[Cloning, expression and characterization of tRNA-isopentenyltransferase genes (tRNA-ipt) from paulownia witches'-broom phytoplasma].

OBJECTIVE: To identify the tRNA-ipt gene of phytoplasmas and analyze the relationship between tRNA-ipt and synthesis of cytokinin as well as pathogenesis in phytoplasmas. METHODS: The paulownia witches'-broom phytoplasma (PaWB) tRNA-ipt gene was expressed in E. coli and specific antibody was prepared. Then the growth curve and cytokinin contents of E. coli with PaWB tRNA-ipt were measured by photodensitometry and ELISA respectively. RESULTS: The length of tRNA-ipt genes from PaWB as well as mulberry dwarf, periwinkle virescence and Chinaberry witches'-broom phytoplasmas were 876 bp. All these genes encoded the proteins consisting of 291 amino acids. They contained and indentical motif (GPTASGKT) at N-terminal region related to the ATP or GTP binding sites. Four phytoplasma tRNA-IPTs shared the 99.1-99.5%, amino acid sequence indentity with each other, shared 95.4-99.3% sequence identity with the same group phytoplasmas, whereas the less than 70% identity with 16SrX apple proliferation and 16SrXII Australia grapevine yellows phytoplasmas. The expression of the tRNA-IPT protein and localization in the phloem in phytoplasma-infected paulownia were confirmed by Western blotting and immunofluorescence detection. The determination of growth curve suggested that the growth rate increase of E. coli was affected by the transformation of exogenous tRNA-ipt gene,which might be associated with the cytokinin accumulation. CONCLUSION: This protein was assumed to be involved in the cytokinin synthesis in phytoplasmas as well as other bacteria, which may play an important role in pathogenic processes of phytoplasmas and symptom development.

[Complete sequence of a full-length DNA and molecular characterization of one plasmid from chinaberry (Melia azedarach Z) witches'-broom phytoplasma].

OBJECTIVE: To clone plasmid from chinaberry witches'-broom phytoplasma and analyse its molecular characterization. METHODS: Fragments of one plasmid (pCWBFq) in chinaberry witches'-broom phytoplasma-Fuqing strain (CWBFq) were amplified with primer pairs which were designed according to plasmid sequences published on NCBI. Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares. Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe. RESULTS: One complete plasmid was sequenced from CWBFq. pCWBFq comprised 4446 bp and had a nucleotide content of 73.5% A + T and encoded six proteins. Protein P2, P3, P4 and P5 of pCWBFq contained 3, 2, 1 and 2 tranmembrane domains respectively, and their predicted signal peptide values were 0.989, 0.505, 0.918 and 0.914 respectively. Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9.6% -85.6% , however, the homology of different SSB proteins was between 74.0% - 89.4%. Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq. In addition, The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy), periwinkle virescence phytoplasma-Hainan stanin (PeVHn), chinaberry witches'-broom phytoplasma-Fuzhou strain (CWBFz) and mulberry dwarf phytoplasma - Puyang strain (MDPy), whereas, no hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain (JWBBj), cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain (BiWBNc). CONCLUSION: The plasmid encoded a replication associated protein (RepA) and a single-stranded DNA binding protein (SSB), which were for the replication of plasmid. Four putative proteins encoded by the plasmid were predicted to contain one or more hydrophobic transmembrane domains, respectively, and presumably to be localized to the membrane. The alignment and homology analysis as well as phylogenetic analysis to the DNA and encoded protein amino acid sequences of the whole plasmids and single ORFs on the known phytoplasmal plasmids showed that the different homologous sequences have distinct variation, among which the repA gene with the largest diversity appeared in all the known plasmids while ssb with less variation were only found in 16SrI plasmids. CWBFq, PaWBNy, PeVHn, CWBFz and MDPy possessed distinct plasmids in terms of number and size, whereas there was no plasmid detected in JWBBj, CLYXc and BiWBNc, perhaps as a result of low homology among repA genes in plasmids of JWBBj, CLYXc and BiWBNc.

[Immunosuppressive triterpenes from Tetraena mongolica].

OBJECTIVE: To study the immunosuppressive constituents from Tetraena mongolica. METHOD: Chemical constituents were isolated and purified by repeated column chromatography( silica gel, Toyopearl HW40C and preparative HPLC). Their structures were elucidated on the basis of spectral data analysis. The MTT assay was applied to evaluate the isolated compounds on the inhibition effect of lymphocyte transformation. RESULT: Six triterpenes were isolated and their structures were identified as follows: 3beta-hydroxy-11alpha, 12alpha:13beta,28-diepoxyoleanane(1), 3beta-(3, 4-dihydroxycinnamoyl)-erythrodi-ol(2), olean-28-al-3beta-yl-caffeate(3), erythrodiol (4), 12-oleanaen-3beta-caffeate(5), 3-O-(E) -coumaroylerythrodiol(6). Compound 24 exhibited the inhibition effects on lymphocyte transformation. CONCLUSION: Compounds 1-6 were isolated from this plant for the first time, and compound 1 was a new nature product. Compound 2-4 showed significant immunosuppressive activity.