杨扇舟蛾颗粒体病毒晚期表达因子的分析

Analysis of the late expression factor genes in Clostera anachoreta granulovirus

【目的】本研究对杨扇舟蛾颗粒体病毒（Clostera anachoreta granulovirus,Clan GV）的晚期表达因子（Late expression factors,LEFs）进行生物信息学分析,并对Clan GV的lefs与其它杆状病毒的lefs进行对比,对鉴定lef基因和研究Clan GV的感染机制具有重要意义。【方法】使用ORF finder在线程序进行开放阅读框（ORFs）分析,BLASTP程序用于蛋白序列的同源分析,DNAStar和DNAClub生物学软件用于对序列进行处理和分析,采用BLAST在公共数据库中对序列进行比对,Clustal W Version 1.8和Gene Doc 2.7用于多序列的比对和分析,用MEGA 6.06的NJ法进行系统发育分析。【结果】Clan GV基因组包括15个lef基因,分别为ie-1、lef-2、39k、lef-11、p35、p47、lef-1、lef-6、lef-5、lef-4、dnapol、lef-3、lef-9、lef-8和lef-10。Clan GV与云杉枞色卷蛾颗粒体病毒（Choristoneura occidentalis granulovirus,Co GV）、黄地老虎颗粒体病毒（Agrotis segetum granulovirus,As GV）、苹果异形小卷蛾颗粒体病毒（Cryptophlebia leucotreta granulovirus,Cl GV）、马铃薯块茎蛾颗粒体病毒（Phthorimaea operculella granulovirus,Po GV）和苹果蠹蛾颗粒体病毒（Cydia pomonella granulovirus,Cp GV）有着较近的亲缘关系。颗粒体病毒中只有Clan GV和分月扇舟蛾颗粒体病毒含有p35。Clan GV的lef-2的转录方向不同于其它12个颗粒体病毒。γ和δ杆状病毒分别只含有7个和3个Clan GV的lef同源基因。Clan GV的lefs跟其它杆状病毒lefs的相似性较低,其中Clan GV与其它颗粒体病毒间的相似性高于Clan GV与核型多角体病毒间的相似性。Clan GV的lef-2、39k、p35、lef-5、dnapol和lef-9在启动子上游拥有TATA box;p47和lef-1在TATA下游20~40 bp处还有一个CACT基序;lef-11、p35、lef-6、lef-5、lef-3、lef-9、lef-8和lef-10这8个基因有晚期启动子基序TAAG;而p35、lef-5、lef-9和lef-10上游既有TATA又有TAAG基序。【结论】结果为研究杆状病毒的基因功能和感染机制提供了数据基础。

[Objectives] To identify late expression factor（LEF） genes and investigate the Clan GV infection mechanism. [Methods] We analyzed the Clan GV LEF family of genes of Clostera anastomosis granulovirus, including gene identification and organization, gene sequences, and structural characteristics at the DNA and protein levels, and compared the data obtained with those on other baculoviruses. Open reading frames（ORFs） were confirmed using the ORF finder online program. Homology searches were carried out using the BLASTP program. Sequence data assembly and analysis were performed using DNAStar and DNAClub software. Sequence comparison in public databases was done using the BLAST program. Multiple sequence analysis was performed using Clustal W Version 1.8 and Gene Doc 2.7. Phylogenetic analysis was carried out with MEGA 4.0 software using the neighbor joining（NJ） distance method（1 000 bootstrap replicates）. [Results] Sequence analysis indicated that the C. anastomosis granulovirus Clan GV genome includes 15 LEF genes, annotated as ie-1, lef-2, 39 k, lef-11, p35, p47, lef-1, lef-6, lef-5, lef-4, dnapol, lef-3, lef-9, lef-8 and lef-10, respectively. C. anastomosis granulovirus Clan GV shares higher homology with the Choristoneura occidentalis granulovirus, Agrotis segetum granulovirus, Cryptophlebia leucotreta granulovirus, Phthorimaea operculella granulovirus, and the Cydia pomonella granulovirus, than with other granuloviruses. With the exception of 9 nucleopolyhedroviruses, P35 was only found in C. anastomosis granulovirus Clan GV and the C. anastomosis granulovirus. C. anastomosis granulovirus Clan GV lef-2 differed from that of the other 12 granuloviruses in transcription orientation. The nucleopolyhedrovirus belonging to gammabaculovirus and deltabaculovirus include 7 and 3 Clan GV lefs, respectively. C. anastomosis granulovirus Clan GV lefs have very low homology with baculoviruses, although their homology with granulovirus lefs is higher than with those of nucleopolyhedroviruses. C. anastomosis granulovirus Clan GV lef-2, 39 k, p35, lef-5, dnapol, and lef-9, possess an early promoter motif（TATA）. p47 and lef-1 have a CACT motif 20-40 bp downstream of TATA, and eight genes; lef-11, p35, lef-6, lef-5, lef-3, lef-9, lef-8, and lef-10, have a late promoter motif TAAG. However, only p35, lef-5, lef-9, and lef-10, possess both the TATA and TAAG. [Conclusion] The results of this study are of considerable importance to research on the Clan GV granulovirus gene function and infection mechanism.