摘 要

dihydroflavonol 4-reductase, DFR）作为将二氢黄酮醇转化为花色素反应的第一个酶，在花色形成过程中意义重大，同时也与类黄酮化合物的合成关系密切。由于类黄酮化合物在抗菌消炎等诸多方面有重要意义，深入探讨DFR基因的作用机理，有助于更深地了解类黄酮化合物的合成机制。本研究通过编制Bioperl生物分析环境，利用本实验室克隆的浙江红山茶DFR基因全长，并收集茶树、银杏、黑核桃、苹果、美洲山杨、杜鹃、西洋梨、葡萄等14个物种的DFR序列，利用编写的脚本将序列格式进行转化，得到了相关生物信息。并使用SOPMA、pfam、TMHMM、SignalP3.0和MEME等软件分析上述序列的二级结构、功能结构与、跨膜位点、信号肽和保守基序等性质。在Bioperl环境下调用ClustalW程序对序列进行比对，使用MEGA6.0构建了系统发育树。分析结果表明木本植物中DFR基因高度保守，上述蛋白均属于Epimerase家族，通过对其信号肽有无的判断，表明不同木本植物DFR蛋白的定位地点存在差异。木本植物DFR蛋白序列均存在三个基序，而黑核桃DFR序列的异常是由于克隆序列的不完整，而非功能突变导致。系统发育树的构建表明浙江红山茶与油茶的亲缘关系最近，其次是杜鹃。

关键词：Bioperl模块；浙江红山茶；DFR基因；生物信息学分析

Construction of Bioperl environment and bioinformatic analysis on DFR genes in Camellia chekiangoleosa

Abstract: As the first enzyme in the anthocyanidin reaction which transforms dihydroflavonol, Dihydroflavonol 4-reductase plays an essential role in the formation of flower colors. It is closely associated with the formation of flavonoid as well. Since flavonoid is useful in antisepsis, anti-inflammation and many other aspects, it’s very important to understand the synthetic mechanism. In this study, we constructed Bioperl environment and chose Camellia chekiangoleosa cloned in our labotory previously as the main subject, then we selected Camellia sinensis, Forsythia×intermedia, Gerbera hybrid cv. ‘Terra Regina’, Ginkgo biloba, Juglans nigra, Malus domestica, Populus tremuloides, Rhododendron simsii, Pyrus communis, Vitis vinifera and Rosa hybrid,all together 14 woody plants and got their DFR sequences through GenBank. We constructed Bioperl analysis environment under windows operating system and transform the sequences using the written scripts to obtain the bioinformatic information. SOPMA, pfam, TMHMM, SignalP3.0 MEME and other softwares were used to analysis the listed sequences’ secondary structures, functional domains, transmembrane regions, conserved motifs and other features. We used ClustalW script under Bioperl environment to search for the sequences’ conserved regions and constructed a phylogenetic tree with MEGA6.0. Analysis results revealed that DFR genes were highly conserved in woody plants and all the proteins belonged to the Epimerase Family. By judging the existence of signal peptides, we may infer that the location regions varied a lot in different woody plants. Three motifs were widely distributed in DFR domains, and the abnormal performance of Juglans nigra was resulted by the incomplete of the sequence instead of functional mutation. The construction of phylogenetic tree revealed that Camellia chekiangoleosa had the closest relationship with Camellia sinensis, the next was Rhododendron simsii.

Key word: Bioperl module; Camellia chekiangoleosa; DFR gene; Bioinformatic analysis

目 录

1 文献综述...................................................................................................................................1

1. 材料与方法..............................................................................................................................3

3.1 Bioperl分析环境的构建..................................................................................................3

2.2 Bioperl环境的构建..........................................................................................................2

2.3 序列选取..........................................................................................................................3

2.4 序列分析..........................................................................................................................4

3 结果与分析...............................................................................................................................5

3.1 脚本编写效果及信息获取..............................................................................................5

3.2 主要林木DFR蛋白质分析............................................................................................6

3.3 基序分析、序列比对及系统发育树构建......................................................................8

4 讨论...........................................................................................................................................10

致谢…………………………………………………………12

参考文献 ……………………………………………………13

图1：length.pl核心代码.............................................................................................5

图2：展示序列信息的常用指令................................................................................5