Study On Molecular Identification And Expression Of Antigenic Membrane Protein Of Paulownia Witches Phytoplasma In China

Paulownia witches'broom(PaWB) was caused by Paulownia witches phytoplasma which spread in most of cultivated Paulownias areas in China.This disease was distributed in Shaan- xi,Hebei,Shandong,Henan,Anhui,Hunan,Hubei,Jiangsu,Zhejiang and Jiangxi provinces,etc. The rate of PaWB which outbreak reached above 80%,so it was the most impo- rtant disease to harm Paulownia production. According to the statistics in 1990,the harmed areas reach to 880,000 hectares,and the direct loss exceed to one billion RMB. Therefore,the study of PaWB is an important problem for a long time.There were some difficulty of culturing phytoplasmas in vitro and the content of phytoplasma in plants are too low which restricted the study. So a study of PaWB phytoplasma was done by molecular methods and clone antigenic membrane protein gene.The results are as follows:(1) A 1.2kb of 16S rDNA length frgement is obtained by nest polymerase chain reaction from paulownia witches phytoplasma samples which were collected from Shaanxi, Shanxi, Gansu, Henan, Hebei, Shandong Province, and also a 850bp DNA fragement of tuf (EF-Tu) gene was amplified from above samples using primers (fTufu/rTufu). Homologic analysis shows that it is the same of 16S rDNA nucleotide sequences of PaWB-Shaanxi, PaWB-Taiwan and that of PaWB from Shanxi, Gansu, Henan, Hebei, Shandong Province. All species of PaWB phytoplasma, from different regions, are believed to be a 16Sr I-D group of phytoplasma. Therefore, we can confirm classified position of PaWB phytoplasma from China.The sequences of 16S rDNA and tuf gene were submitted to the GenBank,the numbers were DQ851169 and DQ851170,respectivly.(2) PCR assays were used to amplified antigenic membrane protein gene from PaWB phytoplasma,then sequenced the gene,the result shows that amp is 696bp,GC% is 32.5%,coding 231aa,the number of GenBank is DQ515794.Homologic analysis of nucleotide is 98.39% among Paulownia witches'broom,Aster yellow and Onion yellow.Through the analysis of ANTHEPROT5.0,the results show the molecular weight of Amp is 24.7KDa,the protein is of good antigenicity. The secondary structure contain helixes,sheets and coils without turns.There are two transmembranous regions and several potential cleavage sites of signal peptide. The assay provide theoretic basis for studying function of antigenic membrane protein and hope that it can reavel disease mechanism.(3) Constructed plasmid pET30-amp,pET30-amp603 and pBV221-amp603 were transform into E.coli.,respectily. Induced in different conditions,the result of SDS-PAGE show that there are no expressed protein which were induced in E.coli..There is maybe some toxin from antigenic membrane protein. Then Constructed plasmid pPIC9K-AMP, and test the sequence with PCR,enzyme and sequencing. Although the expression of Amp was not successful,the results provide another evidence on diversity of phytoplasmas.