文献类型：期刊文献

中文题名：南美油藤PvFAD7基因的克隆及其表达模式和功能分析

英文题名：Homology-based cloning,expression pattern and functional analysis of PvFAD7 gene from Plukenetia volubilis

作者：刘果[1] 吴志华[1] 彭彦[1] 尚秀华[1] 高丽琼[1]

第一作者：刘果

机构：[1]中国林业科学研究院速生树木研究所,广东湛江524022

年份：2023

卷号：45

期号：4

起止页码：745-755

中文期刊名：中国油料作物学报

外文期刊名：Chinese Journal of Oil Crop Sciences

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；

基金：广东省基础与应用基础研究基金(2019A1515110168、2021A1515011021)。

语种：中文

中文关键词：南美油藤;PvFAD7基因;表达模式;亚细胞定位;过表达

外文关键词：Plukenetia volubilis;PvFAD7 gene;expression pattern;subcellular localization;overexpression

分类号：S718.46

摘要：ω-3 FAD基因是α-亚麻酸合成途径中的一个关键限速酶基因,通过调节该基因的过量表达,可提高植物中α-亚麻酸含量。对南美油藤ω-3脂肪酸脱氢酶基因PvFAD7的克隆及其表达模式和功能分析,有利于探明南美油藤种子高含量α-亚麻酸的合成机制,为下一步利用遗传改良生产植物α-亚麻酸奠定理论基础。基于前期南美油藤种子转录组数据,从南美油藤新鲜叶片同源克隆获得南美油藤脂肪酸脱氢酶基因PvFAD7的DNA全长序列,并对其进行生物信息学分析。通过实时荧光定量PCR分析该基因在南美油藤不同器官(成熟叶、新叶、茎、根、果皮、幼嫩种子和成熟种子)中的表达模式,并利用亚细胞定位技术观察该基因的编码蛋白在烟草叶表皮细胞中的定位情况。通过构建过表达载体,介导农杆菌遗传转化获得过表达PvFAD7基因的转基因阳性烟草植株,并对收获的种子利用气相色谱法进行脂肪酸组分的测定。南美油藤PvFAD7基因DNA全长序列为2222 bp,编码461个氨基酸,包括8个外显子和7个内含子。蛋白多序列比对结果显示南美油藤PvFAD7编码蛋白与同科植物蓖麻和橡胶树同源性最高。生物信息学分析结果表明,PvFAD7蛋白有4个组氨酸保守区,是典型的膜结合类脂肪酸脱氢酶,N端具有叶绿体转运肽特征与亚细胞定位结果一致,显示为PvFAD7蛋白定位于叶绿体。RT-qPCR的结果显示PvFAD7基因在南美油藤成熟叶和新叶中相对表达量最高,而种子中的表达量最低且两个不同时期种子中PvFAD7的相对表达量差异不显著。通过构建过表达载体pCAMBIA1301-PvFAD7并转化获得过表达PvFAD7基因的转基因阳性烟草植株,发现烟草种子亚油酸含量降低了6.40%,而α-亚麻酸、棕榈酸和含油量分别增加了0.87%、3.19%和18.09%。因此,PvFAD7为叶绿体型ω-3脂肪酸脱氢酶基因,在催化亚油酸合成α-亚麻酸的反应中发挥了一定的功能,但可能不是南美油藤种子中α-亚麻酸合成的主效基因。
Theω-3 FAD gene is a key rate-limiting enzyme gene inα-linolenic acid synthesis pathway.By regulating the overexpression of this gene,α-linolenic acid content in plants can be increased.The homologybased cloning,expression pattern and functional analysis of PvFAD7 gene from Plukenetia volubilis will help to elu-cidate the biosynthesis mechanism ofα-linolenic acid in Plukenetia volubilis seeds.Based on the transcriptome da-tabase,the PvFAD7 gene was cloned from fresh leaves and analyzed by bioinformatics methods.The expression pat-tern of PvFAD7 in different organs(mature leaves,new leaves,stems,roots,pericarp,young seeds(Pv-1)and ma-ture seeds(Pv-2))was analyzed by real-time quantitative polymerasechain reaction,and the subcellular localiza-tion of the PvFAD7 protein in tobacco leaf epidermal cells was observed.By constructing an overexpression vector,Agrobacterium tumefaciens-mediated genetic transformation was performed to obtain transgenic positive tobacco plants with overexpressing PvFAD7 gene.The fatty acid components of harvested seeds were determined by gas chromatography.The DNA sequence of PvFAD7 gene was 2222 bp,encoding 461 amino acids,including 8 exons and 7 introns.The results of amino acid sequences alignment showed that PvFAD7 protein had the highest homolo-gy with Ricinus communis and Hevea brasiliensis.Bioinformatics analysis indicated that PvFAD7 protein had four histidine conserved regions,which was a typical membrane-bound fatty acid dehydrogenase.The N-terminal of Pv-FAD7 was characterized by chloroplast transport peptide,which was consistent with the results of subcellular local-ization.Results of RT-qPCR showed that PvFAD7 gene had the highest relative expression level in mature and new leaves,the lowest expression level in seeds,and there was no significant difference between the two development stages of seeds.By constructing an overexpression vector pCAMBIA1301-PVFAD7 and Agrobacterium-mediated genetic transformation,positive transgenic tobacco plants with overexpressing PvFAD7 gene were obtained.Fatty ac-id determination of harvested tobacco seeds showed that the linoleic acid content of transgenic positive tobacco seeds was reduced by 6.40%.Theα-linolenic acid,palmitic acid and oil content increased by 0.87%,3.19%and 18.09%,respectively.In conclusion,PvFAD7 is a chloprplastω-3 fatty acid dehydrogenase gene,which plays an active role in catalyzing the biosynthesis ofα-linolenic acid from linoleic acid,but it might not be the major gene forα-linolenic acid biosynthesis in Plukenetia volubilis seeds.