油茶CoFAD2-1基因的克隆、亚细胞定位及组织表达

doi:10.16036/j.issn.1000-2650.2019.04.007

摘要/Abstract

摘要： 【目的】克隆油茶CoFAD2-1基因,并进行亚细胞定位和组织表达研究。【方法】采用CTAB法提取油茶4种组织中的总RNA;运用RT-PCR方法,设计基因特异性引物,从油茶未成熟胚中克隆CoFAD2-1基因;用生物信息学手段分析了CoFAD2-1基因的结构和进化关系;利用实时荧光定量PCR技术研究CoFAD2-1基因在不同组织的表达;同时运用烟草瞬时表达技术对该基因进行亚细胞定位分析。【结果】该基因编码区全长为1 149 bp,共编码382个氨基酸,具有FAD2基因家族保守的3个组氨酸簇。系统树分析显示该基因与种子特异表达的FAD2基因聚在一起,组织表达分析也表明CoFAD2在种子中特异表达,转化烟草叶片验证CoFAD2-1蛋白定位于内质网膜上。【结论】CoFAD2-1基因在种子中特异性表达,且定位在内质网膜上。

关键词: 油茶, CoFAD2-1, 基因克隆, 表达分析, 亚细胞定位

Abstract: 【Objective】 The aim of this study was to clone CoFAD2-1 gene, and to investigate the subcellular localization and tissues expression. 【Method】 The total RNA of Camellia Oleifera Abel. was extracted by CTAB method. RT-PCR was used to clone the CoFAD2-1 gene with gene specific primers from the immature embryo of Camellia Oleifera Abel. The structure and evolution of CoFAD2-1 gene was analyzed by bioinformatics. Meanwhile, real-time PCR was used to check CoFAD2-1 gene expression profile in different tissues and transient tobacco expression system was employed to reveal its subcellular localization. 【Result】 CoFAD2-1 gene was 1 149 bp long in open reading frame. It encoded382 amino acids and contained three conserved His box of CoFAD2-1. Phylogenetic tree analysis showed that the gene was clustered in the same class with seed specific expression FAD2 gene. Tissue expression analysis also suggested that the specific expression of CoFAD2 was found in the embryo and the gene was verified to localize in the endoplasmic reticulum membrane using tobacco leaf. 【Conclusion】 The results indicate that CoFAD2-1 gene is seed-specificity expressed and localized in the endoplasmic reticulum membrane.

Key words: Camellia oleifera Abel, CoFAD2-1, gene cloning, expression analysis, subcellular localization

中图分类号:

S794.4

陈潇潇, 罗红艳, 顾真琪, 陈世品, 曹光球, 曹世江. 油茶CoFAD2-1基因的克隆、亚细胞定位及组织表达[J]. 四川农业大学学报, 2019, 37(04): 475-480.

CHEN Xiaoxiao, LUO Hongyan, GU Zhenqi, CHEN Shipin, CAO Guangqiu, CAO Shijiang. Cloning Subcellular Localization and Tissues Expression of CoFAD2-1 Gene from Camellia oleifera[J]. Journal of Sichuan Agricultural University, 2019, 37(04): 475-480.

参考文献

[1] 庄瑞林. 中国油茶[M]. 北京: 中国林业出版社, 1988.
[2] 郑德勇, 凌宏有. 福建油茶籽在成熟过程中重要成分动态变化的研究[J]. 中国粮油学报, 2014(2): 36-43.
[3] 马超, 尤幸, 王广东. 中国主要木本油料植物开发利用现状及存在问题[J]. 中国农学通报, 2009(24): 330-333.
[4] 陈素传, 肖正东. 安徽省油茶生产现状及发展对策[J]. 湖北林业科技, 2011(1): 47-50.
[5] 邹小玲. 福建油茶建园良种选择及丰产栽培关键技术[J]. 中国农业信息, 2014(2): 61-63.
[6] CAO S J, ZHOU X R, CRAIG C W.et al.A large and functionally diverse family of FAD2 genes in safflower(Carthamus tinctorius L.)[J]. BMC Plant Biology, 2013(13):5.
[7] 邓树丛, 于小飞, 陈军, 等.橄榄油的加工工艺及品质评价[J].林产工业, 2011, 38(1): 62-63.
[8] BALK E M, LICHTENSTEIN A H, CHUNG M, et al. Effects of omega-3 fatty acids on serum markers of cardiovascular disease risk:A systematic review[J]. Atherosclerosis, 2006, 189(1): 19-30.
[9] 张喜雨, 周军, 晁燕, 等. 四种天然食用植物油脂的脂肪酸成分分析[J]. 湖南林业科技, 2015, 42(3): 69-71.
[10] 陈明久. 延平区油茶优良单株选择[J]. 福建林业科技, 2014(1): 97-102.
[11] HEPPARD E P, KINNEY A J, STECCA K L, et al.Developmental and growth temperature regulation of two different microsomal [omega]-6 desaturase genes in soybeans[J]. Physiologia Plantarum, 1996(110): 311-319.
[12] JUNG S, POWELL G, MOORE K, et al. The high oleate trait in the cultivated peanut(Arachis hypogaea L). II. Molecular basis and genetics of the trait[J]. Molecular & General Genetics, 2000(263):796-805.
[13] LOPEZ Y, SMITH O D, CONNELL J P, et al. Isolation and characterization of the h12-fatty acid desaturase in peanut(Arachis hypogaea L.) and search for polymorphisms for the high oleate trait in spanish market-type lines[J]. Theoretical and Applied Genetics, 2000(101): 1131-1138.
[14] 谢禄山, 谭晓风, 张琳, 等.油桐种子FAD2基因全长cDNA序列分析[J]. 经济林研究, 2012(2): 1-9.
[15] 谭晓风, 胡芳名, 谢禄山, 等. 油茶种子EST文库构建及主要表达基因的分析[J]. 林业科学, 2006(1): 43-48.
[16] OKULEY J, LIGHTNER J, FELDMANN K, et al.Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis[J]. Plant Cell, 1994(6): 147158.
[17] 宋志波, 刘敏, 贾宝光, 等.油茶总RNA的提取与内参基因的初选[J].经济林研究, 2014, 32(2): 93-98.
[18] LIVAK K J, SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408.
[19] BROOK K N, XUE C, ANDREAS N.A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants[J]. The Plant Journal, 2007(51): 1126-1136.
[20] 谭晓风, 陈鸿鹏, 张党权, 等. 油茶FAD2基因全长cDNA的克隆和序列分析[J]. 林业科学, 2008(3): 70-75.
[21] ALONSO D L, MAROTO F G, RUIZ R G, et al.Evolution of the membrane-bound fatty acid desaturases[J]. Biochemical Systematics and Ecology, 2003(31): 1111-1124.
[22] 王仲伟, 温强, 汤诗杰, 等. 一个油茶FAD2基因家族新成员的克隆及分析[J]. 分子植物育种, 2017, 15(1): 84-90.
[23] KAWACHI M, KOBAE Y, MIMURA T, et al.Deletion of a histidine-rich loop of AtMTP1, a vacuolar Zn²+/H+ antiporter of Arabidopsis thaliana, stimulates the transport activity[J]. Journal of Biological Chemistry, 2008, 283(13): 8374-8383.
[24] CHEN H P, TAN X F, XIE Y J, et al.Constructingand function of plant expression vector and RNA interference vector of a FAD2 gene from Camellia Oleifera[J]. Bulletin of Botanical Research, 2015, 35(3): 347-354.