Cloning And Functional Analysis Of CsbHLH2 In Tea Plant(Camellia Sinensis)

Due to its great economic and ecological benefits, tea(Camellia sinensis) has become one of the most important economic trees cultured in China. However, the growth and yield of tea trees suffer seriously from metal deficiency, pathogen infection,drought, salinity, and cold. It is important to reveal the molecular mechanisms of regulatory role of metal homeostasis and the stress tolerance in tea plants, especially Fe homeostasis, that will provide a theoretical basis for the molecular breeding of tea.Therefore, The b HLH transcription factor Csb HLH2 was cloned from tea plant(Camellia sinensis) cultivars ‘Shanchayihao’. And its sequence features, subcellular localization, expression patterns in different tissues and expression patterns in different abiotic stress and hormone treatment were analyzed. At last, the overexpression analysis of Csb HLH2 from tea in transgenic Arabidopsis plats showed that tea Csb HLH2 play an important role in both the progress of metal homeostasis and the stress tolerance. This research obtained such experimental results as follows:1. The b HLH transcription factor Csb HLH2 was cloned from tea plant(Camellia sinensis) cultivars ‘Shanchayihao’ by homologous cloning technology using c DNA template. Bioinformatics analysis showed that the Csb HLH2 contains 714 bp ORF and was predicted encoding 297 amino acid, the deduced protein molecular weight was 58.4 k D and its theoretical isoelectric point was 5.14. Amino acid sequence alignment revealed that Csb HLH2 was highly homologous to other higher plant b HLH proteins. Quantitative real-time PCR analysis of the expression profiles showed that the Csb HLH2 gene was constitutively expressed in bud, leaf, stem and root. The highest expression level of the Csb HLH2 was found in the young leaf.2. Real-time PCR was employed to real the expression patterns of CsbHLH2 in young leaves after treated by ETH, SA, ABA, MeJA and abiotic stress of high salinity and low-temperature. The results indicated that CsbHLH2 could be induced by high salinity, ETH, MEJA and SA treatment, respectively, but had different expression patterns.3. Transient expression of recombinant plasmid Csb HLH2/PBI221-GFP in Arabidopsis protoplasts use the method of PEG-Ca2+ showed that Csb HLH2 was located in cell nuclei.4. Plant expression vectors of constructed by ligating the cloned cDNA of Csb HLH2 to vector pBI121(contain GFP).Using flower dipping, the constructed vectors were transformed into Arabidopsis mediated by Agrobacterium tumefaciens. The successfully transformed plants were verified by PCR. Then the expression patterns of Csb HLH2-GFP were determined by Western blot. At last, 9 Arabidopsis transgenic lines were obtained.5. To investigate the function of Csb HLH2 protein, we generated Csb HLH2 over-expression transgenic plants. The result show that, over expression transgenic plants in different growth media have significantly different wild-type, Where in the transgenic plants grown on medium without iron and calcium significantly better than wild-type, but seeds grown in the medium contain ABA show much more intense than the wild type, Illustrate of plant genes involved in the transport of metal ions and may be involved to drought stress resistance in plants. In addition, transgenic Arabidopsis plants more sensitive to high salt, relative to wild-type showed late germination and slow growth, indicating that the gene play a negative regulation role in plants resistance high salt.