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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.
The full-length cDNA clone pUBF52 of beet blackfat-borne virus (BBSV) driven by T7 RNA polymerase promoter was constructed by RT-PCR.After inoculation with Chenopodiumamanticidus, in vitro transcripts resulted in the same Western blotting and Northern blot analysis also proved the transfection product infectivity.The prokaryotic expression vector pECP1 of BBSVp24 gene was constructed, and the induced expression product transformed into E.coli BL21 was able to react specifically with the antisera of BBSV , Indicating that the gene encodes the coat protein (CP) of BBSV.The frameshift mutants of BBSVCP gene and different degrees of deletion mutants were constructed respectively by using pUBF52 as a template.The infectivity test showed that the frameshift mutation of CP gene BBSV caused by Quercetin amaranth symptoms and viral RNA accumulation in the host almost no effect, but most or completely deleted CP gene will greatly reduce the accumulation of viral RNA in vivo, in which the CP gene is completely missing Mutant transcripts were able to produce only very slight symptoms of dry spots when inoculated with amaranthus.According to the green fluorescent protein (GFP) gene and glucuronidase (GUS) Do not fuse with the 5 ’end of BBSVCP gene to construct the expression vectors pBGFP and pBGUS.The expression of GFP or GUS gene was observed after friction inoculation with the leaves of A. amabilis, which laid the foundation for exploring the expression vector using BBSV as foreign protein.