滇牡丹ω-3脂肪酸脱氢酶基因克隆与功能分析
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摘要
ω-3脂肪酸脱氢酶是多不饱和脂肪酸亚麻酸生物合成途径的关键酶。依据转录组数据设计特异性引物,使用RT-PCR方法从滇牡丹克隆得到1个FAD3基因的c DNA全长序列,命名为Pd FAD3(Gene Bank登录号为KX289610)。生物信息学分析结果表明Pd FAD3基因片段序列全长2 134 bp,包含完整的c DNA开放阅读框,编码含有450个氨基酸残基的蛋白质;Blast比对结果显示该蛋白质属于FAD蛋白质家族;系统进化树分析结果显示与芍药属芍药组植物芍药亲缘关系较近;该蛋白质属于跨膜蛋白质,亲水性较低;RT-PCR结果显示其在种子中的表达随着种子成熟出现双峰型的表达量变化,与目前所报道的其他物种的FAD3表达情况相似。为进一步研究其调控机理提供理论依据及获得含高不饱和脂肪酸转基因滇牡丹品种奠定理论基础。
Omega- 3 fatty acid desaturase is the key enzyme in the biosynthesis pathway of linolenic belonging to polyunsaturated fatty acids.A full- length c DNA sequences of a new FAD3 gene was successfully cloned from Paeonia delavayi by RT- PCR method with the specific primers designed based on transcriptome datas,named Pd FAD3( Gene Bank accession number: KX289610).Bioinformatics analysis results showed that full- length sequence of Pd FAD3 gene was 2 134 bp,containing the complete c DNA open reading frame( ORF),encoded a protein with 450 amino acids.Blast comparison results showed that the protein belonged to the FAD family.Analysis results of phylogenetic tree showed that Paeonia delavayi had close genetic relationship with Paeonia lactiflora belonging to Paeonia Sect.Paeonia.The protein belonged to the transmembrane protein and was less hydrophilic.RT- PCR results showed that the expression in the seed as maturation occured bimodal change,similar to the FAD3 reported in other species.The Pd FAD3 gene cloned from Paeonia delavayi provided a scientific basis for further study on its regulation mechanism and obtaining Paeonia delavayi transgenic variety with highly unsaturated fatty acids.
Omega- 3 fatty acid desaturase is the key enzyme in the biosynthesis pathway of linolenic belonging to polyunsaturated fatty acids.A full- length c DNA sequences of a new FAD3 gene was successfully cloned from Paeonia delavayi by RT- PCR method with the specific primers designed based on transcriptome datas,named Pd FAD3( Gene Bank accession number: KX289610).Bioinformatics analysis results showed that full- length sequence of Pd FAD3 gene was 2 134 bp,containing the complete c DNA open reading frame( ORF),encoded a protein with 450 amino acids.Blast comparison results showed that the protein belonged to the FAD family.Analysis results of phylogenetic tree showed that Paeonia delavayi had close genetic relationship with Paeonia lactiflora belonging to Paeonia Sect.Paeonia.The protein belonged to the transmembrane protein and was less hydrophilic.RT- PCR results showed that the expression in the seed as maturation occured bimodal change,similar to the FAD3 reported in other species.The Pd FAD3 gene cloned from Paeonia delavayi provided a scientific basis for further study on its regulation mechanism and obtaining Paeonia delavayi transgenic variety with highly unsaturated fatty acids.
引文
[1]周海梅,马锦琦,苗春雨,等.牡丹籽油的理化指标和脂肪酸成分分析[J].中国油脂,2009,34(7):72-74.
[2]李奎.滇牡丹保护生物学与遗传多样性研究[D].北京:中国林业科学研究院,2013.
[3]杭晓敏,唐涌濂.多不饱和脂肪酸的研究进展[J].中国生物工程杂志,2001,21(4):18-21.
[4]MARTINS L P,SILVA S D M,ALVES R E.Chilling injury physiology in red mombin fruit(Spondias purpurea L.)[J].Rev Bras Frutic,2003,25(1):23-26.
[5]毛峰,秦振华.长链多不饱和脂肪酸与人体健康[J].医药产业资讯,2006,3(3):40-41.
[6]刘红梅.油脂中功能性成分的研究进展[J].轻工科技,2015(8):24-25.
[7]GIBSON S,ARONDEL V,IBA K,et al.Cloning of a temperature-regulated gene encoding a chloroplast omega-3desaturase from Arabidopsis thaliana[J].Plant Physiol,1995,106(4):1615-1621.
