Cloning And Preliminary Function Analysis Of DnWEKY29Gene

Dendrobium is a kind of important medicinal ornamental plants in China, and the germplasm resource is deficient. WRKY superfamily, belonging to plant-specific transcription factors, plays important roles in the regulation of plant growth, development and stresses responses. In the present study, a full-length cDNA sequence of WRKY gene was cloned from Dendrobium officinale. Its nucleotide and protein was analyzed with bioinformatics technical. Moreover, to primarily analyze the function of the WRKY gene under abiotic stress, and also to provide theoretical foundation for Dendrobium growth, a plant expression vector was built to transform the full-length cDNA into tobacco. The main results are as follows:1). PCR primers were designed based on the Phalaenopsis amabilis cDNA library and conserved domain of WRKY transcription factor of other plants. The full-length cDNA sequence which was1276bp in length was cloned from Dendrobium officinale through rapid amplification cDNA end (RACE) and RT-PCR techniques.2). Bioinformatics analysis indicates that the sequence contains an open reading frame of930bp and encodes310amino acids. A protein encoded by the foil long cDNA contains one conserved domain WRKYGQK and the C2H2motif. The WRKY protein molecular weight is78.17KD, and theoretical pI value is5.03. The sequence is highly identity to other WRKY factors, belonged to group Ⅱ WRKY family, and named DnWRKY29.3). The plant expression vector SN1301-WRKY29was built and transformed into a tobacco by Agrobacterium tumefaciens EHA105. The results showed17transgenic tobacco lines through hygromycin resistance screening and PCR-based screening.The lines of transgenic tobacco(T0)were grown,and the T1generation seeds were obtained. The T0-8, T0-13. T0-15lines and their T1generation seeds were choosed for further research of gene function.4). The wild-type and three T1generation seeds were sown in1/2stress medium which contained200mM NaCl and200mM Mannitol.Compared with the wild type plants, germination rates, root lengths and fresh weights of DnWRKY29transgenic plants were more sensitive to NaCl and Mannitol, showed more sensitive by NaCl stress.While there are minor differences during the DnWRKY29 transgenic plants. The DnWRKY29maybe involved in abiotic stresses, and speculated as a transcriptional repressor..5) The wild type and T1generation tobacco seeding which had been grown for four weeks in1/2Hoagland culture medium had been under200mM Mannitol stress for24hours.The seedings were made into a certain proportion of solution,and differences in SOD, POD, CAT activities and MDA content were detected.The results showed that compared with the wild type lines,the SOD、POD、CAT activities of transgenic lines was decreased and the MDA contents was increased. It was suggested that the transgenic tobacco which were more sensitive to stress conditions had accumulated more free radicals,and inhibited the activity of protective enzymes. The protective enzymes could remove free radicals,so the enzymes activity of transgenic plants were decreased.The wild type had more tolerance and was more easily remove the oxygen free radicals,so its MDA content was significantly reduced.