Cloning, Expression Of A Glutathione S-transferase Gene From Ziziphus Jujuba Mill.(ZjGSTU1) And The Functional Analysis Associated With Jujube Witches' Broom (JWB)

Glutathione transferases (GSTs) are a family of multifunctional enzymes which are known to play important roles in combating different biotic and abiotic stresses. Based on the homology comparison,GSTs are distributed into 6 classes, i.e., phi, tau, zeta, lambda, theta and DHARs in plant. Among them, the phi and tau classes are the most represented and are plant specific.Glutathione S-transferase (GST) is one of the important genes involved in protection of plants. It is expressed during normal development as well as in response to different stress conditions exhibiting major transcriptional regulation. So it is very important to study the response mechanism of GSTs to plant development and stress conditions. The glutathione transferase gene (ZjGSTU1, HM345954) was isolated by homologous cloning method and step-out PCR (SO-PCR) from Ziziphus jujuba Mill.. The related function was also studied in this paper. The main results were as follows:1 The Glutathione transferase gene (ZjGSTU1, HM345954) was firstly isolated by homologous cloning method and step-out PCR (SO-PCR) from Ziziphus jujuba Mill.. The 914bp included a complete open reading frame of 669 bp encoding a peptide of 222 amino acid residues and 3' non-coding region of 206 bp.2 Based on the comparison with other amino acids sequences among glutathione transferase family and the analysis of the phylogenetic evolution, this ZjGSTU1 belonged to a glutathione transferase Tau family. Sequence alignment show that ZjGSTU1 is highly homologous to the GSTs from other species, 84%,76%,74%,73% homologus to Populus trichocarpa, Ricinus communis, Arabidopsis thaliana and Capsicum chinense.3 Prokaryotic expression of ZjGSTU1 was carried out, and a protein of 30kDa was detected in bacterium precipitation. The NCBI analysis predicted that ZjGSTU1 encode a protein of 25kDa. And the His Tag of pET-30a was about 5.5kDa. So the expressed protein in bacterium precipitation was ZjGSTU1 expressed protein. Then purification of ZjGSTU1 and preparation of its antibody were carried out, and the ideal experimental result was obtained.4 The plantlets and leaves in field used as material, the western blot was carried out in this study. Those result showed that the expressing level of ZjGSTU1 was different among different cultivars. The expressing level of ZjGSTU1 in diseased trees increased than in healthy trees.'Xingguang', a resistant cultivar to jujube witches'broom (JWB), was also used as material to study the expressing of ZjGSTU1. After infecting by JWB, the expressing of ZjGSTU1 increased dramaticlly in resistant scions of'Xingguang'.