摘要
【目的】柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)是威胁我国柑橘产业稳定发展的主要病毒,但其在柑橘上的侵染和致病机制尚不清楚。本文以CYVCV的外壳蛋白(coat protein,CP)为诱饵筛选尤力克柠檬(Citrus limon Burm.f.)c DNA文库,利用生物信息学方法分析与其互作的寄主因子在病毒侵染和致病过程中可能发挥的作用。【方法】Trizol法提取尤力克柠檬叶片总RNA,用SMART法反转录合成First-Strand c DNA,以其为模板通过Long-Distance PCR获得ds c DNA,均一化处理后与线性化p GADT7质粒重组链接构建尤力克柠檬初级c DNA文库,重组质粒转化大肠杆菌DH10B获得尤力克柠檬大肠杆菌c DNA文库并对文库质量进行鉴定;PCR扩增CYVCV的CP序列并构建到载体p GBKT7上,鉴定诱饵质粒对酵母细胞的毒性以及CP蛋白对酵母报告基因的自激活性。将尤力克柠檬c DNA文库质粒转化含有诱饵质粒p GBKT7-CP的Y2H Gold酵母菌株,共转化子依次涂布SD/-Leu-Trp、SD/-Leu-Trp-His/X-α-Gal和SD/-Leu-Trp-His-Ade/X-α-Gal平板,最终筛选蓝色且长势较好的阳性菌落,提取酵母质粒并测序比对获得候选基因,利用Uniprot在线网站的gene ontology(GO)注释候选基因,分析候选互作蛋白的生物学功能。根据分析结果,选取可能参与寄主抗病或症状发展的候选因子,扩增其CDS全长序列并构建到靶标载体p GADT7后分别与p GBKT7-CP共转化酵母细胞进行点对点酵母双杂交互作验证。【结果】尤力克柠檬大肠杆菌c DNA文库滴度为1.02×10^(8)cfu/m L,库容符合试验标准;成功构建酵母双杂交诱饵载体p GBKT7-CP,该载体没有自激活性且对酵母菌没有毒性;在SD/-Leu-Trp-His-Ade/X-α-Gal平板上筛选得到41个酵母阳性克隆,经序列相似性比对,去除重复,共筛得32个候选寄主因子;GO通路注释结果表明这些寄主因子参与多个叶绿体相关的生物过程,包括碳水化合物代谢、光合作用、光刺激反
【Objective】Citrus yellow vein clearing virus(CYVCV)is one of the viruses mostly threatening the stable development of citrus industry in China,but its infection and pathogenic mechanism in citrus is still unclear.In this study,the coat protein(CP)of CYVCV was used as a bait to screen the Eureka lemon(Citrus limon Burm.f.)cDNA library,and the function of obtained host factors in the interaction between host and virus was analyzed by bioinformatics method.【Method】The total RNA of Eureka lemon leaves was extracted by the Trizol method,and then reversely transcribed to the First-Stand cDNA with SMART method,which was used as a template for obtaining ds cDNA through Long-Distance PCR.After homogenization,the ds cDNA fragments were ligated to pGADT7 plasmid vector by recombination junctions to construct the primary cDNA library of Eureka lemon.The recombinant plasmids were transfected into Escherichia coli DH10B to obtain the E.coli cDNA library of Eureka lemon,and its quality was identified.Simultaneously,the CP sequence of CYVCV was amplified by PCR and ligated into the yeast two-hybrid(Y2H)bait vector pGBKT7,and the plasmids of pGBKT7-CP and pGADT7 were co-transfected into yeast Y2H Gold.The positive yeast clones were grown on the plate of SD/-Trp,SD/-Leu-Trp,SD/-Leu-Trp/X-α-Gal and SD/-Leu-Trp-His medium,respectively,and then the growth situation of the yeast was tested to identify the toxicity of pGBKT7-CP on yeast Y2H Gold and the self-activating effect of pGBKT7-CP on the reporter gene of yeast was analyzed.Then the Y2H Gold containing bait vector pGBKT7-CP was transformed with the primary cDNA library of Eureka lemon,the co-transformed yeasts were coated on the plate of SD/-Leu-Trp,SD/-Leu-Trp-His/X-α-Gal and SD/-Leu-Trp-His-Ade/X-α-Gal medium in turn.Finally,the blue and well grown positive clones were selected.The plasmids of positive yeast clones were extracted and sequenced.The candidate genes were preliminarily compared in the GenBank,and the interacted protein factors were annotated and the pro
作者
宾羽
张琦
王春庆
赵晓春
宋震
周常勇
BIN Yu;ZHANG Qi;WANG ChunQing;ZHAO XiaoChun;SONG Zhen;ZHOU ChangYong(Citrus Research Institute,Southwest University/National Citrus Engineering Research Center,Chongqing 400712)
出处
《中国农业科学》
CAS
CSCD
北大核心
2023年第10期1881-1892,共12页
Scientia Agricultura Sinica
基金
重庆市自然科学基金(CSTB2022NSCQ-MSX0752)
重庆市自然科学基金博士后项目(cstc2021jcyj-bsh X0017)。
关键词
柑橘黄化脉明病毒
尤力克柠檬
酵母双杂交
外壳蛋白
寄主因子
citrus yellow vein clearing virus(CYVCV)
Eureka lemon
yeast two-hybrid
coat protein
host factor