[8]刘文浩,李伟,陈相艳,等.ω-3脂肪酸脱氢酶在高、低油大豆品种中的克隆与序列分析[J].大豆科学,2008,27(4):569-571.
[9]李敏,高翔,陈其皎,等.小麦品种陕253ω-醇溶蛋白基因的克隆及序列分析[J].农业生物技术学报,2010,18(5):853-860.
[10]SHIMADA T,WAKITA Y,OTANI M,et al.Modification of fatty acid composition in rice plants by transformation with a tobacco microsomal.Omega-3 fatty acid desaturase gene(Nt FAD3)[J].Plant Biotechnol,2000,17(1):43-48.
[11]DOMNGUEZ T,HERNNDEZ M L,PENNYCOOKE J C,et al.Increasingω-3 desaturase expression in tomato results in altered aroma profile and enhanced resistance to cold stress[J].Plant Physiol,2010,153(2):655-665.
[12]MURAKAMI Y,TSUYAMA M,KOBAYASHI Y,et al.Trienoic fatty acids and plant tolerance of high temperature[J].Science,2000,287(5452):476-479.
[13]李苏雨,王毅,原晓龙,等.滇牡丹ω-6脂肪酸脱氢酶基因的克隆与功能分析[J].西部林业科学,2016,45(2):22-28.
[14]官玲亮,侯凯,陈郡雯,等.ω-6和ω-3脂肪酸脱氢酶家族系统进化与功能分化[J].遗传,2013,35(5):643-654.
[15]LI S S,WANG L S,SHU Q Y,et al.Fatty acid composition of developing tree peony(Paeonia section Moutan DC.)seeds and transcriptome analysis during seed development[J].BMC Genomics,2015,16(1):1-14.
[2]李奎.滇牡丹保护生物学与遗传多样性研究[D].北京:中国林业科学研究院,2013.
[3]杭晓敏,唐涌濂.多不饱和脂肪酸的研究进展[J].中国生物工程杂志,2001,21(4):18-21.
[4]MARTINS L P,SILVA S D M,ALVES R E.Chilling injury physiology in red mombin fruit(Spondias purpurea L.)[J].Rev Bras Frutic,2003,25(1):23-26.
[5]毛峰,秦振华.长链多不饱和脂肪酸与人体健康[J].医药产业资讯,2006,3(3):40-41.
[6]刘红梅.油脂中功能性成分的研究进展[J].轻工科技,2015(8):24-25.
[7]GIBSON S,ARONDEL V,IBA K,et al.Cloning of a temperature-regulated gene encoding a chloroplast omega-3desaturase from Arabidopsis thaliana[J].Plant Physiol,1995,106(4):1615-1621.
[8]刘文浩,李伟,陈相艳,等.ω-3脂肪酸脱氢酶在高、低油大豆品种中的克隆与序列分析[J].大豆科学,2008,27(4):569-571.
[9]李敏,高翔,陈其皎,等.小麦品种陕253ω-醇溶蛋白基因的克隆及序列分析[J].农业生物技术学报,2010,18(5):853-860.
[10]SHIMADA T,WAKITA Y,OTANI M,et al.Modification of fatty acid composition in rice plants by transformation with a tobacco microsomal.Omega-3 fatty acid desaturase gene(Nt FAD3)[J].Plant Biotechnol,2000,17(1):43-48.
[11]DOMNGUEZ T,HERNNDEZ M L,PENNYCOOKE J C,et al.Increasingω-3 desaturase expression in tomato results in altered aroma profile and enhanced resistance to cold stress[J].Plant Physiol,2010,153(2):655-665.
[12]MURAKAMI Y,TSUYAMA M,KOBAYASHI Y,et al.Trienoic fatty acids and plant tolerance of high temperature[J].Science,2000,287(5452):476-479.
[13]李苏雨,王毅,原晓龙,等.滇牡丹ω-6脂肪酸脱氢酶基因的克隆与功能分析[J].西部林业科学,2016,45(2):22-28.
[14]官玲亮,侯凯,陈郡雯,等.ω-6和ω-3脂肪酸脱氢酶家族系统进化与功能分化[J].遗传,2013,35(5):643-654.
[15]LI S S,WANG L S,SHU Q Y,et al.Fatty acid composition of developing tree peony(Paeonia section Moutan DC.)seeds and transcriptome analysis during seed development[J].BMC Genomics,2015,16(1):1-14.