异源四倍体鲫鲤及其原始父母本细胞周期蛋白B基因克隆与分子遗传分析

Cloning and Genetic Analysis of Cyclin B Gene in Allotetraploids and Their Original Parents

细胞周期蛋白(cyclin)B是真核细胞周期运转中调控G2期至M期转化的关键因子.本实验根据斑马鱼细胞周期蛋白B1基因的剪切方式,设计3对特异于金鱼和银鲫细胞周期蛋白B基因外显子区兼并引物,首次扩增出异源四倍体鲫鲤及其原始亲本红鲫和鲤鱼细胞周期蛋白B基因2条大小分别约为2.4kb和2.1kb的片段.测序及比对分析表明:这3种鱼的细胞周期蛋白B基因2个片段均包含8个外显子和7个内含子.内含子剪切位点符合GT-AG规则,推测细胞周期蛋白B基因2个片段可能是细胞周期蛋白B基因的2种存在形式.异源四倍体鲫鲤与其原始父母本细胞周期蛋白B基因片段序列的比较结果表明:无论是外显子区还是内含子区,异源四倍体鲫鲤与其原始亲本都具有较高的遗传相似性,在1025bp的外显子序列中相同的碱基位点数达963个,为异源四倍体鲫鲤来源于红鲫和鲤鱼提供了分子证据;同时,异源四倍体鲫鲤与其原始亲本差异碱基位点的存在又表明这一独特的多倍体物种与其原始亲本存在着进化上的变异.此外,还分别以细胞周期蛋白B基因外显子和内含子序列构建了包括异源四倍体鲫鲤及其原始父母本在内的系统进化树.结果初步表明:对于亲缘关系较近的物种,用外显子和内含子序列构建的系统进化树与传统的物种进化树一致;而对于亲缘关系较远的物种,用内含子序列构建的进化树与传统的物种进化顺序存在较大差异.

As one of the key regulators in cell cycle, cyclin B regulates the G2-M transition. We amplified two cycline B gene fragments, 2.1 kb and 2.4 kb, from allotetraploid and its original maternal red crucian carp（ Carassius carassius red var. ） and original paternal common carp （ Cyprinus carpio L. ） by PCR. The degenerate primers were designed to be specific for the exon regions of cyclin B in gold fish and silver crucian carp, but based on the two fragments consisted presumed that these two splicing character of cycline B gene in zebrafish. Sequence analysis showed that these of 8 exons and 7 introns and all the introns obeyed ＂GT-AG＂ rule. Therefore, we fragments were different forms of the same gene. Sequence comparison showed that 963 nucleotides against total 1 025 bp in allotetraploids were the same as their original parents, indicating a high genetic similarity between them. It also proved that allotetraploids came from red crucian carp and common carp on the molecular level. On the other hand, there were different nucleotides between allotetraploids and their original parents revealed that there was genetic variability in the evolution process. In addition, the phylogenetic trees of different species including allotetraploids and their parents were constructed based on the exons and introns sequences of cyclin B gene, respectively. This result suggested that among the closely related species, the phylogenetic trees based on the cyclin B exons and introns were consistent with the traditional one. However, among the distant relative species, there were variation between the phylogenetic trees based on the cyclin B introns and the traditional phylogenetic trees.