Cloning Of CBF-like Gene In Cinnamomum Camphora L. And Its Application On Improving Cold Tolerance Of Plants Via Genetic Transformation

Camphor tree(Cinnamomum camphora L.) belonging to subtropics evergreen broadleaf tree is an excellent landscape greening plant species and widely applicated on urban greening in southern city. Recently, with the rapid development of urban construction, botanical graden workers have made numerous attempts to introduce Camphor tree into Northern China, for enriching local evergreen tree species and improving garden landscape in winter. However, the winter in Northern China is long and extremely cold, thus dramatically limits the successful introduction, polularity and landscape application of Camphor tree. Improving cultivation and management or selecting cold-tolerance camphor tree is the key to solving the above problem, while the initiation of strategies to improve cold tolerance of camphor tree through transgenic approaches is the most essential way.As an important transcription factor, C-repeat binding factor(CBF), also called dehydration-responsive element binding factor 1(DREB1) is considered to play a key role in enhancing plants tolerance to abotic environmental stresses such as low temperature, drought and salinity. Researchers have isolated and functionally characterized numbers of CBF-Like genes encoding CBF/DREB1 transcription factors from various plant species, in recent years. It has been observed that, CBF/DREB1 genes from different plants share a great similarity in sequences and structure. Moreover, overexpression of CBF/DREB1 genes conferred an increased tolerance to one or multiple stresses in transgenic plants. Those results provide new ideas for enhancing the capability of cold resistance of camphor tree through genetic transformation.Based on the previous findings about CBF/DREB1 transcription factors, extensive work including molecular cloning, expression analysis of CBF-like genes in camphor tree, and stress resistance analysis of transgenic tobacco plants were carried out. The major results of this study were summarized as follows:1. Four actin genes fragments named CcACTa, CcACTb, CcACTc and CcACTd(accession number:KM086736, KM086737, KM086738 and KM086739)and one EF1α gene fragment named CcEF1α(accession number:KM086740)were isolated. The expression levels of Cc ACTc gene and CcEF1α gene in leaves of camphor tree under chilling stress were relatively stable according to the quantitative real-time PCR results. Both of the two genes could serve as reference gene in quantitative real-time PCR analysis, with CcEF1α being the better one.2. Four full length cDNA fragments encoding CBF transcription factor were isolated from camphor tree.They were named Cc CBFa, CcCBFb, CcCBFc and CcCBFd and submitted to the GenBank with accession numbers of KJ958932, KJ958933, KP336741 and KP336742. Results of quantitative real-time PCR showed that, the four CcCBF genes were involved in response to various abiotic stresses including cold, drought and salinity, and the gene transcription levels were also active in ABA-responsive signaling pathway.3. The coding regions of the four CcCBF genes were ligated into the modified pCAMBIA2300 vector under the control of CaMV 35 S promoter, respectively. The recombined plasmid(CcCBFb-P2300) was introduced into Agrobacterium tumefaciens EHA105, and tobacco(Nicotiana tabacum L.)was transformed using leaf-disk method. Stress resistance analysis indicated that, T1 generation of transgenic tobacco plants exhibited increased tolerance to various stresses comparied to control(wild type taobacco).This study is just the fist report of isolation and characterization of CBF/DERB1 genes, which controls the expression of a large number of stress realted genes in camphpr tree. However, this subject is an indispensible part of improving cold tolerance of camphor tree by genetic transformation.