Molecular Identification Of Some Phytoplasma Diseases And Cloning The Imp Gene Of Jujube Witches' Broom Phytoplasma

Phytoplasma is a kind of important prokaryotic pathogen which caused many plant diseases,and it is also an obligate parasitism in plant phloem.There were nearly thousand of plants endangered by phytoplasma around the world,which caused great loss to economic crops,food crops and ornamental plants.Due to the wide spread of the phytoplasm,it was necessary to strengthen the detection and identification of phytoplasma In this study,the pathogens of several plants suspected to be phytoplasma were investigated and identified,and the immunodominant membrane protein gene imp was cloned and heterologously expressed.The main results were as follows:1.About twenty plants with symptoms of witches' broom,yellowing,and leaf rolling were investigated and sampled.The pathogens were detected by PCR method with universal 16 S rRNA gene primers of phytoplasma.As a results the samples of mulberry witches' broom ?Robinia pseudoacacia witches' broom(RpWB)?Euonymus bungeanus witches' broom(EbWB)?Robinia pseudoacacia cv.Honghuahuai witches' broom(RpWB-HHH)?apricot leafroll and poinsettia branch-inducing were detected with phytoplasma,and they were also judged to be phytoplasma disease.2.The mulberry witches'-broom(MWB)phytoplasmas were identified by PCR method and sequence analysis with 16SrDNA?ribosomal protein gene rp?elongation factor tuf gene and transport channel protein gene secY.The results showed that the 16 S rDNA sequence of MWB phytoplasma had the highest similar rate with 16Sr?group B subgroup members which got to 99.12%.The other sequence analysis results indicated that the MWB should belong to rp?-B subgroup,tuf?-B subgroup and secY?-B subgroup simultaneously,which had higest similar rate with these members.These conclusion were also supported by phylogenetic and simulating RFLP analysis.As a conclusion,the MWB phytoplasma should belong to the members of 16Sr?-B subgroup?rp?-B,tuf?-B and secY?-B subgroup.3.By using the 16 S rRNA,secY,rp,himA,tmkB and imp gene sequences,the relationship among the jujube witches' broom(JWB),EbWB,RpWB and RpWB-HHH were investigated.As a result,the16 S rDNA sequences of the four phytoplasmas were so closely related and all should belong to16Sr?-B subgroup.The phylogenetic analyses of the secY and rp gene sequences revealed that they all belong to the rp?-C subgroup and secY?-C subgroup.Even in the comparison with himA and imp gene sequences,the 4 phytoplasma had a high similarity rate with each other which more than 95%.However,when the tmkB gene sequences were used,the EbWB phytoplasm were separated from other three phytoplasma,which had only 74% similarity rate between them.4.The immunodominant membrane protein gene imp was amplified and cloned,and the transmembrane domain was predicted.The prokaryotic expression recombinant vector was constructed for expressing Imp protein.As a result,the Imp protein was predicted with one transmembrane domain,and the Imp protein could't expressed unless the transmembrane domain was removed.