西方蜜蜂表皮蛋白基因apd-like转录起始位点的定位及cDNA序列的分析

TSS identification and cDNA cloning of a cuticular protein gene apd-like from the Western honeybee(Apis mellifera)

Apidermin蛋白家族是根据蜜蜂表皮蛋白apidermin1-3(APD1-3)而命名的一个新型的昆虫结构性表皮蛋白家族。为了鉴定西方蜜蜂Apis mellifera基因组序列上毗邻基因簇apd1-3的一个预测基因座LOC727145是否为一个新的apd基因,本研究在用5′LongSAGE标签定位该基因的转录起始位点(TSS)的基础上,利用其中的3条5′LongSAGE标签序列作为上游引物,通过RT-PCR方法克隆了该基因的cDNA序列(GenBank登录号:GU358197,GU358199,GU358198)。生物信息学分析发现,基因座LOC727145含有2个外显子和1个"GT-AG"型内含子,其cDNA序列富含GC(70%),可编码一条长152aa残基的高度疏水性多肽。此多肽序列的氨基酸组成与蜜蜂APD1-3表皮蛋白类似,富含Ala,Gly,Pro,Leu和Val5种氨基酸(占77%),其中Ala残基含量最高(29%)。该多肽序列与蜜蜂APD-1表皮蛋白序列的相似性为50%,且其N末端的预测信号肽序列与APD蛋白的信号肽序列类似。5′LongSAGE标签的基因组定位结果显示,基因座LOC727145在雄蜂头部中表达丰度很高,RNA PolⅡ可从6个不同的TSS上以不同效率起始转录,其中由一个优势TSS上起始了90%的转录。本研究为apidermin表皮蛋白家族增添了一个新成员,命名为apidermin-like(apd-like)。

The apidermin（APD）family,named after three cuticular proteins（apidermin 1-3,APD 1-3）in the Western honeybee,Apis mellifera,is a novel insect structural cuticular protein family.A hypothetical gene locus（LOC727145）was found to be located at the upstream of the reported apd gene cluster in the honeybee genomic sequence.In order to characterize this gene,we mapped its transcription start sites（TSSs）by a group of 5＇ LongSAGE tag sequences,and subsequently cloned its cDNA sequences through RT-PCR by using three different 5＇ LongSAGE tag sequences as the up-stream primers.The cloned cDNA sequences were submitted to GenBank under the accession numbers GU358197,GU358198 and GU358199.Bioinformatics analysis revealed that the genomic DNA sequence LOC727145 contains two exons and one intron,where the boundary conforms to the GU/AG rule.The cDNA sequence is extremely GC-rich（70%）,and the deduced protein is highly hydrophobic.Five amino acids（Ala,Gly,Pro,Leu and Val）account for 77% of the deduced polypeptide sequence,and Ala is the most abundant residue（29%）.These characters are comparative to APD 1-3.Moreover,the deduced polypeptide sequence has 50% identity with APD-1 protein,and both possess a similar predicted hydrophobic signal peptide at the N-terminal.These results suggest that LOC727145 encodes a novel APD protein.The mapping result of 5＇ LongSAGE tag sequences on genome sequence showed that LOC727145 was highly expressed in drone head,and RNA PolⅡ initiated transcription of this gene at six alternative TSSs with different efficiency,and 90% of the mRNA molecules came from a dominant TSS.This research identified a novel apidermin protein,named apidermin-like（apd-like）.