摘要
通过抑制性差减杂交技术建立了包含对虾白斑综合征病毒表达性基因的差减文库,并用cDNA微阵列技术进行了鉴定,得到255个正向克隆。对其中的184个正向阳性克隆进行了测序,测序结果通过BLAST与GenBank中序列进行比对,共得到WSSV(whitespotsyndromevirus,WSSV)基因30个。此次首次鉴定了5个,其中WSV184具有调控蛋白的结构特征(Cys2/Cys2型锌指),WSV321和WSV322含跨膜结构,且存在可能的糖基化位点。有3个被其它研究推断无polyA结构的阅读框所处的mRNA应有polyA结构。进一步用DotNorthernblot对克隆号PCI118(含WSSV阅读框WSV321和WSV322)进行鉴定,表明确实存在该基因的转录。进而根据已报道的WSSV基因序列设计两条引物,用快速扩增cDNA末端技术,扩增WSV321和WSV322两个阅读框所处的cD NA的5′端片段和3′端片段,分析得到其全长共1109bp,与已报道的WSSV全基因序列(AF332093)的相关序列完全相同。该mRNA存在polyA,并有加尾信号AATAAA;两个阅读框都没有自己的TATA盒,但都有病毒RNA聚合酶Ⅱ的结合位点-CCAAT盒;它们编码的蛋白质分别有117和227个氨基酸,都存在可能的糖基化位点,其中WSV321一个,WSV322两个。
cDNA library including partial expressed genes of white spot syndrome virus(WSSV) was constructed by suppression subtractive hybridization.255 positive clones from forward subtraction library were identified by cDNA microarray.184 of the 255 positive clones were sequenced using the Pinpoint-T vector sequence as primer.The sequences were aligned with GenBank using the BLASTn program.30 ORFs of WSSV were detected including five new genes (WSV294,WSV184,WSV321,WSV322,WSV053).WSV184 had a Cys2/Cys2-type Zinc finger motif which had been shown to be involved in DNA-protein interaction and in regulation of transcriptional activation.WSV321 and WSV322 were found to be expressed in the same mRNA,which had transmembrane domains and potential sites for glycosylation.According to the sequence analysis,WSV294,WSV321 and WSV322,which had been considered to be no potential polyadenylation site(AATAAA),had a potential polyadenylation site downstream of the mRNA.To further understand the expression of WSV321 and WSV322,the SSH clone PCI 118(including WSV321 and WSV322) was identified by Dot Northern blot.The result was coincident with that by cDNA microarray.According to the sequence of WSSV,two primers were designed.The 5′ end and 3′ end of the cDNA containing two ORFs (WSV321,WSV322) were amplified respectively.A fragment of 1 109bp was obtained and had identities with partial sequences of WSSV in the GenBank.There existed a poly A tail in the mRNA and a virus RNA polymerase Ⅱ polyadenylation signal AATAAA at the 3′ end.Neither of the ORFs had a TATA box,but both had CCAAT boxes for virus RNA polymerase Ⅱ conjugation.The deduced proteins of ORF WSV321 and ORF WSV322 contained 117 and 227 amino acids respectively.There were three potential sites for glycosylation,one in the ORF WSV321 and two in the ORF WSV322.
出处
《病毒学报》
CAS
CSCD
北大核心
2004年第3期255-260,共6页
Chinese Journal of Virology
基金
国家自然科学基金(30170729)
瑞典国际科学基金(A/3362-1)。
关键词
对虾白斑综合征病毒
表达
基因
斑点杂交
CDNA微阵列
white spot syndrome virus
expressed gene
Dot Northern blot
rapid amplification of cDNA end
