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广西2种甘薯病毒(SPFMV与SPCSV)的CP基因序列分析及SPCSV抗血清制备

发布时间:2018-05-23 23:55

  本文选题:甘薯羽状斑驳病毒 + 甘薯褪绿矮化病毒 ; 参考:《广西大学》2016年硕士论文


【摘要】:甘薯病毒病是广西甘薯的主要病害之一。从广西南宁、崇左、北海、玉林的甘薯种植区采集疑似甘薯病毒病样品,通过RT-PCR法进行甘薯羽状斑驳病毒(Sweet potato feathery mottle virus, SPFMV)与甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus, SPCSV)的检测,发现SPFMV、SPCSV在广西普遍存在,当甘薯单独感染SPFMV与SPCSV时,表现的病毒病症状轻微,但甘薯同时感染SPFMV和SPCSV时,表现出植株矮化、叶片皱缩等严重症状。设计扩增SPFMV与SPCSV的CP基因的引物,通过克隆并测序分别得到18个SPFMV广西分离物的CP基因序列和14个SPCSV广西分离物的CP基因序列。SPFMV广西分离物CP基因的核苷酸序列945bp,编码315个氨基酸;SPCSV广西分离物CP基因的核苷酸序列774bp,编码257个氨基酸。其CP基因的序列分析结果表明:SPFMV广西分离物存在O株系、RC株系、EA株系,不存在C株系,其中O株系是SPFMV广西分离物的优势株系;SPCSV广西分离物只有WA株系,无EA株系。将RT-PCR扩增得到SPCSV的CP基因克隆到pET30a(+)载体上,转化于大肠杆菌BL21(DE3)pLysS, IPTG诱导表达得到与预期大小一致的SPCSV CP的目的蛋白。经纯化回收后免疫兔子,获得了SPCSV CP的抗血清。通过ELISA和Western Blot检测,抗血清的效价达到1:64000,,特异性好。SPCSV CP抗血清为生产上监测SPCSV的侵染提供技术条件。
[Abstract]:Sweet potato virus disease is one of the major diseases of sweet potato in Guangxi. Samples of suspected sweet potato virus were collected from sweet potato planting areas in Nanning, Chongzuo, Beihai and Yulin, Guangxi. Sweet potato feather mottle virus (Sweet potato feathery mottle virus, SPFMV) and sweet potato chlorotic dwarf virus (Sweet potato chlorotic stunt virus, SPCSV) were detected by RT-PCR method. It was found that SPFMVN SPCSV was common in Guangxi. When sweet potato was infected with SPFMV and SPCSV alone, it showed mild viral symptoms, but when sweet potato was infected with SPFMV and SPCSV, it showed plant dwarfing, leaf shrinkage and other serious symptoms. A primer was designed to amplify the CP gene of SPFMV and SPCSV. The CP gene sequences of 18 SPFMV Guangxi isolates and 14 SPCSV Guangxi isolates were obtained by cloning and sequencing. The nucleotide sequence of CP gene of SPFMV Guangxi isolate was 945bp, encoding 315 amino acids. The nucleotide sequence of the gene was 774 BP, encoding 257 amino acids. The results of sequence analysis of CP gene showed that there were O lines, RC lines, and no lines C, and O lines were the dominant line of SPFMV Guangxi isolate, only WA line and no EA line were found in the Guangxi isolate. The CP gene of SPCSV was cloned into pET30a () vector by RT-PCR amplification and transformed into E. coli BL21DE3pLysS. the target protein of SPCSV CP was induced by IPTG. The antiserum of SPCSV CP was obtained by immunizing rabbits after purification and recovery. The titer of antiserum reached 1: 64000 by ELISA and Western Blot detection. The antiserum of SPCSV with good specificity provided technical conditions for monitoring the infection of SPCSV in production.
【学位授予单位】:广西大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:S435.661

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