雌核发育二倍体鲫鲤及其他倍性鱼cdc2基因cDNA全序列克隆及表达分析

为研究雌核发育二倍体鲫鲤产生二倍体卵子的分子机制,实验采用PCR和cDNA末端快速分离法,克隆获得了雌核发育二倍体鲫鲤第三代(G3)、二倍体红鲫、三倍体湘云鲫和四倍体鲫鲤的细胞周期相关基因——cdc2基因cDNA全序列.结果显示,4种不同倍性鱼cdc2基因均编码含有302个氨基酸蛋白,而且编码的蛋白都含有与其他CDK激酶相当保守的序列PSTAVRE;同源性分析发现,4种鱼cdc2基因编码的氨基酸序列之间的相似度大于97.6％,说明Cdc2蛋白在这4种不同倍性鱼中具有高度保守性.采用实时荧光定量PCR(real-time PCR)对cdc2基因在G3、二倍体红鲫、三倍体湘云鲫及四倍体鲫鲤早期卵巢中的表达进行分析,结果发现,G3cdc2基因比普通二倍体红鲫和三倍体湘云鲫表达要高,比四倍体鲫鲤的表达水平低.该研究从分子水平证明了G3早期性腺中存在着大量的多倍体卵原细胞.同时,研究表明,cdc2基因在雌核发育二倍体鲫鲤早期卵巢的高表达暗示着G3多次进入S期却不经历M期导致二倍体配子的产生.

The cloning of cdc2 cDNAs and a comparative study of its expression in different ploidy fishes including the diploid gynogenetic hybrid of red crucian carp×common carp

Cdc2(Cyclin Dependent Kinase, namely CDK1) encoded by cdc2 gene and CyclinB combination regulates G2/M transition. To find out molecular mechanism that diploid hybrid fish could produce diploid gamete, the full length cDNAs of cdc2 in the third gynogenetic generation (G3), red crucian carp( Carassius auratus red var.), triploid crucian carp and allotetraploid were obtained by PCR and rapid amplification of cDNA ends. Our data showed that all the cDNAs of cdc2 gene in the four different ploidy fishes encode a protein of 302 amino acids containing a domain (PSTAVRE) which combinate Cyclins. A high homology of 97.6% of the Cdc2 protein can be drawn by comparing the amino acid sequences in this four fishes, which indicating the higher conservative function and evolution of Cdc2 protein in this four fishes. A comparative expression pattern of cdc2 in early-stage gonads of G3 and different ploidy fishes was carried out by Real-time PCR using specific primers against the same sequences of coding regions in the four fishes. The results showed that the expression of cdc2 in the ovary of G3 was higher than those of red crucian carp and triploid crucian carp, while lower than that of allotetraploid, which, at the molecular level, indicates existence of polyploid oogonia in early-stage gonads of G3. The higher expression of cdc2 in G3 suggests that consecutive S-phase replication may occur without intervening mitosis, which might be related to the formation mechanisms for the diploid eggs generated by diploid hybrids.