家蚕乳糖酶-根皮苷水解酶基因BmLPH014192的表达和序列选择性剪接分析

乳糖酶-根皮苷水解酶(lactase-phlorizin hydrolase,LPH)是肠组织上皮细胞微绒膜上的一种糖蛋白,对乳糖和根皮苷有水解活性。鉴定了家蚕基因组中的一个LPH基因,命名为BmLPH014192。该基因CDS长1 305 bp,编码434个氨基酸,预测蛋白质分子质量为50.8 kD,等电点(pI)为5.35。将该基因的cDNA与家蚕基因组序列比对,显示其具有7个外显子,外显子/内含子边界处均符合GT-AG规则。将BmLPH014192与哺乳动物人、褐鼠、家兔和昆虫黑腹果蝇、冈比亚按蚊、二化螟、佛罗里达弓背蚁、大红斑蝶等的LPH进行氨基酸序列比对,相似度都在60%左右;聚类分析中BmLPH014192与昆虫类LPH聚在一起。芯片数据分析结果表明BmLPH014192在家蚕5龄第3天幼虫的9个组织中,只在中肠中有表达,并且表达量很高。在绿茧品种大造和白茧品种19-710幼虫的中肠中扩增到的特异性条带其长度分别为1 000 bp和750 bp,存在序列选择性剪接;在其它白茧品种的中肠中没有扩增出特异性条带。推测BmLPH014192的特殊表达方式可能与其在不同家蚕品种中肠组织中的特异性功能有关。

Expression and Sequence Alternative Splicing Analysis of the Lactase-phlorizin Hydrolase Gene BmLPH 014192 in Bombyx mori

Lactase-phlorizin hydrolase(LPH) is a glycoprotein in microvillus membrane of the intestinal epithelial cell.It has hydrolytic activity to lactose and phlorizin.We identified one of the silkworm(Bombyx mori) LPH genes and named it as BmLPH 014192.CDS(coding sequence) of this gene is 1 305 bp long and encodes 434 amino acids,with predicted molecular weight of 50.8 kD and isoelectric point of 5.35.An alignment of the cDNA sequence with silkworm genomic sequence revealed that there are 7 exons in BmLPH 014192 and all of the exon /intron boundaries conformed to the GTAG rule.Sequence comparison between BmLPH 014192 and LPH genes from other species including Homo sapiens,Rattus norvegicus and Oryctolagus cuniculus of mammal and Drosophila melanogaster,Anopheles gambiae,Chilo suppressalis,Camponotus floridanus and Danaus plexippus of insect revealed that their amino acids all shared about 60% similarity.Phylogenetic analysis showed that BmLPH 014192 was clustered with other insect LPH genes.Microarray ana-lyzing results indicated that BmLPH 014192 was expressed only in midgut among 9 tissues of day 3 silkworm larvae of the 5th instar,and the expression level was very high.Fragments amplified from midgut cDNAs of Dazao,a variety spinning green cocoons,and of 19-710,a variety spinning white cocoons,were 1 000 and 750 bp long respectively,indicating their origination from alternative splicing.No specific band was amplified from midgut of other white cocoon species.It is suggested that the special expression pattern of BmLPH 014192 is related to the specific function of this gene in midgut tissues of different silkworm varieties.