Molecular biological studies on multipulication of BmNPV and viral gene

BmNPV与病毒基因增殖的分子生物学研究

• 批准号: 09460032
• 负责人: TAKEI Ryuzo
• 金额: $ 5.38万
• 依托单位: SHINSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460032/
• 关键词: NPV; polyhedra; polyhedrin; 核多角体病ウイルス

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

测定了家蚕核型多角体病毒（BmNPV）25 K基因的核苷酸序列，并定位了其体内转录产物的末端。在BmNPV中，尚未鉴定出由25 K基因突变引起的少数多角体（FP）突变体。在本研究中，我们已经证实，BmNPV 25 K基因的破坏诱导其FP突变表型。我们已经克隆并测序了三个BmNPV分离物的25 K基因; BmSNPV-H，其产生野生型多角体，BmSNPV-T，其产生立方体多角体，封闭相对少量的病毒，和BmMNP V-La，其在中肠细胞中产生许多不寻常的多角体。BmNPV-T和BmNPV-La的25 K基因的全长cDNA在poly（A）尾的起始处有一个645 bp的蛋白质编码区，其中5'端非翻译区有126 bp，3'端非翻译区有135 bp。从BmNPV-T和BmNPV-La的25 K基因核苷酸序列推导的氨基酸序列相同，表明在BmNPV-T和BmNPV-La之间观察到的多面体形成的差异。 ...更多信息 BmNPV-T和-La与25 K基因突变无关。与BmNPV-T和-La的氨基酸序列比较，发现BmNPV-H的氨基酸序列中有一个氨基酸发生了替换，另一个氨基酸发生了缺失。我们的引物延伸实验表明BmNPV 25 K基因的转录起始位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系统中25 K基因的转录起始位点位于杆状病毒晚期启动子ATAAG元件内。测定了感染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，以研究多角体在这些细胞中的不良产生。当通过北方印迹分析研究多角体蛋白mRNA在中肠上皮细胞中的出现和积累时，早在感染后6 h（p.L.）就检测到多角体蛋白mRNA。感染后72 h，中肠上皮细胞和脂肪体细胞中病毒DNA的定量PCR。结果表明，中肠上皮细胞中病毒DNA的含量与脂肪体细胞中病毒DNA的含量接近，表明病毒DNA的合成发生在中肠上皮细胞中。感染后72 h，中肠上皮细胞和脂肪体细胞中多角体蛋白mRNA的定量RT-PCR。结果表明，两种细胞中多角体蛋白mRNA的表达量无明显差异，提示多角体蛋白基因的转录主要发生在中肠上皮细胞中。Westernblot分析显示，多角体蛋白在中肠上皮细胞中有表达，但表达量远低于脂肪体细胞。结论：多角体蛋白基因at翻译水平低可能与中肠上皮细胞多角体形成不良有关。少

项目成果

H.Tsuda, M.Nakagaki, T.Hashimoto, Z.Kajiura, R.Takei and Y.Iwashita: "Multiplication of NPV and expression of polyhedrin gene in the midgut epithelium of silkworm, Bombyx mori" J.Sericult.Sci.Jpn.Vol.68, No.1. 19-25 (1999)

H.Tsuda、M.Nakagaki、T.Hashimoto、Z.Kajiura、R.Takei 和 Y.Iwashita：“在家蚕中肠上皮细胞中 NPV 的增殖和多角体蛋白基因的表达” J.Sericult.Sci.Jpn

H.Tsuda,M.Nakagak 他: "Determination of nucleotide sequence and transcription initiation site of 25K gene of Bombyx mori Nucleopolyhedrovirus." 日本蚕糸学 雑誌. 68. 27-39 (1999)

H. Tsuda、M. Nakagak 等：“家蚕核多角体病毒 25K 基因的核苷酸序列和转录起始位点的测定”，日本血清学杂志 68. 27-39 (1999)。

津田・中垣 他: "カイコ中腸におけるNPVの増殖とポリヘドリン遺伝子の発現" 日本蚕糸学雑誌. 68. 19-25 (1999)

Tsuda, Nakagaki 等：“家蚕中肠中 NPV 的增殖和多角体蛋白基因的表达”，日本蚕丝学杂志 68. 19-25 (1999)。

H.Tsuda, M.Nakagaki, T.Hashimoto, Z.Kajiura and R.Takei: "Determination of nucleotide sequence and transcription initiation site of 25K gene of Bombyx mori Nucleopolyhedrovirus" J.Sericult.Sci.Jpn.Vol.68, No.1. 27-39 (1999)

H.Tsuda、M.Nakagaki、T.Hashimoto、Z.Kajiura 和 R.Takei：“家蚕核多角体病毒 25K 基因的核苷酸序列和转录起始位点的测定”J.Sericult.Sci.Jpn.Vol.68，No

H.Tsuda, M.Nakagaki 他: "Determination of nucleotile sequence and Transcription initiation site of 25K gene of Bombyx mori Nucleopolyhedrovirus." 日本蚕糸学雑誌. 68. 27-39 (1999)

H. Tsuda、M. Nakagaki 等人：“家蚕核多角体病毒 25K 基因的核序列和转录起始位点的测定”，日本血清学杂志 68. 27-39 (1999)。