家蚕铁蛋白Ferritin家族基因的鉴定及序列特征与表达谱分析

Identification of Bom byx mori Ferritin Gene and Analysis on Its Sequence Features and Expression Patterns

机体内的铁蛋白Ferritin能存储和释放铁离子,在调节细胞的铁离子平衡中发挥重要作用。采用生物信息学方法分析家蚕基因组中Ferritin基因家族成员Bm Fer1、Bm Fer2、Bm Fer3和Bm Fer4的序列特征,并通过转录组芯片数据和实时定量PCR分析其时空表达谱,为研究蚕体内的铁元素代谢机制提供基础数据。Bm Fer1、Bm Fer2、Bm Fer3具有Ferritin蛋白家族典型的5个α螺旋结构,但铁离子结合位点的种类和数量相对较少。Bm Fer1和Bm Fer3为线粒体铁蛋白,具有O糖基化位点;Bm Fer2和Bm Fer4为细胞质铁蛋白,具有N糖基化位点。Bm Fer1、Bm Fer4为重链型铁蛋白,具有典型的亚铁氧化酶活性中心,在系统进化上与代表物种的重链型铁蛋白聚为一支;而Bm Fer2为轻链型铁蛋白,无亚铁氧化酶活性中心,聚类于轻链型分支。转录组芯片数据表明,家蚕5龄幼虫中肠中Bm Fer1、Bm Fer2的表达量明显高于其他各组织,而且在头部、脂肪体、血细胞、马氏管中的表达量是Bm Fer1的平均值高于Bm Fer2。在家蚕幼虫5龄4 d至蛾期的发育过程中,Bm Fer1和Bm Fer2的表达量均明显上调,尤以上蔟后期发育至蛾期的上调最为明显。实时定量PCR分析显示,Bm Fer1和Bm Fer2在家蚕5龄幼虫丝腺组织的表达量明显高于其他组织。Western blotting分析发现Bm Fer2在家蚕胚胎细胞中有表达,并在催青后的蚕卵中上调表达,推测Bm Fer2基因在家蚕早期胚胎发育中已开始转录、表达行使其功能。

Ferritin,an iron storage protein,plays a key role in regulating iron balance by storing and releasing ferrous ions.In this paper,sequence features of silkworm Bm Fer1,Bm Fer2,Bm Fer3 and Bm Fer4 genes of Ferritin gene from the silkworm（Bombyx mori）were analyzed by bioinformatic measures,and temporal-and spatial-expression profiles of these genes were detected by microarray and qRT-PCR to provide useful basic data for investigating iron metabolism in silkworm.Bm Fer1,Bm Fer2 and Bm Fer3 have the typical five alpha-helical structures as in most Ferritin family members,while the types and numbers of iron binding sites are relatively less.Bm Fer1 and Bm Fer3 are mitochondrial Ferritins with O-glycosylation sites,while Bm Fer2 and BmFer4 are cytoplasmic Ferritins with N-glycosylation sites.Bm Fer1 and Bm Fer4 are heavy-chain Ferritins with typical ferroxidase active centers,and cluster in one phyletic clade with other heavy-chain Ferritins from representative species.Bm Fer2 is a light-chain Ferritin without ferroxidase active center,and cluster with other light-chain ferritins.Micro-array data of transcriptome show that expression levels of Bm Fer1 and Bm Fer2 genes in mid gut of the 5th instar silkworm larvae are obviously higher than those in other tissues.In addition,Bm Fer1 gene presents higher expression level in head,fat body,hemocyte and Malpighian tubule than Bm Fer2 gene.From day 4 of the 5th larval instar to adult stage,both Bm Fer1 and Bm Fer2 genes exhibit an obvious up-regulation,especially during late stage of cocooning to emergence of moths.Real-time quantitative PCR analysis showed that Bm Fer1 and Bm Fer2 genes had obviously higher expression in silk gland of the 5th instar silkworm larvae than those in other tissues.Western blotting analysis indicated that Bm Fer2 protein was expressed in silkworm embryonic cells and up-regulated in silkworm eggs around the incubation stage.It is suggested that Bm Fer2 gene starts transcription/expression and plays its role in early stage of silkworm embryo development.