油菜种子发育早期的油体发生与调控
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摘要
以甘蓝型油菜(Brassica napus L.)品种‘Westar’和‘Topas’为材料,通过超微结构观察和荧光定量PCR技术对油菜胚胎发育早期油体的发生、油体蛋白及脂肪酸合成转录因子基因的表达情况进行分析。结果显示:油体出现在油菜胚胎发育早期,在授粉9～11 d后(球形胚时期)的胚体和胚柄中均存在直径小于0.5μm的油体;荧光定量实验结果表明,除Bn CLO3的表达量在整个胚胎发育阶段无明显变化外,其他油体蛋白基因Oleosins、Steroleosins和Bn CLO1的表达量在心形胚时期就明显增多并持续增长;脂肪酸合成转录因子Bn LEC1、Bn L1L、Bn WRI1和Bn FUS3在胚胎发育阶段,基因表达规律均呈先上升再下降的趋势,但达到最高值的时间存在差异,其中Bn LEC1最早,Bn L1L其次,Bn WRI1和Bn FUS3较晚。研究结果表明甘蓝型油菜在球形胚时期出现油体,其结构蛋白和转录调控因子基因的表达自心形胚开始明显增多。
Rapeseed is one of the most important oil crops in China. Most oil in rapeseed is stored in the oil bodies. In this paper,the biogenesis of oil bodies and gene expression level variations in oil body proteins and fatty acid synthesis transcription factors were investigated during embryogenesis in two Brassica napus L. cultivars( ‘Westar ’ and ‘Topas ’) via ultrastructural observation and real-time fluorescence quantitative PCR. Results showed that oil bodies existed in early embryos of B. napus,even in the embryo proper and suspensor,9-11 d after pollination( globular embryo stage). The gene expression level of all oil body proteins( oleosins,steroleosins,and Bn CLO1),except Bn CLO3,increased at the heart embryo stage and continued to increase during the embryonic development stage. The gene expression levels of fatty acid synthesis transcription factors( Bn LEC1,Bn L1 L,Bn WRI1,and Bn FUS3) increased at the early stages and decreased at the late stages,but their peak times were different. The gene expression level of Bn LEC1 peaked first, followed by Bn L1 L,Bn WRI1,and Bn FUS3 last. Our results showed that oil bodies exist in early embryos of B. napus,even at the globular-embryo stage,and the gene expression level of their structural proteins and transcription factors increases from the heart-embryo stage.
Rapeseed is one of the most important oil crops in China. Most oil in rapeseed is stored in the oil bodies. In this paper,the biogenesis of oil bodies and gene expression level variations in oil body proteins and fatty acid synthesis transcription factors were investigated during embryogenesis in two Brassica napus L. cultivars( ‘Westar ’ and ‘Topas ’) via ultrastructural observation and real-time fluorescence quantitative PCR. Results showed that oil bodies existed in early embryos of B. napus,even in the embryo proper and suspensor,9-11 d after pollination( globular embryo stage). The gene expression level of all oil body proteins( oleosins,steroleosins,and Bn CLO1),except Bn CLO3,increased at the heart embryo stage and continued to increase during the embryonic development stage. The gene expression levels of fatty acid synthesis transcription factors( Bn LEC1,Bn L1 L,Bn WRI1,and Bn FUS3) increased at the early stages and decreased at the late stages,but their peak times were different. The gene expression level of Bn LEC1 peaked first, followed by Bn L1 L,Bn WRI1,and Bn FUS3 last. Our results showed that oil bodies exist in early embryos of B. napus,even at the globular-embryo stage,and the gene expression level of their structural proteins and transcription factors increases from the heart-embryo stage.
引文
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[13] Cernac A,Benning C. WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis[J]. Plant J,2004,40(4):575-585.
[14] Li Q,Shao J,Tang S,Shen Q,Wang T,et al. Wrinkled1accelerates flowering and regulates lipid homeostasis between oil accumulation and membrane lipid anabolism in Brassica napus[J]. Front Plant Sci,2015,6:1015.
[15] Kanai M,Mano S,Kondo M,Hayashi M,Nishimura M.Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds[J]. Plant Biotechnol J,2016,14(5):1241-1250.
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[17] Elahi N,Duncan RW,Stasolla C. Decreased seed oil production in FUSCA3 Brassica napus mutant plants[J].Plant Physiol Biochem,2015,96:222-230.
[18] He YQ,Wu Y. Oil body biogenesis during Brassica napus embryogenesis[J]. J Integr Plant Biol,2009,51(8):792-799.
[19] Boulard C,Bardet M,Chardot T,Dubreucq B,Gromova M,et al. The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes[J]. Planta,2015,242(1):53-68.
[20] Holbrook LA,van Rooijen GJ,Wilen RW,Moloney MM.Oilbody proteins in microspore-derived embryos of Brassica napus:hormonal,osmotic,and developmental regulation of synthesis[J]. Plant Physiol,1991,97(3):1051-1058.
