甜菜黑色焦枯病毒外壳蛋白与病毒致病性的关系

Effect of Beet black scorch virus Coat Protein on Viral Pathogenicity

利用RT-PCR方法,构建获得了由T7RNA聚合酶启动子驱动的甜菜黑色焦枯病毒(BBSV)全长cDNA克隆pUBF52.摩擦接种苋色藜(Chenopodiumamaranticolor)后,体外转录产物可导致与野生病毒相同的枯斑症状,蛋白质印迹和RNA印迹检测也都证明了转录产物的侵染活性.构建了BBSVp24基因的原核表达载体pECP1,转化大肠杆菌BL21后的诱导表达产物能够与BBSV的抗血清呈现特异性反应,表明该基因编码产生BBSV的外壳蛋白(CP).以pUBF52为模板,分别构建了BBSVCP基因的移码突变体和不同程度的缺失突变体.侵染性检测表明,CP基因的移码突变对BBSV在苋色藜上所导致的枯斑症状及病毒RNA在寄主体内的积累基本没有影响,但CP基因的大部或完全缺失会使体内病毒RNA的积累水平大大降低,其中CP基因完全缺失的突变体转录物接种苋色藜后仅能够产生很轻的枯斑症状.将绿色荧光蛋白(GFP)基因和葡糖苷酸酶(GUS)基因分别与BBSVCP基因的5′端融合,构建了表达载体pBGFP和pBGUS.摩擦接种苋色藜叶片后可观察到GFP或GUS基因的表达,为探索利用BBSV作为外源蛋白的表达载体奠定了基础.

A full-length Beet black scorch virus （BBSV） cDNA clone （pUBF52） was constructed by RT-PCR. The clone contains an upstream T7 RNA polymerase promoter designed for in vitro transcription of infectious RNAs from the linearized plasmid that faithfully represent the viral cDNA. Leaves of Chenopodium amaranticolor inoculated with in vitro transcripts developed the same symptoms and disease phenotype as the wild type virus. The presence of BBSV RNA and coat protein in the leaves was confirmed respectively by Northern blotting and Western blotting. Comparisons of specific immunoreactions between the expression product of the BBSV p24 gene in E. coli and antiserum against purified BBSV virions demonstrated that the p24 gene encodes the coat protein. Based on the sequence of the pUBF52 cDNA, a flame-shift mutant and two deletion mutants were generated. One of the deletions encompasses the entire CP ORF and the other truncates 174 amino acids from the central region of the protein. Transcripts derived from the frame-shift CP mutant, which terminates the CP after the first 23 amino acids, elicited the same symptom phenotype and levels of RNA accumulation as the wild type virus, but the leaves infected with the CP deletion mutants exhibited greatly reduced RNA accumulation. In addition, leaves inoculated with in vitro transcripts of the mutant in which the entire CP gene was deleted had lower local lesions than wild type virus transcripts. Two expression vectors, pBGFP and pBGUS, were constructed by fusing the GFP and GUS genes to the 23 N-terminal amino acids of the CP gene, respectively. Leaves infected with in vitro transcripts of pBGFP and pBGUS exhibited expression of GFP and GUS proteins as assessed by laser confocal microscopy and histochemical staining, respectively. The high levels of expression of the GFP and GUS proteins provide tools that can be used for studies of replication and movement of the virus, and indicate that BBSV has considerable biotechnology potential as a plant virus expression vehicle.