姜黄苯丙氨酸解氨酶基因的克隆与序列分析

Cloning and sequence analysis of phenylalanine ammonia-lyase gene from Curcuma longa

目的对姜黄Curcuma longa苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)基因(Cur PAL)全长进行克隆并开展生物信息学分析,为姜黄次生代谢产物的生物合成机制研究奠定基础。方法利用RT-PCR和RACE相结合的方法,以姜黄叶片c DNA为模板,克隆获得Cur PAL全长,进行生物信息学分析。结果克隆的Cur PAL(登录号为KJ780359)全长c DNA为1 293 bp,其中包括5’-UTR 243 bp,3’-UTR 123 bp,含有1个927 bp的完整开放阅读框(opening reading frame,ORF),编码308个氨基酸;预测蛋白质相对分子质量为33 000,理论等电点p I为5.76;Cur PAL蛋白性质稳定,为可溶性蛋白;Cur PAL编码的蛋白质序列包含与夹竹桃PAL蛋白质相同的脱氨基位点和催化活性位点,氨基酸序列多重比较发现,Cur PAL编码的氨基酸序列与中国李、浙江红山茶、拟南芥、辣椒、小果野蕉PAL氨基酸序列的同源性达到75%以上;与姜目类的植物(如小果野蕉)的PAL聚为一支,说明两者亲缘关系较近;通过蛋白质二维、三维结构的预测,表明Cur PAL蛋白为全α型蛋白,由同源四聚体构成。结论首次克隆并获得Cur PAL基因全长c DNA,为姜黄素生物合成途径的阐明和改善中药材品质提供科学依据。

Objective This study aimed at cloning the phenylalanine ammonia-lyase （PAL） gene from Curcuma longa （CurPAL） and analyzing the bioinformatics. Methods PAL gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from C. longa leaves. The bioinformatic analysis of this gene and its corresponding protein were performed. Results One unique sequence of PAL, named as CurPAL （GenBank NO. KA780359）, was cloned from C. longa. The full-length of CurPAL cDNA was 1 293 bp, including 243 bp of 5＇-UTR, 123 bp of 3＇-UTR, and 927 bp of ORF encoding 308 amino acids. The molecular weight and theroretical isoelectric point （pI） of the deduced CurPAL protein were 33 000 and 5.76, respectively. The protein of CurPAL was stable and soluble. The domination sites and catalytic active sites in PAL protein of Nerium oleander were also found in CurPAL. It was found that the amino acid sequence of CurPAL had more than 75% homology with PAL of Prunus salicina, Camellia chekiangoleosa, Capsicum chinense, and Musa acuminate via multiple alignments. It revealed that CurPAL had closer relationship with PALs from Zingiberales plants than from other plants by phylogenetic tree analysis. Secondary and tertiary structures indicate that CurPAL is a full ct protein contained by homotetramer. Conclusion The cDNA encoding PAL from C. longa is cloned and reported for the first time. This work provides a scientific basis for exploring the biosynthetic pathway of the medicinal ingredient and improving its quality in C. longa.