牡丹5个管家基因的克隆及其在系统进化分析中的应用

Cloning of Five House-keeping Genes and Their Application in Phylogenetic Analysis of Tree Peony

利用RT-PCR技术从牡丹组7个野生种和1个栽培种中分别克隆得到泛素延伸蛋白基因（ubiquitin,UBI）、素环蛋白基因（cyclophilin,CYP）、肌动蛋白基因（Actin）、葡萄糖六磷酸脱氢酶基因（glucose-6-phosphate 1-dehydrogenase,G6PD）和β–微管蛋白基因（beta-tubulin,TUB）等5个管家基因的编码序列（coding sequence,CDS）。序列比对分析发现,5个管家基因序列均相对保守,其在牡丹7个野生种和1个栽培种中的保守性在99.04%~99.69%。利用5个管家基因分别构建了其在牡丹组8份材料中的系统进化树。5个系统进化树均将8份材料分成两组,即革质花盘亚组和肉质花盘亚组;由于5个管家基因核苷酸序列各自的保守性不同,在区分亚组成员间略有差异。综合分析5个管家基因构建的系统进化树可知,杨山牡丹（Paeonia ostii）和栽培牡丹（P.suffruticosa‘Luoyanghong’）常聚为一支,二者亲缘关系较近,因而推测杨山牡丹可能参与了栽培牡丹的形成;紫斑牡丹（P.rockii）和四川牡丹（P.decomposita）一直聚为一支,推测二者亲缘关系较近;黄牡丹（P.lutea）和紫牡丹（P.delavayi）一直聚为一支,推测这二者亲缘关系较近。狭叶牡丹（P.potaninii）与黄牡丹和紫牡丹分化较早,结合前人研究结果建议将狭叶牡丹作为一个独立的种。

Coding sequences（CDS）of five house-keeping genes i.e. Ubiquitin（UBI）,Cyclophilin（CYP）,Actin,Glucose-6-phosphate 1-dehydrogenase（G6PD）and Beta-tubulin（TUB）were obtained by Reverse Transcription Polymerase Chain Reaction（RT-PCR）from 7 wild and 1 cultivated tree peonies. Sequence comparative analysis demonstrated that nucleotide sequences of five genes were relatively conservative and the similarity of nucleotide sequences was about 99.04% to 99.69%. Furthermore,5 phylogenetic trees were constructed by using nucleotide sequences of these house-keeping genes from the 8 tree peonies. The phylogenetic tree analysis results showed that the 8 tree peonies could be clearly divided into two groups,viz. subsect. Vagiantae and Delavayanae. While each phylogenetic relationship of wild species in different sect. had a few differences,because of the degree of conservatism from 5 kinds of house-keeping genes. Considering to the five phylogenetic trees,it was found that Paeonia ostii and P. suffruticosa always cluster together. And these results indicated that there was close relationship between P. ostii and P. suffruticosa and it was deduced that P. ostii may take part in the origin of tree peony cultivars. In addition,P. rockii and P. decomposita was always clustered together,and it was demonstrated that the relationship between P. rockii and P. decomposita was very close;P. lutea and P. delavayi was always clustered together,and it was demonstrated that their relationship was also close. P. potaninii divergence time is earlier than P. lutea and P. delavayi. According to previous phylogenetic relationships of P. potaninii,P. lutea and P. delavayi,we recommend P. potaninii as a separate species.