Molecular biological studies on multipulication of BmNPV and viral gene

我们测定了家蚕核型多角体病毒(BmNPV)25K基因的核苷酸序列，并对其体内转录末端进行了定位。在BmNPV中，由于25K基因突变而导致的多角体(FP)突变体很少。在本研究中，我们证实了BmNPV 25K基因的破坏导致了其FP突变表型。我们已经克隆并测序了三个BmNPV分离物的25K基因：BmSNPV-H产生野生型多角体，BmSNPV-T产生相对较少数量的立方多角体，BmMNP V-La在中肠细胞产生许多不寻常的多角体。BmNPV-T和-La的25K基因全长为645bp的蛋白编码区，5‘端非翻译区为126bp，3’端非翻译区为135bp。从BmNPV-T和BmNPV-La的25K基因核苷酸序列推导出的氨基酸序列与BmNPV-La的氨基酸序列相同，表明…-T和BmNPV-La在多面体形成方面存在差异更多的BmNPV-tand-La与25K基因突变无关。与BmNPV-T和-La的氨基酸序列相比，BmNPV-H的氨基酸序列中存在一个氨基酸的替换和另一个氨基酸的缺失。我们的引物延伸实验表明，BmNPV 25K基因的转录起始点位于基因编码区之前的早期启动子CAGT元件附近，而不像其他NPV系统那样，25K基因的转录起始点位于杆状病毒晚期启动子ATAAG元件内。检测感染BmNPV的中肠上皮细胞中病毒DNA、多角体蛋白mRNA和多角体蛋白的含量，以研究中肠上皮细胞中多角体产生不足的原因。用Northern印迹法检测中肠上皮细胞中多角体蛋白基因的表达，发现感染后6h即可检测到多角体蛋白基因的表达(P。L，12h后，L发现，中肠上皮细胞中多角体蛋白基因的含量增加。中肠上皮细胞和脂肪体细胞中病毒DNA的定量聚合酶链式反应显示，中肠上皮细胞中病毒DNA的量与脂肪体细胞中的病毒DNA量接近，提示病毒DNA的合成发生在中肠上皮细胞中。72h后，对中肠上皮细胞和脂肪体细胞中多角体蛋白mRNAs的定量RT-PCR检测表明，中肠上皮细胞和脂肪体细胞中多角体蛋白基因的转录数量没有显著差异，提示多角体蛋白基因的转录在中肠上皮细胞中发生了显著的变化。Western印迹分析表明，中肠上皮细胞产生多角体蛋白，但其含量远低于脂肪小体细胞。结论：多角体基因AT翻译水平低可能与中肠上皮细胞多角体形成不良有关。较少

We have determined the nucleotide sequence and located the in vivo transcript termini of Bombyx mori nucleopolyhedroviruses (BmNPV) 25K gene. In BmNPV, few polyhedra(FP) mutants by mutation of the 25K gene have not been identified. In this study, we have confirmed that the disruption of BmNPV 25K gene induces its FP mutant phenotype. We have cloned and sequenced the 25K gene of three BmNPV isolates ; BmSNPV-H which produces wild type polyhedra, BmSNPV-T which produces cubic polyhedra occluding relatively small number of viruses, and BmMNP V-La which produces a number of unusual polyhedra in midgut cells. The full-length cDNA of the 25K gene of BmNPV-T and -La has a 645 bp protein-coding region with 126 bp of 5' untranslated region and 135bp of 3' untranslated region before the start of the poly(A) tail. The amino acid sequence deduced from nucleotide sequence of 25K gene of BmNPV-T was the same as that of BmNPV-La, indicating that the variations in polyhedron formation observed between … More BmNPV-Tand -La are not associated with mutation of the 25K gene. The substitution of one amino acid and the deletion of another amino acid were recognized in the deduced amino acid sequence of BmNPV-H, compared with sequences of BmNPV-T and -La. Our primer extension experiment indicated that transcription initiation site ofthe BmNPV 25K gene is at a position adjacent to the early promoter CAGT element located prior to the gene coding region, unlike other NPV systems where transcription initiation sites of 25K gene are within the baculovirus late promoter ATAAG element.We. determined the amounts of viral DNA, polyhedrin mRNA and polyhedrin in the midgut epithelial cells infected with BmNPV, in order to study the poor production of polyhedra in these cells. When the appearance and accumulation of polyhedrin mRNA in the midgut epithelial cells were investigated by northern blot analysis, polyhedrin mRNA were detected as early as 6 h post-infection (p. L At 12 h p. L, amount of polyhedrin mRNA increased in the midgut epithelial cells. Quantitative PCR of virus DNA in the midgut epithelial and fat body cells at 72 h p. i. indicated that amount of the virus DNA in midgut epithelial cells was close to that in fat body cells, suggesting that virus DNA synthesis occurred in midgut epithelial cells. Quantitative RT-PCR for polyhedrin mRNAs in the midgut epithelial and fat body cells at 72 h p. i. indicated that amounts of the mRNA in both cells did not differ signficantly each other, suggesting that the transcription of polyhedrin gene occurred markedly in midgut epithelial cells. Western blot analysis showed that polyhedrin was produced in midgutepithelial cells, but its amountwas much lower than thatin fatbody cells. It is concluded that low level of at translation of polyhedrin gene may be related to poor polyhedron formation in midgut epithelial cells. Less