油用牡丹PEPC基因的克隆及表达分析  

Cloning and expression analysis of PEPC gene from Paeonia ostii

作　　者：袁秀云[1] 田云芳 张燕[1] 马杰[1] 崔波[1] YUAN Xiu-yun;TIAN Yun-fang;ZHANG Yan;MA Jie;CUI Bo(Institute of Bioengineering,Zhengzhou Normal University,Zhengzhou 450044,China;School of Life Sciences,Zhengzhou Normal University,Zhengzhou 450044,China)

机构地区：[1]郑州师范学院生物工程研究所,河南郑州450044 [2]郑州师范学院生命科学学院,河南郑州450044

出　　处：《中国油料作物学报》2019年第6期878-886,共9页Chinese Journal of Oil Crop Sciences

基　　金：郑州师范学院科技创新团队支持计划;郑州市都市生态农业产业技术体系(20170106)

摘　　要：磷酸烯醇式丙酮酸羧化酶(PEPC)是调控植物种子中蛋白质/油脂含量比例的关键酶。利用RT-PCR和RACE技术,从油用牡丹(Paeonia ostii)叶片中克隆得到1个PEPC基因PaPEPC2(GenBank登录号为MK606450)。其cDNA全长为3201 bp,包含2898 bp开放阅读框,编码965个氨基酸。PaPEPC2编码蛋白分子量为110.3 kD,理论等电点为5.65,属于酸性亲水性蛋白;二级结构预测表明,该蛋白的主要结构为α螺旋和不规则卷曲。氨基酸序列比对和进化树分析表明,PaPEPC2基因编码的蛋白属于C3型PEPC,与葡萄的C3型PEPC蛋白(XP002280569.1)亲缘关系最近。实时荧光定量分析表明,PaPEPC2基因在茎、叶、花芽和子房中均有表达,其中在茎和子房中的表达水平最高,在花芽中表达水平最低;在种子发育过程中,该基因表达水平呈现先升高,后降低的趋势,其中在发育42 d时表达水平最高;在种子收获后,常温存放7 d时,该基因表达水平最高,随后其表达水平逐渐下降。由此推测,PaPEPC2基因在油用牡丹种子发育和成熟过程中对油脂和蛋白的合成起阶段性调控作用。Phosphoenolpyruvate carboxylase(PEPC) is the key enzyme that regulates the protein/oil content ratio in plant seed. The phosphoenolpyruvate carboxylase gene PaPEPC2(accession number in GenBank is MK606450) was cloned from the leaves of Paeonia ostii by the method of RT-PCR and RACE. The full-length cDNA of PaPEPC2 gene was 3201 bp with an open reading frame of 2898 bp encoding 965 amino acids. PaPEPC2 gene encoded protein which belongs to an acidic and hydrophilic protein with the molecular weight of 110.3 kD and the academic isoelectric point of 5.65. The results of secondary structure prediction showed that the main structural element for PaPEPC2 protein was α-helix and random coil. Furthermore, the sequence alignment and phylogenetic analyses indicated that the protein encoded by PaPEPC2 gene belongs to C3 type PEPC, which was most closely related to C3 type PEPC protein of Vitis vinifera(XP002280569.1). qRT-PCR showed that PaPEPC2 was expressed in all of tissues, and expression level of the PaPEPC2 gene was the highest in stem and ovary, and the lowest in bud. During the seed development, the expression level of PaPEPC2 gene showed increasing trend firstly, and then decreasd, with the highest expression level at 42 d. Moreover, the expression of PaPEPC2 gene in the seed was increased after harvest with the peak level after harvest at 7 d after storage under room temperature, and then decreased gradually. These findings implied that PaPEPC2 gene played a periodic regulate role in synthesis of oil and protein during seed development and maturation in Paeonia ostii.

关 键 词：油用牡丹 磷酸烯醇式丙酮酸羧化酶 种子 基因克隆 表达分析 

分 类 号：S567.15[农业科学—中草药栽培]