Expression And Purification Of Citrus Huanglongbing Serralysin And Molecular Detection Of Two Important Pathogens

Citrus is an important worldwide subtropical fruit which has the largest planting area and production, but there are some serious problems in citrus production process, among which the widespread of citrus diseases is one of the most important factors affecting citrus industry.1. Citrus Huanglongbing (HLB) is the most devastating disease affecting the world citrus production, at present its pathogenesis is not clear. According to the submitted Serralysin protein sequences information of Asian citrus Huanglongbing (HLB) pathogen in GenBank, the article cloned a N-terminal fragment gene of Serralysin protein, named SerN; we constructed the pMD19-T-SerN recombinant plasmid which containing SerN gene, sequencing results showed that there was a100%homology compared with the original sequences in GenBank; then we constructed the recombinant expression vector pET24a-SerN which was expressed in E. coli and purified by Ni2+-NTA affinity column to choose optimization expression and purification conditions.2. The expression of SerN protein in different strains showed that it was better expressed in E. coli pLysS and SerN protein could be expressed in both supernatant and precipitation after ultrasonic broken, most of which was existed in the form of inclusion body; by using different IPTG concentration to induce SerN protein expression, we found that there was a highest protein expression in the supernatant when the IPTG concentration was0.05mM; by using different Binding buffer in protein purification process we found that there was not obvious effect for protein purification and the protein solubility was not significantly improved; Reducing salt concentration in Binding buffer could not improve the solubility of SerN protein and there was a significantly higher protein expression in supernatant when NaC1concentration in PBS buffer was500mM protein than other cases.3. We used conventional PCR and real-time quantitative PCR (QPCR) to detect whether the101samples from different citrus productions infected citrus HLB. The conventional PCR detection results showed that4samples had infected citrus HLB, but the QPCR detection results showed that28samples was suspected to carry citrus HLB. Comparing the two detected methods we found that QPCR detection had a much higher sensitivity than the conventional PCR detection, which might be associated with pathogens content plant carried. 4. The article cloned p23protein (GenBank accession number: AFD33289.1) gene of Citrus tristeza virus (CTV) pathogen and constructed the recombinant plasmid pMD19-T-p23, sequencing results showed that there was a100%homology compared with the submitted sequences in GenBank which could be used as a positive control in the detection of CTV. The CTVdetected results of81samples from different citrus productions showed that75samples carried CTV virus and only6samples did not carry CTV virus.