灰毡毛忍冬苯丙氨酸解氨酶(LmPAL2)基因的克隆与表达分析

Cloning and expression of phenylalanine ammonia-lyase 2 gene in Lonicera macranthoides

目的对灰毡毛忍冬苯丙氨酸解氨酶(LmPAL2)基因进行克隆与表达分析。方法以灰毡毛忍冬花总RNA为模板,在灰毡毛忍冬转录组数据的基础上,利用RT-PCR与RACE技术克隆LmPAL2基因的cDNA序列全长,并通过一系列软件对LmPAL2基因进行生物信息学分析。应用qRT-PCR技术测定其在灰毡毛忍冬不同花期及不同器官(茎、叶)中的相对表达量。结果克隆得到LmPAL2(GenBank:MH779889)基因,开放阅读框(ORF)长度为2124 bp,编码707个氨基酸,具有苯丙氨酸解氨酶(PAL)保守结构域和活性中心,与其他植物PAL基因同源性较高;qRT-PCR结果显示,LmPAL2基因在绿白色花蕾期的相对表达量最高;在不同器官中,叶>白色花蕾期花>茎。结论克隆得到LmPAL2基因,并探索其在不同器官的表达模式,推测其可能与灰毡毛忍冬绿原酸的生物合成有关。

Objective To clone the phenylalanine ammonia lyase(LmPAL2)gene of Lonicera macranthoides and analyze its expression.Methods The total RNA served as a template to clone the cDNA full-length sequence of LmPAL2 gene by RT-PCR and RACE,which was based on the transcriptome data of Lonicera macranthoides,and the genome sequence of LmPAL2 were analyzed by bioinformatics software.The relative expression at different flowering periods and in various organa(stem and leaf)was determined by qRT-PCR technique.Results The LmPAL2(GenBank:MH779889)gene was cloned,and the open reading frame was 2124 bp encoding 707 amino acids.It contained conserved domains and active sites of PAL,which was highly homologous to PALs of other plant.qRT-PCR showed that the relative expression level of the green and white flower periods was the highest.In different organs,the relative expression in the white bud period was higher than the stem and lower than the leaf.Conclusion PAL2 gene of Lonicera macranthoides is cloned and its expression patterns in different organs are determined,whose mechanism may be related to the biosynthesis of chlorogenic acid in Lonicera macranthoides.