGA5software, we found that ClCBF1and ClCBF4are in branch of DREB-A1, however CICBF2is in branch of DREB-A4.4. Based on the vector PBI121with promoter CaMV35S, plant expression vector pBI121--CBF was constructed successfully. Plant expression vector of ClCBF1and CICBF4were introduced intointo Arabidopsis thaliana by the method of Agrobacterium tumefaciens infection. We screened6transgenic lines of ClCBF1and13transgenic lines of CICBF4by kana screening to the transgenic arabidopsis. We have transferred the genes of ClCBF1and CICBF4into Arabidopsis thaliana successfully by RT-PCR detection to the transgenic Arabidopsis thaliana.