紫苏Pfgpat9基因的鉴定与功能分析

Identification and Function Analysis of Pfgpat9 Gene from Perilla frntescens

甘油三磷酸酰基转移酶(GPAT)是三酰甘油(TAG)合成过程中的关键酶。本研究从油料作物紫苏中筛选鉴定得到甘油三磷酸酰基转移酶基因Pfgpat9,并揭示其编码蛋白特征及基因功能。从紫苏转录组数据库中筛选获得gpat9基因片段,以优选品种‘晋紫苏1号’为材料,通过RT-PCR技术克隆获得该基因的开放阅读框(ORF)全长序列,命名为Pfgpat9;利用实时荧光定量PCR方法分析Pfgpat9基因在紫苏根、茎、叶、花及不同发育时期种子(开花后10、20、30、40 d)中的组织表达特性;通过双酶切将紫苏Pfgpat9基因完整的ORF序列连接到酵母表达载体pYES2.0上,将重组质粒转化至野生型酵母菌株INVSc1和缺陷型酵母gat1,并验证该基因的功能。结果显示:紫苏Pfgpat9基因ORF为1116 bp,共编码371个氨基酸,理论等电点pI为8.96,不稳定系数为46.67,亲水性系数为-0.108,是一种亲水性不稳定蛋白。紫苏Pfgpat9基因具有典型的GPAT9功能结构域,包含3个典型的跨膜螺旋区;多序列比对分析表明,紫苏Pf GPAT9蛋白与油橄榄、茶树和向日葵GPAT9蛋白的亲缘关系较近;qRT-PCR分析显示,Pfgpat9基因在紫苏不同组织中均表达,在种子发育不同时期表达量呈先升高后降低趋势,在开花后20 d表达量达到最高;酵母转化结果表明,转Pfgpat9基因野生型酵母和转Pfgpat9基因缺陷型酵母总脂含量均提高,且C16∶0、C16∶1含量均有所提高。研究表明,Pfgpat9基因在紫苏种子油脂合成积累过程中发挥重要作用,这为进一步通过基因工程及转基因技术改良紫苏品质提供了新的方向。

Glycerol-3-phosphate acyltransferase(GPAT)is a key enzyme in the synthesis of TAG.The objective of this study was to screen and identify the glyceryl triphosphate acyltransferase 9 gene from perilla and reveal this protein characteristics and functions.The Pfgpat9 gene fragment was screened from the perilla transcriptome database,with the preferred variety‘Jinzisu 1’as material,the full-length ORF sequence was cloned by RT-PCR.It was named Pfgpat9.The tissue expression characteristics of Pfgpat9 gene was analyzed by real-time PCR in root,stem,leaf,flower and seeds at different developmental stages(10,20,30,40 days after flowering)of perilla.The complete ORF sequence of Pfgpat9 gene was ligated into the yeast expression vector pYES2.0 by double digestion,and the recombinant plasmid was transformed into the wild type yeast strain INVSc1 and the GPAT deficient gat1 yeast strain.The results showed that the Pfgpat9 gene ORF was 1116 bp,encoding a total of 371 amino acids.The theoretical isoelectric point(pI)was 8.96,the instability coefficient was 46.67,and the hydrophilicity coefficient was-0.108.These results showed that Pf GPAT9 protein was a hydrophilic and labile protein.The Pfgpat9 gene had a typical GPAT9 functional domain and contained three typical transmembrane helix regions.Multiple sequence alignment analysis showed that the Pf GPAT9 protein was closely related to Olea europaea,Camellia sinensis and Helianthus annuus GPAT9 protein.qRT-PCR analysis showed that Pfgpat9 gene was expressed in different tissues of perilla,and the expression level increased firstly and then decreased at different stages of seed development,and reached the highest level at 20 days after flowering.The yeast transformation results indicated that the total lipid content of Pfgpat9 gene-transformed wild type yeast and Pfgpat9 gene-deficient yeast were increased,and the C16∶0,C16∶1 were all increased.The study indicated that Pfgpat9 gene plays an important role in the oil synthesis and accumulation of perilla seeds,and can provide genetic element and technology system for creating other ordinary oil crop new lines.