GUO Xinya,ZHANG Wenhua,LIN Feng.Functional analysis of soybean oleosin gene GmOLE2[J].Journal of Nanjing Agricultural University,2021,44(3):477-486.[doi:10.7685/jnau.202008027]





Functional analysis of soybean oleosin gene GmOLE2
郭新亚 章文华 林峰
南京农业大学生命科学学院, 江苏 南京 210095
GUO Xinya ZHANG Wenhua LIN Feng
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
oleosinfatty acidabiotic stressGlycine max
[目的] 本文旨在对编码栽培大豆(Glycine max)油体蛋白(oleosin,OLE)的GmOLE2基因进行功能研究,分析其蛋白结构、在油脂合成中的调控作用以及对非生物胁迫的响应。[方法] 运用生物信息学方法,分析其蛋白保守结构、亲疏水性以及进化关系;利用农杆菌侵染获得转基因拟南芥纯合株系,并利用气相色谱检测转基因拟南芥种子脂肪酸组分;对转基因拟南芥进行多种非生物胁迫处理,观察并统计相关性状和指标。[结果] GmOLE2蛋白具有OLE蛋白家族高度保守的结构域——脯氨酸结,且蛋白中间部分是一个由约24个氨基酸组成的超长疏水性结构域。GmOLE2是种子特异性表达的基因,过表达GmOLE2导致拟南芥种子内总蛋白含量增加约25%,总可溶糖含量下降10%,油脂含量下降约10%,饱和脂肪酸和三不饱和脂肪酸含量减少,单不饱和脂肪酸含量增加。过表达GmOLE2拟南芥种子可以缓解冻害,-20℃处理后的种子萌发率仍达到75%,是野生型萌发率的2倍。GmOLE2 mRNA积累受MeJA和ABA调控:10 μmol·L-1 MeJA处理GmOLE2过表达拟南芥幼苗,其主根生长受到抑制的情况下,侧根丰度及长度显著大于未处理组,且在24 h内GmOLE2基因表达量显著增加,是未处理组的8~10倍。将GmOLE2过表达拟南芥的种子播种在含0.5 μmol·L-1 ABA的培养基上,萌发3 d后,经RT-qPCR检测发现GmOLE2基因表达量显著升高,是未处理组的8倍。[结论] GmOLE2基因具有典型油体蛋白结构特征。在拟南芥中过表达GmOLE2基因,虽然减少种子总油含量但可以调节脂肪酸构成,且其种子具有较好的抗冻性,可受MeJA和ABA诱导表达参与植物对非生物胁迫的响应。
[Objectives] The objectives of this article were to investigate the function of soybean oleosin gene GmOLE2 by analyzing its protein structure,the regulation of oil synthesis and its response to abiotic stress,and thus provide genetic sources for the improvement of soybean seed quality and seed oil content. [Methods] The conserved protein structure,hydrophilicity and hydrophobicity and evolutionary relationship were analyzed by using bioinformatics tools;the fatty acid composition of Arabidopsis thaliana seeds was detected by gas chromatography. Transgenic A.thaliana seeds were treated under various abiotic stress,and related traits were observed and quantified. [Results] The GmOLE2 protein had a conserved domain "proline knot",and the middle part was an ultra-long hydrophobic structure composed of about 24 amino acids. GmOLE2 was highly expressed in seeds. Overexpression of GmOLE2 resulted in an about 25% increase in total protein in A.thaliana seeds,an about 10% decrease in total soluble sugar,and an about 10% decrease in oil,in which monounsaturated fatty acids increased accompanied by reduction of saturated fatty acids and triunsaturated fatty acids. The overexpression of GmOLE2 in A.thaliana enhanced the tolerance to freezing,and the seed germination rate still reached 75% at -20℃,twice higher than those of wild-type. GmOLE2 was regulated by MeJA and ABA:when the primary roots were inhibited,the abundance and length of the GmOLE2 seedling lateral roots were significantly longer than those of the untreated group upon 10 μmol·L-1 MeJA treatment,and also the expression level of GmOLE2 increased by 8 to 10 times within 24 h. The germination rate of GmOLE2-OEs seeds on 0.5 μmol·L-1 ABA medium was significantly higher than that of wild type,and also the expression level of GmOLE2 in GmOLE2-OEs was 8 times higher than that of the control group. [Conclusions] GmOLE2 had typical OLE protein structural characteristics. The overexpression of the GmOLE2 in A.thaliana could regulate fatty acid composition,reduce the total oil content in the seed and confer resistance to freezing. GmOLE2 was regulated by MeJA and ABA.


