柑橘黄龙病菌分泌蛋白04580基因的克隆及原核表达  

The cloning and prokaryotic expression of Candidatus Liberibacter asiaticus CLIBASIA-04580 gene

作　　者：邓杰夫 李魏[1,2] 雷玲 洪艳云 刘双清[1,2] 戴良英 易图永[1,2] DENG Jiefu;LI Wei;LEI Ling;HONG Yanyun;LIU Shuangqing;DAI Liangying;YI Tuyong(College of Plant Protection,Hunan Agricultural University,Changsha,Hunan 410128,China;Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Pests,Changsha,Hunan 410128,China)

机构地区：[1]湖南农业大学植物保护学院,湖南长沙410128 [2]植物病虫害生物学与防控湖南省重点实验室,湖南长沙410128

出　　处：《湖南农业大学学报（自然科学版）》2022年第3期294-297,共4页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：科学技术部国家重点研发计划(2021YFD1400800);湖南省科学技术厅重点研发计划项目(2022NK2052);湖南农业大学双一流学科建设项目(SYL2019027)。

摘　　要：为进一步了解柑橘黄龙病菌亚洲种CLas的致病机理,从Prasad预测的166种分泌蛋白中选取1个在柑橘植株中表达量较高的CLIBASIA-04580基因进行研究。提取感染柑橘黄龙病的柑橘叶片总DNA,经PCR扩增得到CLIBASIA-04580基因,切胶回收PCR产物,双酶切连接到pET-28α载体上,使用菌落PCR和限制性内切酶双酶切鉴定筛选阳性克隆;转化大肠杆菌BL21(DE3),用0.5、1.0、1.5、2.0 mmol/L的异丙基硫代半乳糖苷(IPTG)诱导重组蛋白表达融合组氨酸标签的CLIBASIA-04580重组蛋白,聚丙烯酰氨凝胶电泳(SDS-PAGE)检测重组蛋白的表达水平,再用蛋白纯化镍柱进行纯化。结果表明,携带组氨酸标签的CLIBASIA-04580重组蛋白得到表达,且异丙基硫代半乳糖苷(IPTG)浓度为1.5mmol/L时相对表达量达到峰值,通过SDS-PAGE凝胶分析,与未经过镍柱纯化的蛋白原液比较,可知纯化了CLIBASIA-04580重组蛋白。CLIBASIA-04580,which highly expressed in citrus plants,was selected from 166 secreted proteins predicted by Prasad and was investigated to further understand the pathogenesis of Candidatus Liberibacter asiaticus(CLas).The total DNA of citrus leaves identified as infected with citrus Huanglongbing(HLB)was extracted,and the CLIBASIA-04580 gene was obtained by PCR.The PCR product was extracted and connected to the pET-28αvector by double enzyme digestion,and the positive clones were screened by colony PCR and restriction endonuclease double digestion.The recombinant Escherichia coli BL21(DE3)was induced to express the fusion histidine-labeled recombinant protein 04580 by isopropyl thiogalactoside(IPTG)(0.5,1.0,1.5,2.0 mmol/L).The expression level of the recombinant protein was detected by polyacrylamide gel electrophoresis(SDS-PAGE),and then purified by protein purification nickel column.The results showed that the recombinant protein 04580 with histidine label was successfully expressed,and the expression reached the peak when the concentration of IPTG was 1.5 mmol/L.By SDS-PAGE gel analysis,the recombinant protein 04580 was successfully purified compared with the original protein solution which was not purified by nickel column.

关 键 词：柑橘黄龙病菌 分泌蛋白 原核表达 蛋白纯化 

分 类 号：S436.661.12[农业科学—农业昆虫与害虫防治] Q786[农业科学—植物保护]