【摘要】 【目的】克隆家蚕Arylphorin的cDNA全长序列,利用生物信息学对该序列进行分析,检测该基因在家蚕感染质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,BmCPV)后的表达量变化模式,为进一步研究其生物学功能奠定基础。【方法】以家蚕中肠总RNA为模板反转录合成cDNA,利用cDNA末端快速扩增(RACE)技术克隆家蚕Arylphorin的全长cDNA;通过实时荧光定量PCR(qRT-PCR)分析其组织表达模式和在家蚕感染BmCPV后的表达差异。【结果】克隆得到了家蚕Arylphorin的全长cDNA(命名为BmAryl),在GenBank登录号为JN581664。该基因的cDNA全长为2 260 bp,包含1个26 bp 5′非翻译区和1个143 bp的3′非翻译区,3′非翻译区包含有终止密码子、多聚腺嘌呤信号AATAAA和poly(A)尾。其最大开放阅读框(ORF)为2 091 bp,编码的蛋白由696个氨基酸组成,预测蛋白分子量为82.8 kD,等电点为6.07。多序列比对和系统进化分析发现,家蚕BmAryl蛋白与家蚕的性别专一贮藏蛋白2相似性最高,为66%。BmAryl主要在家蚕的脂肪体和血淋巴中表达,精巢和卵巢中的表达量无明显差别;而在家蚕感染BmCPV后该基因在中肠的表达量明显下调。【结论】成功克隆获得BmAryl的全长cDNA,其编码的蛋白质属于芳香蛋白家族,BmAryl主要在家蚕的脂肪体和血淋巴中表达,不存在性别专一性,且家蚕感染BmCPV后BmAryl在中肠的表达明显下调。更多还原

【Abstract】 【Objective】 The objective of this study is to clone the full-length cDNA sequence of Arylphorin in silkworm(Bombyx mori) to analyze the gene sequence by bioinformatics and to investigate the mRNA expression level after the B. mori infected by cytoplasmic polyhedrosis virus(BmCPV), and to provide a new foundation for the further study of its biological function.【Method】 Rapid amplification of cDNA ends(RACE) approach was employed to clone the full-length cDNA of Arylphorin from the total RNA of the silkworm midgut. Fluorescent quantitative real-time polymerase chain reaction(qRT-PCR) was used to analyze the expression profiling of Arylphorin in different tissues and at different time points after the BmCPV infection. 【Result】 The full-length cDNA of Arylphorin was obtained and named as BmAryl with accession number of JN581664 in GenBank. It consisted of a 5′-terminal untranslated region(UTR) of 26 bp and a 3′-UTR of 143 bp with polyadenylation signal sequences AATAAA and a poly(A) tail. The longest open reading frame(ORF) encoded a polypeptide of 696 amino acids with a theoretical isoelectric point of 6.07and the predicted molecular weight of 82.8 kD. Sequence comparison showed that BmAryl was 66% identical to B. mori sex-specific storage-protein 2 precursor. mRNA transcripts of BmAryl were detected mainly in fat body and hemolymph and the expression level had no difference between the testicle and ovary. For the silkworm larvae infected by BmCPV, the relative expression level of BmAryl was significantly down-regulated in the midgut. 【Conclusion】 The full-length cDNA of BmAryl was successfully cloned from silkworm, which encodes a protein that belongs to the aromatic protein family. BmAryl expression were detected mainly in fat body and hemolymph, not sex-specific but down-regulated in the midgut due to BmCPV infection. These results has provided not only helpful information for further studying the function of BmAryl in silkworm but also be helpful for understanding of the role in response to BmCPV infection.更多还原