地涌金莲MlCYP734A6基因的克隆与表达分析

Cloning and Expression Analysis of MlCYP734A6 in Musella lasiocarpa

[目的]为揭示CYP734A6基因在地涌金莲生长发育过程中的调控作用提供分子数据。[方法]从地涌金莲转录组数据库中筛选到1个注释为CYP734A6的差异基因片段,通过3′和5′RACE技术克隆得到该基因的全长序列,命名为MlCYP734A6(登录号为MW013148)。利用生物信息学技术对MlCYP734A6进行序列比对、分子特征分析、系统进化分析等。应用实时荧光定量PCR方法比较分析该基因在不同地涌金莲类型及不同组织部位中的表达量。利用高效液相色谱-质谱联用技术测定地涌金莲不同组织器官中油菜素内酯的含量。[结果]克隆得到1条包含c DNA全长为1 584 bp的MLCYP734A6基因序列,其编码527个氨基酸,相对分子量为60 038.84 Da,等电点为9.44。所编码的氨基酸序列通过序列比对及系统进化分析,结果显示:与小果野蕉亚种的同源序列相似度最高,亲缘关系最近。qRT-PCR分析结果显示:MlCYP734A6基因在地涌金莲的所有组织中均有表达,且表达量最低的部位是叶片,表达量最高的2个组织部位是花序轴和根尖。地涌金莲的各个组织器官中都能检测到油菜素内酯,且与MlCYP734A6的表达水平无显著相关性,矮杆类型的株形可能是由于过量内源油菜素内酯所致。[结论]从地涌金莲中成功克隆了油菜素内酯的失活基因MlCYP734A6,推测由于该基因参与了地涌金莲中油菜素内酯的代谢过程,从而调控了活性油菜素内酯的含量。本研究结果可为进一步分析CYP734As对地涌金莲及其他植物的生长发育的调控机理提供理论支持。

[Objective]To provide data in molecular-level for revealing the regulatory role of MlCYP734A6 in the growth and development of Musella lasiocarpa. [Method]The 3′ and 5′ RACE technology was used to clone the full length cDNA of MlCYP734A6 gene. The software of bioinformatics was used to analyze the nucleotides and protein sequence. Real-time PCR method was used to analyze the gene expression level in different types and tissues of Musella lasiocarpa. HPLC-MS/MS was used to detect the brassinolide content in different tissues. [Result]The full length cDNA of MlCYP734A6 is 1 584 bp, encoding 527 amino acids. The relative molecular mass of coding protein is 60 038.84 Da and the isoelectric point is 6.61. Sequence comparison and phylogenetic tree analysis showed that the amino acid sequence of MlCYP734A6 had the closest evolutionary relationship with the CYP734A6 protein of Musa acuminata subsp. malaccensis. Real-time PCR analysis showed that MlCYP734A6 could be detected in all tissues.The two tissues with the highest expression level were rachis and root tip, and the lowest expression level was in leaf.No significant correlation was found between the brassinolide content and the expression pattern of MlCYP734A6,and excessive brassinosteroids in RD05 was a possible cause of dwarfed phenotype. [Conclusion]The study indicated that MlCYP734A6 may participate in the metabolism of brassinosteroids, and balance the bioactive brassinosteroids content in Musella lasiocarpa. These results provide further theoretical support for the role of CYP734 As in plant growth and development in the future.