栉孔扇贝骨形态发生蛋白2基因的克隆及表达分析

Cloning and Expression Analysis of Bone Morphogenetic Protein 2 Gene of Chlamys farreri

骨形态发生蛋白(Bone morphogenetic protein,BMP)家族成员在动物的骨骼发育及器官形成中发挥着重要作用。本文利用同源克隆策略获得栉孔扇贝(Chlamys farreri)BMP2基因(CfBMP2)的全长cDNA和基因组DNA序列。其中,DNA序列长度为11 252bp,由3个外显子和2个内含子组成;cDNA序列全长3 562bp,含有1 221bp开放阅读框,编码406个氨基酸,与合浦珠母贝(Pinctadafucata)、长牡蛎(Crassostrea gigas)、斑马鱼(Danio rerio)和人(Homo sapiens)的同源蛋白相似性分别为67%、51%、59%和57%。运用实时荧光定量反转录PCR技术(qRT-PCR)对CfBMP2基因的时空表达进行分析。结果表明,在栉孔扇贝早期胚胎中几乎检测不到CfBMP2mRNA,从囊胚期开始有少量该基因表达,在原肠胚时期表达量显著上升,至幼虫期表达量又显著下降。除原肠胚外,在检测的其它发育时期,包括受精卵、4细胞期、担轮幼虫、D型幼虫和壳顶幼虫间,CfBMP2基因表达量没有显著差异。在取样的成体组织中均检测到CfBMP2基因的表达。其中,鳃中CfBMP2的表达量显著高于其它组织,外套膜次之。在横纹肌、性腺、肾和消化腺中,CfBMP2表达量较低且在这4种组织间无显著差异。上述研究结果提示,CfBMP2可能参与栉孔扇贝的器官组织发生和壳的生长。

Members of bone morphogenetic protein （BMP） play important roles in animal organogenesis and bone development. In this study, the Zhikong scallop （Chlamys farreri） BMP2 gene （CfBMP2） was cloned and characterized. The genomic sequence of this gene was 11 252 bp in length with three exons and two introns. The full-length cDNA sequence （3 562 bp） contained an open reading frame （ORF） of 1 221 bp. The encoded amino acid sequence exhibited 67%, 51%, 59% and 57% similarities with BMP2 from Pinctada fucata, Crassostrea gigas, Danio rerio and Homo sapiens, respectively. Expression analysis u sing real-time quantitative reverse transcription PCR （qRT-PCR） showed that CfBMP2 mRNA was unde tected in fertilized eggs and 4-cell embryos, but slightly detectable from blastulae. Significantly more CfBMP2 was found in gastrulae than in other stages. In the following larva stages, including trochophore larva, D-shaped larva and veliger larva, CfBMP2 expression level was sharply down-regulated. CfBMP2 transcripts were detected in all the adult tissues examined, with significantly higher expression level in gill than in other tissues. Relatively more mRNA were also found in mantle, while in gonad, kidney, digestive gland and striated muscle, the expression levels of CfBMP2 were similar and relatively low. The higher expression levels of CfBMP2 in the gastrulae and mantle suggested that this gene might be involved in scallop organogenesis and shell growth.