Effect Of Bacillus Subtilis On Citrus Hlb Infected Nursery Stocks And Screening Of Crystallization Conditions Of GMPS From Candidatus Liberibacter Asiaticus

Citrus Huanglongbing（HLB）is the most serious citrus disease in the world,which seriously affects the development of citrus industry in the world.It’s difficult to isolate and culture the pathogen of HLB,there is no effective control method.The use of biocontrol agent for prevention and control is a green way.Further,guanosine 5’-monophosphate synthesis（GMPS）is a key enzyme in purine nucleotide synthesis,inhibiting its activity can inhibit growth and reproduction of pathogen.In this experiment,the infected nursery stocks of citrus HLB were treated with Bacillus subtilis microbial agents to explore its inhibitory effect on citrus HLB and its influence on physiological indexes of nursery stocks.On the other hand,the functional domain（ATP pyrophosphatase,ATPPase）of GMPS was cloned,and heterologous expression,protein purification and crystallization screening were performed.The results provide a basis for the screening of inhibitors based on structural information.The results are as follows:1.Effects of Bacillus subtilis microbial agent on citrus HLB infected nursery stocks.The citrus HLB infected nursery stocks were treated with Bacillus subtilis microbial agent,and those in the control group were treated with water.It was found that the positive rate of nursery stocks treated with microbial agent was 60%,which was lower than 66.7%of the control group.The chlorophyll content,net photosynthetic rate,polyphenol content,mineral element content and leaf vein callose content of the nursery stocks in the treatment group and the control group were measured.It was found that the chlorophyll content of the treatment group increased by 1.1 times,the polyphenol content increased by 30.93%,the callose content decreased by 13.8%,and the content of most mineral elements increased significantly.The activities of enzymes related to resistance,such as SOD、POD and so on,were increased in a short period of time after treatment.The expression levels of six resistance-related genes GST1,GAPC2,NHO1,WRKY22,WRKY24 and HSP90 were up-regulated,and the expression level of HSP90 changed the most,which was 12.7 times higher than that of the control group after 12 h treatment.The above results indicated that the microbial agent treatment could promote plant growth and induce resistance.2.Expression,purification and crystal screening of the C-terminal functional domain of Clas GMPS.According to the Clas GMPS gene sequence in Gen Bank,the C-terminal sequence of GMPS protein was cloned and named as GMPS₃₁₈;The expression vector p ET28atplus-GMPS₃₁₈ was successfully constructed and heterologously expressed in E.coli.Uniform and stable protein was obtained by nickel column purification and molecular exclusion chromatography.Screening of protein crystallization conditions,crystals appeared under the conditions of 0.2 M Lithium citrate tribasic tetrahydrate,20%PEG3350 and 0.2 M Sodium citrate tribasic dihydrate,20%PEG3350.Finally,salt concentration and precipitant concentration gradient optimization was carried out,protein crystal did not change.