[1] Slocombe S P,Cornah J,Pinfield-Wells H,et al. Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways[J]. Plant Biotechnology Journal,2009,7(7):694-703.
[2] Murphy D J. The dynamic roles of intracellular lipid droplets:from archaea to mammals[J]. Protoplasma,2012,249(3):541-585.
[3] Capuano F,Beaudoin F,Napier J A,et al. Properties and exploitation of oleosins[J]. Biotechnology Advances,2007,25(2):203-206.
[4] Xu M Y,Liu D H,Li G Q,et al. Cloning of soybean 24 kDa oleosin gene and its transient expression as a carrier for foreign protein[J]. Agricultural Sciences in China,2004,3(5):321-329.
[5] Keddie J S,Hübner G,Slocombe S P,et al. Cloning and characterisation of an oleosin gene from Brassica napus[J]. Plant Molecular Biology,1992,19(3):443-453.
[6] Roux E,Baumberger S,Axelos M A V,et al. Oleosins of Arabidopsis thaliana:expression in Escherichia coli,purification,and functional properties[J]. Journal of Agricultural and Food Chemistry,2004,52(16):5245-5249.
[7] Huang M D,Huang A H. Bioinformatics reveal five lineages of oleosins and the mechanism of lineage evolution related to structure/function from green algae to seed plants[J]. Plant Physiology,2015,169(1):453-470.
[8] Abell B M,Holbrook L A,Abenes M,et al. Role of the proline knot motif in oleosin endoplasmic Reticulum topology and oil body targeting[J]. The Plant Cell,1997,9(8):1481-1493.
[9] Huang A H C. Plant lipid droplets and their associated proteins:potential for rapid advances[J]. Plant Physiology,2018,176(3):1894-1918.
[10] Wu Y Y,Chou Y R,Wang C S,et al. Different effects on triacylglycerol packaging to oil bodies in transgenic rice seeds by specifically eliminating one of their two oleosin isoforms[J]. Plant Physiology and Biochemistry,2010,48(2/3):81-89.
[11] Siloto R M,Findlay K,Lopez-Villalobos A,et al. The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis[J]. The Plant Cell,2006,18(8):1961-1974.
[12] Shimada T L,Hara-Nishimura I. Oil-body-membrane proteins and their physiological functions in plants[J]. Biological and Pharmaceutical Bulletin,2010,33(3):360-363.
[13] Liu W X,Liu H L,Qu L Q. Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds[J]. Theoretical and Applied Genetics,2013,126(9):2289-2297.
[14] Zhang D,Zhang H,Hu Z,et al. Artificial selection on GmOLEO1 contributes to the increase in seed oil during soybean domestication[J]. PLoS Genetics,2019,15(7):e1008267.
[15] Shimada T L,Shimada T,Takahashi H,et al. A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana[J]. The Plant Journal,2008,55(5):798-809.
[16] Buchanan C D,Lim S,Salzman R A,et al. Sorghum bicolor’s transcriptome response to dehydration,high salinity and ABA[J]. Plant Molecular Biology,2005,58(5):699-720.
[17] 蒋茂双,元香梅,刘晓东,等. 谷子Oleosin基因家族及其对干旱响应的分析[J]. 山西农业大学学报(自然科学版),2018,38(1):16-20. Jiang M S,Yuan X M,Liu X D,et al. Analysis of oleosingene family and its response to drought in foxtail millet[J]. Journal of Shanxi Agricultural University(Natural Science Edition),2018,38(1):16-20(in Chinese with English abstract).
[18] 宫宇,毛卓卓,史贵霞,等. 大豆E3泛素连接酶基因GmPUB1的克隆及功能研究[J]. 南京农业大学学报,2020,43(5):820-828. DOI:10.7685/jnau.201912063. Gong Y,Mao Z Z,Shi G X,et al. Cloning and functional analysis of soybean E3 ubiquitin ligase gene GmPUB1[J]. Journal of Nanjing Agricultural University,2020,43(5):820-828(in Chinese with English abstract).
[19] 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.
[20] 李双飞,黄颜众,轩慧冬,等. 大豆GmTIP1-1基因克隆及功能研究[J]. 南京农业大学学报,2019,42(5):793-801. DOI:10.7685/jnau.201901024. Li S F,Huang Y Z,Xuan H D,et al. Cloning and function study of soybean GmTIP1-1 gene[J]. Journal of Nanjing Agricultural University,2019,42(5):793-801(in Chinese with English abstract).
[21] Li Y,Beisson F,Pollard M,et al. Oil content of Arabidopsis seeds:the influence of seed anatomy,light and plant-to-plant variation[J]. Phytochemistry,2006,67(9):904-915.
[22] 逯玉,谭建宁,杨蕊娟,等. 蒽酮比色法测定油菜籽中硫代葡萄糖苷条件优化[J]. 中兽医医药杂志,2014,33(4):60-61. Lu Y,Tan J N,Yang R J,et al. Optimization on the determination of glucosinolates in rapeseed with anthrone colorimetry[J]. Journal of Traditional Chinese Veterinary Medicine,2014,33(4):60-61(in Chinese with English abstract).
[23] Kim S,Yamaoka Y,Ono H,et al. AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum[J]. Proc Natl Acad Sci USA,2013,110(2):773-778.
[24] Parthibane V,Iyappan R,Vijayakumar A,et al. Serine/threonine/tyrosine protein kinase phosphorylates oleosin,a regulator of lipid metabolic functions[J]. Plant Physiol,2012,159:95-104.
[25] Parthibane V,Rajakumari S,Venkateshwari V,et al. Oleosin is bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities[J]. Plant Physiology,2012,159(1):95-104.
[26] Tzen J T C,Chuang R L C,Chen J C F,et al. Coexistence of both oleosin isoforms on the surface of seed oil bodies and their individual stabilization to the organelles[J]. Journal of Biochemistry,1998,123(2):318-323.
[27] Hays D B,Wilen R W,Sheng C,et al. Embryo-specific gene expression in microspore-derived embryos of Brassica napus. An interaction between abscisic acid and jasmonic acid1,2[J]. Plant Physiology,1999,119(3):1065-1072.
[28] Chen C,Wu C,Miao J,et al. Arabidopsis SAG protein containing the MDN1 domain participates in seed germination and seedling development by negatively regulating ABI3 and ABI5[J]. Journal of Experimental Botany,2014,65(1):35-45.


更新日期/Last Update: 1900-01-01