坛紫菜有丝分裂阻滞缺陷蛋白2基因(Ph-mad2)的克隆及其在单性生殖过程中的表达

为探究坛紫菜雌配子体单性生殖过程的细胞二倍化机制,实验基于坛紫菜雌性配子体的转录组信息,克隆和分析了坛紫菜的有丝分裂阻滞缺陷蛋白2(PhMAD2)基因(Phmad2),同时对其在坛紫菜单性生殖发生过程中处于不同发育时期的细胞中的表达特征进行了初步研究。结果显示,坛紫菜Ph-mad2基因的cDNA的完整开放阅读框为672 bp,编码223个氨基酸,分子量为23.8 ku。从氨基酸序列比对结果可知,坛紫菜PhMAD2蛋白中所具有的典型的HORMA结构域和保守的丝氨酸和苏氨酸磷酸化位点,均与团藻和莱茵衣藻MAD2蛋白相同。系统发育分析表明,坛紫菜与藻状菌纲的异丝水霉和寄生水霉的亲缘关系最近。实时荧光定量PCR检测显示,与可进行正常有丝分裂的营养细胞相比,在坛紫菜单性生殖过程中发生细胞二倍化的生殖细胞和单性孢子时期,Ph-mad2基因的表达量显著下降;但是,在随后可进行正常有丝分裂的单性孢子萌发体细胞和处于营养生长期的单性孢子体细胞中则表达上调。这一结果暗示Ph-mad2基因的表达下调可能阻碍了坛紫菜单性生殖初期单倍体细胞进行有丝分裂时纺锤体组装检验点的形成,在一定程度上致使它们发生了染色体加倍。

Cloning and characterization of homologue of mitotic arrest deficient 2 from Pyropia haitanensis and its expression analysis during parthenogenesis

To investigate the role and molecular mechanism of diploidization of cells during the parthenogenetic development of female gametophyte in Pyropia haitanensis, a cDNA sequence of homologue of mad2 was isolated and its expression profile at different developmental stages of parthenogenesis was characterized. In this study, the cDNA of Ph-mad2 spans 672 bp that encodes a protein of 223 amino acids (aa) with a predicted molecular weight of 23.8 ku. The deduced protein sequence of PhMAD2 has a typical HORMA domain and conservative serine and threonine phosphorylation sites. Phylogenetic analysis revealed that evolutionary status of P. haitanensis was extremely related to the fungi from Phycomycetes, such as Saprolegnia diclina and S. parasitica. Quantitative real-time PCR (qRT-PCR) analysis showed that the expressions of Ph-mad2 at reproductive cell development stage (RDS) and parthenospore development stage (PDS) with diploid carpogonium-like cells and carpospore-like cells were significantly down-regulated compared with that at vegetative cell proliferation stage (VPS) with haploid vegetative cells, while its expression profiles were up-regulated in the later stages of parthenosporophyte formation stage (PFS) and parthenosporophyte growth stage (PGS), in which the diploid carpospore-like germlings and parthenosporophytes underwent normal mitosis. These results suggested that Ph-mad2 reduction potentially prevented the formation of spindle assembly checkpoint (SAC) at mitotic cells during the early stages of parthenogenesis, and triggered their development of diploidization. These results provide important information for revealing the mechanism of spontaneous chromosome doubling in the parthenogenesis of P. haitanensis.