槟榔黄叶病毒1外壳蛋白的互作蛋白筛选

Screening of Interacting Proteins in the Coat Protein of Areca Palm Yellowing Leaf Virus 1

为明确槟榔黄化病致病机理,探究槟榔黄叶病毒1(areca palm leaf yellowing virus 1,APLYV 1)病毒与宿主蛋白之间相互作用的机制,本研究以槟榔叶片为材料,构建槟榔cDNA酵母双杂交文库,以APLYV 1的外壳蛋白(coat protein,CP)为诱饵,构建诱饵载体pGBKT7-CP,采用酵母双杂交技术,从槟榔cDNA文库中筛选与其互作的蛋白。结果表明,成功构建槟榔酵母文库,文库容量达1.1×10^(7)以上,重组率为100%,插入片段平均长度>1000 bp。成功构建诱饵载体pGBKT7-CP,通过自激活实验检验,阳性对照在四缺培养基平板上能正常长菌且显蓝,阴性对照和实验组在二缺培养基平板上能正常长菌,在三缺和四缺培养基平板上未长菌,表示构建成功的重组诱饵载体无自激活活性,可用于筛库实验。共筛选出13个阳性克隆,经NCBI网站与槟榔转录组库进行比对,最终得到10个候选槟榔蛋白,包含sHSP(small Heat Shock Protein)与DnaJ蛋白等。选择DnaJ蛋白,通过理化性质分析和蛋白二级结构预测,发现该槟榔基因全长1035 bp,蛋白理论分子量为37.9 kD,等电点pI约为5.05,疏水性蛋白,其二级结构由无规则卷曲(49.13%)和α-螺旋(25.87%)构成,不存在信号肽,无跨膜结构域,主要定位在细胞核内。本研究结果为进一步研究APLYV 1蛋白功能和表达调控机制提供依据。

In order to identify the pathogenesis of Areca palm leaf yellowing virus 1(APLYV 1)and explore the interaction between APLYV 1 and its host protein,a areca leaf yeast two-hybrid cDNA library was constructed.Using coat protein(CP)of APLYV 1 as bait,the decoy vector pGBKT7-CP was constructed,and the interacting proteins were screened from areca cDNA library by yeast two-hybridization.The results showed that the areca yeast library was successfully constructed with a library capacity of more than 1.1×10^(7),a recombination rate of 100%,and an average insert length of more than 1000 bp.The bait vector pGBKT7-CP was successfully constructed.Through the self-activation test,the positive control could grow bacteria normally and show blue color on the four-deficiency medium plate,while the negative control and experimental group could grow bacteria normally on the two-deficiency medium plate,but did not grow bacteria on the three-deficiency and four-deficiency medium plate,indicating that the successfully constructed recombinant bait vector had no self-activation activity and could be used in the screen library experiment.A total of 13 positive clones were screened,and 10 candidate areca proteins,including sHSP(small Heat Shock Protein)and DnaJ protein,were obtained by comparison between NCBI website and areca transcriptome library.DnaJ protein was selected and predicted by physical and chemical properties analysis and secondary structure analysis.The total length ofthe aele gene was 1035 bp,the theoretical molecular weight of the protein was 37.9 kD,the isoelectric point pl was about 5.05,and the secondary structure was com posed of random curl(49.13%)andα-helix(25.87%),without any signal peptide.There is no transmembrane domain,mainly located in the nucleus.The results of this study provide a basis for further research on the function and expression regulation mechanism of APLYV 1 protein.