[21]何宇清,操春燕,沈文忠,黄冬,马胜,吴燕.甘蓝型油菜种子中油体的超微结构及蛋白质组分析[J].植物科学学报,2017,35(4):566-573.He YQ,Cao CY,Shen WZ,Huang D,Ma S,et al. Study on the ultrastructure and proteome of oil bodies in Brassica napus L. seeds[J]. Plant Science Journal,2017,35(4):566-573.
[22] Gu J,Chao H,Wang H,Li Y,Li D,et al. Identification of the relationship between oil body morphology and oil content by microstructure comparison combining with QTL analysis in Brassica napus[J]. Front Plant Sci,2017,7:1989.
[23] Borisjuk L,Neuberger T,Schwender J,Nicolas H,Sunderhaus S,et al. Seed architecture shapes embryo metabolism in oilseed rape[J]. Plant Cell,2013,25(5):1625-1640.
[24] Tzen JT,Cao YZ,Laurent P,Ratnayake C,Huang AH.Lipids,proteins,and structure of seed oil bodies from diverse species[J]. Plant Physiol,1993,101:267-276.
[25] Murphy DJ. The biogenesis and functions of lipid bodies in animals,plants and microorganisms[J]. Prog Lipid Res,2001,40(5):325-438.
[26] Mantese AI,Medan D,Hall AJ. Achene structure,development and lipid accumulation in sunflower cultivars differing in oil content at maturity[J]. Ann Bot Lond,2006,97(6):999-1010.
[27] Miquel M,Trigui G,d'Andréa S,Kelemen Z,Baud S,et al. Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds[J]. Plant Physiol,2014,164(4):1866-1878.
[28] Kim HU,Hsieh K,Ratnayake C,Huang AH. A novel group of oleosins is present inside the pollen of Arabidopsis[J]. J Biol Chem,2002,277(25):22677-22684.
[29] Hyun TK,Kumar D,Cho YY,Hyun HN,Kim JS. Computational identification and phylogenetic analysis of the oilbody structural proteins,oleosin and caleosin,in castor bean and flax[J]. Gene,2013,515(2):454-460.
[30] Lu C,Fulda M,Wallis JG,Browse J. A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis[J].Plant J,2006,45(5):847-856.
[31] Siloto RM,Findlay K,Lopez-Villalobos A,Yeung EC,Nykiforuk CL,et al. The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis[J]. Plant Cell,2006,18(8):1961-1974.
[32] Hernandez-Pinzon I,Patel K,Murphy DJ. The Brassica napus calcium-binding protein,caleosin,has distinct endoplasmic reticulum-and lipid body-associated isoforms[J]. Plant Physiol Biochem,2001,39(7-8):615-622.
[2] Tzen JTC. Integral proteins in plant oil bodies[J]. ISRN Botany,2012,2012:173954.
[3] Jolivet P,Boulard C,Bellamy A,Valot B,d'Andréa S,et al. Oil body proteins sequentially accumulate throughout seed development in Brassica napus[J]. J Plant Physiol,2011,168(17):2015-2020.
[4] Obermeier C,Hosseini B,Friedt W,Snowdon R. Gene expression profiling via LongSAGE in a non-model plant species:a case study in seeds of Brassica napus[J].BMC Genomics,2009,10:295.
[5]闵文莉,曹喜涛,季更生,张国政.调控脂肪酸合成植物转录因子的研究进展[J].发酵科技通讯,2017,46(2):107-112.Min WL,Cao XT,Ji GS,Zhang GZ. Researches of plant transcription factors involving in fatty acid synthesis[J].Bulletin of Fermentation Science and Technolgy,2017,46(2):107-112.
[6]宋雷,范成明,陈宇红,张新永,胡赞民.植物油脂合成的分子调控机制[J].分子植物育种,2016,14(8):2178-2187.Song L,Fan CM,Chen YH,Zhang XY,Hu ZM. The molecular regulation mechanism of the plant lipid biosynthesis[J]. Molecular Plant Breeding,2016,14(8):2178-2187.
[7]李玉兰,孙勤富,王幼平.植物油脂合成的转录调控研究进展[J].分子植物育种,2016,14(9):2509-2518.Li YL,Sun QF,Wang YP. Research advance in transcriptional regulation of lipid synthesis and accumulation in plant[J]. Molecular Plant Breeding,2016,14(9):2509-2518.
[8] Pelletier JM,Kwong RW,Park S,Le BH,Baden R,et al.LEC1 sequentially regulates the transcription of genes involved in diverse developmental processes during seed development[J]. Proc Natl Acad Sci USA,2017,114(32):6710-6719.
[9] Tao Z,Shen L,Gu X,Wang Y,Yu H,et al. Embryonic epigenetic reprogramming by a pioneer transcription factor in plants[J]. Nature,2017,551(7678):124-128.
