Cloning And Analyzing Of HaDREB2C And The Establishing On Regeneration System Of Haloxylon

Haloxylon is a kind of vegetation types widely distributed in Central Asia desert, with the strong ability of against of drought and salt. It is an effective way to obtain high-quality and neat Haloxylon seedlings by using culture and rapid propagation technique, which has a positive sense to protect and reproduce endangered species and molecular research in Haloxylon. Assimilation branches, stem,hypocotyl,cotyledon are usually used in Haloxylon regeneration system. A good foundation is been laid in establishing a genetic transformation system with Haloxylon assimilating shoots as explants in this study.DREB transcription factors are widely found in Arabidopsis, maize, tomato, tobacco and rice and other plants,which involve in the regulation of gene expression on various functions,but current researches on Haloxylon molecular biology are still in its infancy. In this study, a new Haloxylon transcription factor gene was cloned and studied on its basic characteristics and biological function, which proved that the gene had same basic characteristics with DREB transcription factor genes. With these results, a theoretical foundation was provided to insight the molecular mechanism on plant stress response reaction and excellent candidate genes were supplied on plants improvement of using genetically engineered manner.The main findings are as follows:(1) Establishing Haloxylon Callus Induction and Plant Regeneration System. With MS as the basic medium and different groups of Plant Growth Regulator and concentration ratio, the effects of growth regulators on Haloxylon callus formation and differentiation were studied. The results show that the Haloxylon assimilating shoots were optimal explants in callus formation, 1.0 mg / L 2, 4-dichloro-acetic acid(2, 4-D) and 1.0 mg / L KT inducible formation of Haloxylon tissue.(2) With high-throughput transcriptome sequencing on drought stress Haloxylon materials in early study, a differentially expressed sequences HaDREB2C was screened out according to the differents in expressing. A HaDREB2C transcription factor genes(accession number KU523927) was cloned with gene-specific primers, and was identified as typical DREB2 transcription factor genes after amino acid sequences alignments.(3) According to bioinformatics analysis, the ORF length of HaDREB2C was 1125 bp, which encoded a protein of 347 amino acid. Online prediction showed that the gene was a non hydrophilic secretory protein and there was no transmembrane structure. The results showed that the expression of the gene were subject to drought, high salt stress, after tests on the expression of the gene in different stress conditions by semi-quantitative RT-PCR.(4) A yeast recombinant plasmid was constructed and transferred into the yeast AH109. Positive transformants were screened out on SD-Trp and SD-Trp-His-Ade medium. Experimental results showed that HaDREB2C yeast could grow on SD-Trp-His-Ade medium and ?-galactosidase activity color reaction presented blue, which means that HaDREB2C had the transcriptional activation function.(5) Transgenic tobaccos were obtained by Agrobacterium mediated transformation of tobaccos after constructing a plant expression vector of pCAMBIA1304-HaDREB2C.