Molecular Characterization Of The Multi-locus Genes And Preservation Research Associated With Phytoplasmal Diseases In Yuanmou County Of Yunnan Province

Phytoplasmas are phytopathogenic prokaryotes which lacking cell walls and inhibit phloem sieveelements in infected plants.There are hundreds of kinds of plants have been infected byphytoplasma,and the diseases have caused great damage.Today, phytoplasma still cannot be cultured invitro. Using advantages of abundant phytoplasma resources in Yuanmou county of YunnanProvince.This study major in three aspects.The first, by adopting the method of molecular biology.Extracting the total DNA of plants which have could been infected phytoplasma in Yuanmou county,using universal primers and specific primers to amplify phytoplasmal16SrDNA gene, ribosomal protein(rp) gene, transport protein (secY) gene and the immune membrane protein (Imp) gene which areconservative in the process of evolution,then cloned and sequenced.Through the multi-locus sequencesanalysis associated with phytoplasmal diseases in yuanmou of Yunnan, Further clear about theirtaxonomic status.The second, the characterization and structure prediction of protein of Imp, which wasin the sec protein translocation system and cell membrane system respectively, were analyzed and to dothe prokaryotic expression.Thirdly, preliminarily research the method of preserving phytoplasma at thelow temperature in a long time.The main results are as follows:1）This study detecting and identifying seven phytoplasmas diseases at molecular level. For thefirst time to find and identify clearly the disease associated with sword bean witches’-broom. In china,cauliflower witches’-broom diseas and cowpea virescence diseas is also first detected and identified,andtomato witches’-broom diseas, peanut witches’-broom diseas, parthenium virescence diseas is first timein Yuanmou county of Yunnan province., The results of molecular identification is that cauliflower yetbe infected by phytoplasma belong to16SrII group,except the16SrI, III and XIII groups havereported.Based on the16S rDNA、rp and secY gene,molecular identification and the multi-locussequences analysis associated with these seven phytoplasmal diseases to point out that they wasclassified in16SrII group, subgroup A,related to candidatus Phytoplasma australasiae.Sequence analysisand the phylogenetic of rp gene show that these phytoplasmas was clustered in the rp subclade（iii）with PnWB、 SPWB、 SEPN、 SEPT and alfalfa witches’-broom phytoplasma related to theCa.P.australasia.About secY gene they have high homology with PnWB，SEPT，SEPN，SPWB of16SrII-A and TBB of16SrII-D.All of the three genes have similar analysis results.2） The Imp genes of seven phytoplasmas were amplified using IMP(II)F and IMP(II)Rprimers,cloned and sequenced,The obtained fragments all are519bp.The comparation of sequences bybiology software and the prediction of the characterization and structure of protein IMP indicates thatthe IMP protein belongs to Type I of immunodominant membrane proteins.The prediction of transmembrane domain discover that immunodominant membrane proteins can be divided into TypeI-A and B based on the different locations of N-terminal and C-terminal that inside and outside ofmembrane.The Imp obtained in this study all belong to Type I-B.The predictions of leading signalpeptide and surface antigenicity demonstrate that the protein from seven phytoplasmas possesse aN-terminal hydrophobic transmembrane region without export leader signal sequence,and there are ninesignificant antigenic determinant.Meanwhile,to do prokaryotic expression of IMP from cauliflowerwitches’-broom phytoplasma and parthenium hysterophorus Virescence(PHV-YNym).Constructingrecombinant plasmids of prokaryotic expression as expression vector named pET30a-Imp.To obtainexpectant about25kDa protein by using SDS-PAGE.And the recombinant plasmids of other fivephytoplasmas is also constructed,but not expressing successfully.Our research lays the groundwork forpreparation of antiserum and detection of phytoplasma.3）The research for tissue culture of cauliflower and crab cactus associated with phytoplasmasmakes clear that the mothed of keeping phytoplasma in living host body for a long time by using tissueculture is available and it is good for using phytoplasmal material anytime and anywhere. Subculture inthe appropriate low temperature can make tissue culture seedling grow slowly and maintain its growingability as well. we can use them to do reseach whenever we need.The characteristics of the materialitself and the limitations of the conditions make this method still existing many shortcomings liketime-consuming, labor-intensive, cost savings, and a lot of uncertainty, and even may affectphytoplasma concentration of host plant and stability of phytoplasma strains in the process of tissueculture.So select the most appropriate plants which be suitable for tissue culture in long-term atlow-temperature as a host of the research object is a good mothed that can be worthy of considerationand tested.Whether can we establish an effective and long-term mechanism to preserve phytoplasma byplant tissue culture will require further in-depth study and discussion.