[10] Gnesutta N,Saad D,Chaves-Sanjuan A,Mantovani R,Nardini M. Crystal structure of the Arabidopsis thaliana L1L/NF-YC3 histone-fold dimer reveals specificities of the LEC1 family of NF-Y subunits in plants[J]. Mol Plant,2017,10(4):645-648.
[11] Tan H,Yang X,Zhang F,Zheng X,Qu C,et al. Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds[J]. Plant Physiol,2011,156(3):1577-88.
[12] Mu J,Tan H,Zheng Q,Fu F,Liang Y,et al. LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis[J]. Plant Physiol,2008,148(2):1042-1054.
[13] Cernac A,Benning C. WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis[J]. Plant J,2004,40(4):575-585.
[14] Li Q,Shao J,Tang S,Shen Q,Wang T,et al. Wrinkled1accelerates flowering and regulates lipid homeostasis between oil accumulation and membrane lipid anabolism in Brassica napus[J]. Front Plant Sci,2015,6:1015.
[15] Kanai M,Mano S,Kondo M,Hayashi M,Nishimura M.Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds[J]. Plant Biotechnol J,2016,14(5):1241-1250.
[16] Zhang M,Cao X,Jia Q,Ohlrogge J. FUSCA3 activates triacylglycerol accumulation in Arabidopsis seedlings and tobacco BY2 cells[J]. Plant J,2016,88(1):95-107.
[17] Elahi N,Duncan RW,Stasolla C. Decreased seed oil production in FUSCA3 Brassica napus mutant plants[J].Plant Physiol Biochem,2015,96:222-230.
[18] He YQ,Wu Y. Oil body biogenesis during Brassica napus embryogenesis[J]. J Integr Plant Biol,2009,51(8):792-799.
[19] Boulard C,Bardet M,Chardot T,Dubreucq B,Gromova M,et al. The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes[J]. Planta,2015,242(1):53-68.
[20] Holbrook LA,van Rooijen GJ,Wilen RW,Moloney MM.Oilbody proteins in microspore-derived embryos of Brassica napus:hormonal,osmotic,and developmental regulation of synthesis[J]. Plant Physiol,1991,97(3):1051-1058.
[21]何宇清,操春燕,沈文忠,黄冬,马胜,吴燕.甘蓝型油菜种子中油体的超微结构及蛋白质组分析[J].植物科学学报,2017,35(4):566-573.He YQ,Cao CY,Shen WZ,Huang D,Ma S,et al. Study on the ultrastructure and proteome of oil bodies in Brassica napus L. seeds[J]. Plant Science Journal,2017,35(4):566-573.
[22] Gu J,Chao H,Wang H,Li Y,Li D,et al. Identification of the relationship between oil body morphology and oil content by microstructure comparison combining with QTL analysis in Brassica napus[J]. Front Plant Sci,2017,7:1989.
[23] Borisjuk L,Neuberger T,Schwender J,Nicolas H,Sunderhaus S,et al. Seed architecture shapes embryo metabolism in oilseed rape[J]. Plant Cell,2013,25(5):1625-1640.
[24] Tzen JT,Cao YZ,Laurent P,Ratnayake C,Huang AH.Lipids,proteins,and structure of seed oil bodies from diverse species[J]. Plant Physiol,1993,101:267-276.
[25] Murphy DJ. The biogenesis and functions of lipid bodies in animals,plants and microorganisms[J]. Prog Lipid Res,2001,40(5):325-438.
[26] Mantese AI,Medan D,Hall AJ. Achene structure,development and lipid accumulation in sunflower cultivars differing in oil content at maturity[J]. Ann Bot Lond,2006,97(6):999-1010.
[27] Miquel M,Trigui G,d'Andréa S,Kelemen Z,Baud S,et al. Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds[J]. Plant Physiol,2014,164(4):1866-1878.
[28] Kim HU,Hsieh K,Ratnayake C,Huang AH. A novel group of oleosins is present inside the pollen of Arabidopsis[J]. J Biol Chem,2002,277(25):22677-22684.
[29] Hyun TK,Kumar D,Cho YY,Hyun HN,Kim JS. Computational identification and phylogenetic analysis of the oilbody structural proteins,oleosin and caleosin,in castor bean and flax[J]. Gene,2013,515(2):454-460.
[30] Lu C,Fulda M,Wallis JG,Browse J. A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis[J].Plant J,2006,45(5):847-856.
[31] Siloto RM,Findlay K,Lopez-Villalobos A,Yeung EC,Nykiforuk CL,et al. The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis[J]. Plant Cell,2006,18(8):1961-1974.
[32] Hernandez-Pinzon I,Patel K,Murphy DJ. The Brassica napus calcium-binding protein,caleosin,has distinct endoplasmic reticulum-and lipid body-associated isoforms[J]. Plant Physiol Biochem,2001,39(7-8):615-622.