The Effect Of Phytoplasma Of Jujube Witches’ Broom Disease On The Diversity Of Endophytic Bacteria In Jujube Trees And A Preliminary Study On Its Effector Protein

Red jujube is an important agricultural economic product in northern Shaanxi.In recent years,due to the influence of jujube witches’broom（JWB）disease,the output of jujube has dropped sharply,which has a great impact on the agricultural economy.At present,there is very little research on JWB,and its pathogenic mechanism is still unclear.Certain research methods are urgently needed to prevent and control JWB disease.In this experiment,the phytoplasma of JWB was used as the research object.The high-throughput sequencing technology was used to analyze the diversity of the endophytic bacterial community in the roots and branches of jujube tree suffering from JWB disease.The 16S rDNA sequencing and plate counting method isolate and colonize the bacteria,use the SignalP v.3.0 program and the transmembrane domain prediction program TMHMM to screen the effector proteins of the phytoplasma of JWB,and construct the cDNA library of Gouzou jujube tissue culture seedlings by reverse transcription technology,and the construction of a bait carrier will lay a certain scientific foundation for the next yeast two-hybrid technology to screen target proteins in plants,and at the same time provide data support and theoretical basis for the study of the pathogenic mechanism of phytoplasma of JWB.The research results are as follows:1 Through high-throughput sequencing technology,we analyzed the community diversity of endophytic bacteria in the roots and branches of the same jujube infected with JWB,and compared the endophytic bacteria in the roots and branches of healthy jujube.The community diversity of bacteria in the roots of diseased jujube trees is less than that of healthy jujube trees,and there are differences in common flora;the community diversity of bacteria in the branches of diseased jujube trees is greater than that of healthy jujube trees,and there are obvious differences between the dominant flora and common flora,which shows that the phytoplasma of diseased jujube trees has obvious influence on the bacterial community in the roots and branches of jujube trees.The community diversity of endophytic bacteria in different parts of the same jujube infected with JWB was analyzed.It was found that the community diversity of bacteria in the roots of the infected jujube was greater than that in the branches of the infected jujube,and there were differences between the dominant bacteria and the common bacteria,which indicated that the community diversity of bacteria in different parts of the infected jujube was different.2 Twenty-one strains of endophytic bacteria were isolated and colonized from jujube infected with JWB.Among them,there are 7 strains of kosakonia:kosakonia（X）,kosakonia（Y）,kosakonia（N）,kosakonia（YD）,kosakonia（GH）,kosakonia（NB）,kosakonia（G）;There are 7 strains of Pseudomonas:Pseudomonas（Z）,Pseudomonas（ZH）,Pseudomonas（YX）,Pseudomonas（C₂）,Pseudomonas（C₁）,Pseudomonas（F₁）,Pseudomonas（23）;3 strains of Pantoea:Pantoea（XD）,Pantoea（NH）,Pantoea（10）;3strains of Enterobacter:Enterobacter（GB）,Enterobacter（F₂）,Enterococcus（D₁）;trabuisiella（D₂）.Eleven of them were selected.pBBR-gfpmut3 and pBBR1MCS-5plasmids were introduced into 11 genera by the method of electric stimulation.The results showed that the Pseudomonas（ZH）of 11 strains could be colonized in the tissue culture of jujube.3 Tissue culture seedlings of jujube tree with JWB were cultivated into tissue culture seedlings through plant tissue culture technology.Through nested PCR detection,sequencing and comparison,the tissue culture seedlings we cultivated were determined to be phytoplasma with jujube disease tissue culture seedlings.4 The yeast two-hybrid cDNA library of jujube tissue culture seedlings was constructed by reverse transcription technology.We selected monoclones with inserted fragments for electrophoresis detection by PCR:The average inserted fragment was more than 1200 bp,and the library capacity was more than 1.15*10⁷CFU,which shows that the library we constructed is qualified.We screened 11 effective factors of phytoplasma of JWB through bioinformatics:CWO85-00790、CWO85-00795、CWO85-00800、CWO85-00890、CWO85-01400、CWO85-02040、CWO85-02055、CWO85-02325、CWO85-03070、CWO85-03075、CWO85-03080,and constructed 9 kinds of bait vectors:pGBKT7-00790、pGBKT7-00800、pGBKT7-00890、pGBKT7-03070、pGBKT7-02325、pGBKT7-00795、pGBKT7-01400、pGBKT7-02040、pGBKT7-03080.