Cloning And Expression Application Of 408 And PAP Protein Genes Of Candidatus Liberibacter Asiaticus

Citrus Huanglongbing(HLB)seriously threatens the development of the world citrus industry and causes huge economic losses.It takes at least several months to cause the symptoms of yellowing from the infection of the pathogen.Therefore,it is necessary to establish an efficient,sensitive and rapid detection method for the healthy development of the citrus industry.Secreted protein is an effector produced by bacteria and plays an important role in the pathogenesis of bacteria.In this study,10 genes from secreted protein systems and related secreted protein genes were analyzed from the Candidatus Liberibacter asiaticus,and 6 genes were screened by fluorescence quantitative RT-PCR(qRT-PCR).Two genes with high RNA content were screened out from 6 secreted protein genes by RT-qPCR.The function of two proteins was studied by subcellular localization,and the prokaryotic expression of two genes was carried out.The specific antiserum was prepared from immunized mice.This study provides the basis for protein function research and development of HLB protein detection products.The specific results are as follows:(1)DNA and cDNA was prepared by extracting total DNA and RNA from green orange of HLB infected by Qiongzhong and Qionghoi in Hainan Province.Based on the six genes of the secreted protein system and related secreted proteins genes analyzed in the genome of Candidatus Liberibacter asiaticus,a quantitative PCR(qRT-PCR)primers was designed to test the mRNA amount of six genes in the HLB samples of Qionghai and Qiongzhong.The results showed that the mRNA content of 408 and PAP genes in Qionghai and Qiongzhong samples were higher transcribed,and the Ct values were between 15 and 35.(2)Using the extracted DNA as a template,the fuli-length genes of 408 and PAP were amplified.The result of gene sequence analysis showed that the homology of 408 protein to 408 protein of Candidatus Liberibacter asiaticus strain psy62 was over 99%,And the amino acid sequence is identical to the 408 and PAP proteins of this strain.Further predictive analysis,408 protein contains a highly conserved N-terminal domain,the function of which may be related to pilus formation protein N terminal domain,and the C-terminus contains two highly conserved motify(Motif 1 and Motif 2).The PAP protein contains two highly trusted domains associated with secretion function,namely the CPaC domain(AA22-474)and the Secretrin domain(AA262-419).(3)According to the result of RT-qPCR,two secreted protein genes of 408 and PAP with high RNA content were obtained,and specific primers were designed.The GFP fluorescent expression vector GV1300 was constructed,and the correctly sequenced plasmids were named GV1300-408 and GV 1300-PAP.The recombinant plasmid and the control plasmid were transformed into GV1301 Agrobacterium,and injected to Nicotiana benthamiana separately for subcellular localization study.The experimental results show that 408 protein is localized to the nucleus and cytoplasm of tobacco mesophyll cells,and PAP protein is also located in the nucleus and cytoplasm of tobacco mesophyll cells.(4)The full length ORFs of 408 and PAP secreted proteins gene were cloned and constructed into prokaryotic expression plasmid pET32a(+),which was transformed into expressing bacteria E.coli BL21.After induction with a final concentration of 1 mmol/L IPTG,the fusion protein was highly expressed.Soluble analysis indicated that 408 and PAP proteins were less abundant in the supernatant,but most were in the precipitation,indicating that 408 and PAP proteins were mainly expressed in the form of inclusion bodies.After the fusion protein was purified by Ni2+-NTA column,the 408 protein was mainly in the elution buffer of 150 mmol/L imidazole,and the most of PAP protein was in the elution buffer of 100 mmol/L and 150 mmol/L imidazole.(5)Antisera were obtained after multiple immunization of the purified 408 and PAP fusion proteins.The titer test results showed that the serum titer of 408 antiserum was between 1:500 and 1:10000,and the serum titer of PAP polyclonal antibody was between 1:500 and 1:1000.Western blot detection showed that the specific bands were detected.Using the extracted protein from HLB-infected green orange sample for detection,the result was obvious positive.