西文蜜蜂LOC724521基因座的cDNA序列克隆及TSS的分析

cDNA Cloning and Transcription Start Site Analysis of a Gene LOC724521 from Apis Mellifera

5’LongSAGE标签得自于全长mRNA分子的5’末端的前19 nt.该研究利用定位在西方蜜蜂基因组中的一个预测基因座LOC724521的2条5’LongSAGE标签序列作为5’引物,通过RT-PCR克隆了该预测基因座的2条长335 bp和337 bp的cDNA序列(GenBank登录号:GU358205,GU358204).此cDNA编码一条长88个氨基酸残基的多肽.用所克隆的cDNA序列对基因座LOC724521进行功能注释发现,该基因含有3个外显子和2个"GT-AG"型内含子.5’LongSAGE标签序列的基因组定位结果显示:基因座LOC724521在雄蜂的头部中表达丰度很高,RNA PolⅡ可从5个转录起始位点(TSS)上以不同效率起始转录,该基因的59%和31%的转录是从2个优势TSS上起始.有趣的是,有一条5’LongSAGE标签序列被定位在内含子区,暗示该基因存在外显子的可变性选择现象.该研究结果不仅在转录水平上证实了软件预测的基因座LOC724521确实存在,同时揭示了该基因存在可变性转录起始位点和可变性外显子选择等转录调控机制.

The 5＇LongSAGE tags are the first 19 nt sequences derived from 5＇-end of full-length mRNA molecules.In this study,we cloned the cDNA sequence of a predicted gene locus in the honeybee genome by using 5＇LongSAGE tag sequence as PCR primer.A group of five different 5＇LongSAGE tags,which came from a drone（Apis mellifera）head 5＇LongSAGE library,were mapped around the transcription initiation start site（TSS）of a hypothetical gene locus LOC724521.By using two of these LongSAGE tag sequences as up-stream PCR primers,we cloned two cDNA sequences that were integrated at 5＇-end through RT-PCR method（access no.：GU358205,GU358204）.The cDNA sequences were 335 bp and 337 bp in length and encoding 88 amino acid residues.The gene structure of LOC724521 was annotated with the cloned cDNA sequences.It was shown the genomic DNA of LOC724521 contains three exons and two introns,where all boundaries conform to the GU/AG rule.Mapping result of 5＇LongSAGE tag sequences on LOC724521 shows that this gene is highly expressed in drone head,and RNA PolⅡ initiates transcription at five alternative TSSs with different efficiency.It was found 59% and 31% transcription of LOC724521 were initiated from two dominant TSS.Intriguing,there was one tag sequences being mapping in the first intron,indicating the existence of ＂alternative selection of exon ＂machinery.The results of this investigation not only provide transcriptional evidence for the in silicon predicted locus LOC724521,but also reveal the complex regulation machinery of this gene,such as alternative TSSs and alternative selection of exon.