十字花科多肽激素类基因RALF的同源基因克隆及进化分析

多肽激素类基因是对植物生长发育起重要作用的一类基因,RALF是其中的重要一员,而十字花科在中国蔬菜作物中是重要的一大类群。为了摸清十字花科多种蔬菜作物的RALF同源基因信息,该文根据前期研究从油菜中扩增到的多肽激素类基因RALFbn的序列设计引物,从提取的十字花科芸薹属、萝卜属、蔊菜属、山芥属的7份重要蔬菜作物的基因组DNA中分别克隆到了RALF的同源序列。结果表明:7种蔬菜作物的RALF同源基因编码区均在300 bp左右,且无内含子,编码的蛋白质由79个氨基酸组成,说明RALF在十字花科4个属内的保守性较强;对RALF同源基因在十字花科4个属中的表达分析表明,该类基因在根、茎、叶、花序轴等营养器官中不表达或弱表达,但主要在生殖器官中表达,其中在总花蕾和开放花中的表达量普遍高于嫩角果中的表达量,表明该类基因在十字花科中的生理活跃期是花发育时期。同时,构建了十字花科4个属中RALF同源基因的系统树,芸薹属的油菜、甘蓝和芥蓝形成一个分支,而茎瘤芥和分蘖芥形成另外一个分支,且与萝卜属、山芥属、蔊菜属的材料聚在一支,该基因的进化途径在一定程度上也反映出这几种作物的遗传背景关系。该研究结果丰富了RALF家族信息,增添了十字花科植物的分子进化数据。

Cloning and evolutionary analysis of homologous sequences of RALF in Cruciferae

Peptide hormones genes are important in plant growth and development and RALF(rapid alkalinization factor)is one of them. Cruciferae is a big family in Chinese vegetable crops. Polypeptide hormone gene RALFbn was cloned from Brassica campestris(rape)in the authors'' previous work. In order to find out the information of RALF homologous genes in Cruciferae vegetable crops,this experiment was done. At this time,the primers were designed according to the sequence of RALFbn gene. The genomic DNAs of 7 important vegetable crops were extracted in advance. The vegetable materials were selected from Brassica,Raphanus,Rorippa and Barbarea and they all belonged to the same family of Cruciferae. Seven sequences homologous to RALF were cloned from the 7 materials from important vegetable crops with the primers designed from RALFbn. Then the 7 DNA sequences together with the RALFbn gene were analyzed. The results showed that all the DNA sequences'' open reading frames were about 300 bp long. There was no intron in the 7 genes. The 7 proteins encoded by them were all made by 73 amino acids. All these indicated that the RALF homologous genes were very conservative in the 4 genera of cruciferous species. At the same time,the 7 RALF homologous genes expression analyses were done in four genera in Cruciferae. The result indicated that these genes were not or weakly expressed in root,stem,leaf,inflorescence axis and other vegetative organs,while mainly expressed in reproductive organs,in which,the total amounts of the expression in flower buds and open flowers were generally higher than those in tender siliques. All the expression indicated that the physiological active period of these genes in Cruciferae was in the flower growth period. The system tree was constructed with the 7 RALF homologous genes in the 4 genera of Cruciferae. The NJ tree of RALF homologous genes reflected that RALFbn,BoRALFjx and BoRALFzh formed a branch while BjRALFdn4 and BjRALFxlh formed another branch with the rest RALF homologous genes from Raphanus,Rorippa,Barbarea clustered in the same branch. The evolutionary pathway of these RALF homologous genes in a certain extent also reflected the genetic background relationships between these crops. In all,this study enriched the information of the rapid alkalinization factor(RALF)family and expanded the knowledge of the molecular evolution data of Cruciferae.