南美油藤PvWRI1转录因子的克隆及其在油脂合成中的功能分析  

Cloning of PvWRI1 transcription factor and analysis of its function in oil synthesis of Plukenetia volubilis

作　　者：李晓明[1] 吴志华 尚秀华[1] 刘果 LI Xiao-ming;WU Zhi-hua;SHANG Xiu-hua;LIU Guo(Research Institute of Fast-growing Trees,Chinese Academy of Forestry,Zhanjiang 524022,China)

机构地区：[1]中国林业科学研究院速生树木研究所,广东湛江524022

出　　处：《中国油料作物学报》2024年第5期1048-1057,共10页Chinese Journal of Oil Crop Sciences

基　　金：广东省基础与应用基础研究基金(2021A1515011021)。

摘　　要：WRI1是一类具有APETALA2结构域的转录因子,参与油脂生物合成过程。本研究从新鲜南美油藤叶片中克隆PvWRI1基因组序列,并对其进行生物信息学分析。通过构建过表达载体,将PvWRI1基因转化到烟草中获得转基因阳性烟草植株,收获成熟种子,利用气相色谱法进行脂肪酸组分测定。并通过实时荧光定量PCR分析PvWRI1基因下游脂肪酸途径基因的表达水平。PvWRI1基因组DNA全长5072 bp,编码区长度为1312 bp,编码436个氨基酸,共存在7个外显子和6个内含子。疏水性分析显示该蛋白为亲水性蛋白,结构域分析该蛋白含有2个AP2/EREBP结构区和4个固有无序蛋白区,二级结构分析表明该蛋白相对保守。磷酸化位点预测N端含叶绿体转运肽特征。氨基酸同源性分析在不同物种中AP2结构域同源性较高,系统进化树显示PvWRI1与蓖麻同源性最高。脂肪酸组分测定结果表明,硬脂酸和亚麻酸含量显著增加,分别增加16.8%和7.5%。转基因种子的含油量由12.84%增加到24.06%,达1.87倍。同时,亚油酸/亚麻酸平衡比例下降7.8%。RT-qPCR结果显示过表达PvWRI1导致下游脂肪酸途径基因表达量均出现上调,其中WRI1基因极显著上调,FATA、KASI和FAD3基因显著上调。因此,PvWRI1作为调控脂肪酸生物合成的关键因子,对改良油料植物含油量和脂肪酸平衡具有指导作用。WRI1 is a kind of transcription factors with APETALA2 domain,which is essential for the oil bio-synthesis.In this study,the genomic sequence of PvWRI1 was cloned from fresh leaves of Plukenetia volubilis and analyzed by bioinformatics methods.Through the construction of overexpression vector,PvWRI1 gene was trans-formed into tobacco to obtain transgenic positive tobacco plants.Wild type and transgenic mature seeds were har-vested and the fatty acid components were determined by gas chromatography.The expression level of downstream fatty acid pathway genes of PvWRI1 was analyzed by real-time PCR.The DNA sequence of PvWRI1 was 5072 bp,the coding region is 1312 bp,encoding 436 amino acids,and including 7 exons and 6 introns.Hydrophobicity analy-sis showed that the protein was hydrophilic.Domain analysis showed that the protein contained two AP2/EREBP structural regions and four inherently disordered protein regions,and the secondary structure analysis showed that the protein was relatively conserved.Phosphorylation predicts the characteristics of N-terminal chloroplast trans-port peptides.Amino acid homology analysis showed that the AP2 domain had high homology in several species,and the phylogenetic tree showed that PvWRI1 had the highest homology with WRI1 of castor.According to the analysis of fatty acid components,the contents of stearic acid and linolenic acid increased significantly,by 16.8%and 7.5%,respectively.The total oil content of transgenic seeds increased from 12.84%to 24.06%,and it was up to 1.87 times.At the same time,the linoleic acid/linolenic acid balance ratio decreased by 7.8%.RT-qPCR results showed that overexpression of PvWRI1 gene led to the up-regulation of fatty acid pathway genes,in which WRI1 gene was most significant,and FATA,KASI and FAD3 genes were significantly up-regulated.Therefore,as a crucial regulator in fatty acid biosynthesis,PvWRI1 plays a guiding effect on improving oil content and fatty acid balance in oil plants.

关 键 词：南美油藤 PvWRI1 基因克隆 过表达 脂肪酸测定 表达量 

分 类 号：S718.46[农业科学—林学]