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

Abstract:

【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 protein’s biological functions were analyzed with gene ontology (GO) pathway of Uniprot online websites. According to the results of the analysis, candidate factors that may be involved in host disease resistance or symptom development were selected, their CDS full-length sequences were amplified and constructed into the target vector pGADT7 and then were verified with pGBKT7-CP by one for one in yeast, respectively.【Result】The titer of the Eureka lemon-E. coli cDNA library was 1.02×10⁸ cfu/mL, and it demonstrated that the cDNA library measured up to the experiment standard. The bait vector of pGBKT7-CP was constructed, which had no ability to activate the reporter gene and had no virulence to the yeast. The 41 positive clones were finally selected by using the SD/-Leu-Trp-His-Ade/X-α-Gal medium. After sequence similarity comparison, removing the repetitive sequences, the vector sequences and the frameshift sequences, the 32 host factors that interacted with CYVCV CP were screened. The GO pathway annotation results indicated that these host factors were involved in several chloroplast-related biological processes, including photosynthesis, metabolic process, carbohydrate metabolic process, response to light stimulus, etc. The molecular functions of the 32 host factors were multiple, including catalytic activity, hydrolase activity, transferase activity, protein binding, DNA-binding transcription factor activity, and translation factor activity, etc. Moreover, the cell components of the 32 host factors were involved in the cytoplasm, membrane, chloroplast, thylakoid, nucleolus, and golgi apparatus, etc. Validation of one-to-one yeast two-hybrid crosses of 14 important proteins selected from candidate host factors with CP showed that CP interacted with all 14 proteins.【Conclusion】The cDNA library of Eureka lemon was constructed successfully, and 32 host factors of Eureka lemon interacted with CYVCV CP were preliminarily screened. According to the results, the function of the important proteins was analyzed. It was presumed that CYVCV CP affects photosystem stability, thylakoid structure and chlorophyll synthesis by interacting with multiple chloroplast-related proteins such as oxygen-evolving enhancer protein 2 (PsbP), chlorophyll a-b binding protein 8 (Lhca3) and ribulose bisphosphate carboxylase small subunit (RbcS), resulting in reduced photosynthesis and damaged chloroplast morphology and function. The interaction of CP with these chloroplast-associated factors was separately verified one-to-one in yeast, which will provide a theoretical basis for revealing the molecular mechanism of CYVCV CP in viral pathogenicity.

Key words: citrus yellow vein clearing virus (CYVCV), Eureka lemon, yeast two-hybrid, coat protein, host factor