甘蓝型油菜皖油20号种子不同部位油脂合成的转录调控分析
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摘要
甘蓝型油菜是主要的油料作物之一,种子含油量一般在35%～50%。油脂主要储存于油菜种子胚中,胚主要由子叶[包括外子叶(OC)和内子叶(IC)和胚轴(EA)]组成。低芥酸油菜品种皖油20号(WY20)种子不同部位的含油量存在显著差异。WY20的胚中, OC含油量最高, EA含油量最低。同时,脂肪酸组成在种子不同部位也存在差异, EA中棕榈酸(C16:0)、亚油酸(C18:2)及二十碳酸(C20:0)的比例均显著高于子叶,特别是C16:0在EA中的比例约为子叶的2倍。而油酸(C18:1)及二十碳烯酸(C20:1)在子叶中的比例均显著高于EA。硬脂酸(C18:0)在OC中含量最低,在IC和EA中无差别。亚麻酸(C18:3)则在OC中含量最高,在IC和EA中无差异。对发育34d种子的IC、OC和EA进行转录组分析,将三个部位中基因表达定量分析的结果两两比较后共发掘出7192个差异表达基因,其中OC和IC之间差异表达基因数目较少,子叶和EA间有较多的差异表达基因。子叶和胚轴中的差异表达基因富集在光合作用、脂肪酸代谢和叶绿素合成等生物学过程。基因功能注释显示,差异表达基因中有355个和脂质代谢相关,且多集中在质体中脂肪酸从头合成途径。本研究表明油脂合成途径关键基因的差异调控是造成油菜种子不同部位含油量和脂肪酸组成差异的主要因素。
Brassica napus is one of the main oil crops and the seed oil content is generally between 35% and 50%. Oil is mainly stored in the seed embryo. Embryo is composed of cotyledons(including outer and inner cotyledons) and embryonic axis. The oil content and fatty acid composition of different parts of low erucic Brassica napus WY20’s seed were analyzed. There was a significant difference in oil content in different parts of the seed. The oil content in the outer cotyledon was the highest while embryonic axis had the lowest oil content. At the same time, the fatty acid composition also showed significant difference in different parts of the seed. The ratio of C16:0, C18:2, and C20:0 in embryonic axis was significantly higher than that in cotyledon. The ratio of C16:0 in the embryonic axis was about twice more than that of the cotyledons. The ratio of C18:1 and C20:1 in cotyledons was significantly higher than that in embryonic axis. C18:0 had the lowest content in the outer cotyledon and no difference in the inner cotyledons and embryonic axis. C18:3 had the highest content in the outer cotyledons and no difference between inner cotyledons and embryonic axis. Transcriptome analysis was performed for the inner cotyledon, outer cotyledon and embryonic axis of the34-day-old seed. A total of 7192 differentially expressed genes(DEGs) were identified after pairwise comparison of gene expression of the three parts. There were much fewer DEGs between cotyledons and more DEGs between cotyledon and embryonic axis.These DEGs were enriched in biological processes such as photosynthesis, fatty acid metabolism and chlorophyll metabolism.Gene function annotations revealed that there were 355 genes involved in lipid metabolism, especially in the de novo fatty acid biosynthesis in plastid. This study suggests that transcriptional regulation of key genes involved in oil biosynthesis results in different oil contents and fatty acid compositions in different parts of seed in Brassica napus.
Brassica napus is one of the main oil crops and the seed oil content is generally between 35% and 50%. Oil is mainly stored in the seed embryo. Embryo is composed of cotyledons(including outer and inner cotyledons) and embryonic axis. The oil content and fatty acid composition of different parts of low erucic Brassica napus WY20’s seed were analyzed. There was a significant difference in oil content in different parts of the seed. The oil content in the outer cotyledon was the highest while embryonic axis had the lowest oil content. At the same time, the fatty acid composition also showed significant difference in different parts of the seed. The ratio of C16:0, C18:2, and C20:0 in embryonic axis was significantly higher than that in cotyledon. The ratio of C16:0 in the embryonic axis was about twice more than that of the cotyledons. The ratio of C18:1 and C20:1 in cotyledons was significantly higher than that in embryonic axis. C18:0 had the lowest content in the outer cotyledon and no difference in the inner cotyledons and embryonic axis. C18:3 had the highest content in the outer cotyledons and no difference between inner cotyledons and embryonic axis. Transcriptome analysis was performed for the inner cotyledon, outer cotyledon and embryonic axis of the34-day-old seed. A total of 7192 differentially expressed genes(DEGs) were identified after pairwise comparison of gene expression of the three parts. There were much fewer DEGs between cotyledons and more DEGs between cotyledon and embryonic axis.These DEGs were enriched in biological processes such as photosynthesis, fatty acid metabolism and chlorophyll metabolism.Gene function annotations revealed that there were 355 genes involved in lipid metabolism, especially in the de novo fatty acid biosynthesis in plastid. This study suggests that transcriptional regulation of key genes involved in oil biosynthesis results in different oil contents and fatty acid compositions in different parts of seed in Brassica napus.
引文
[1]沈金雄,傅廷栋.我国油菜生产、改良与食用油供给安全.中国农业科技导报,2011,13(1):1-8Shen J X,Fu T D.Rapeseed production improvement and edible oil supply in China.J Agric Sci Technol,2011,13(1):1-8(in Chinese with English abstract)
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[3]李殿荣,田建华,陈文杰,张文学,李永红,王灏.甘蓝型油菜特高含油量育种技术与资源创新.西北农业学报,2011,20(12):83-87Li D R,Tian J H,Chen W J,Zhang W X,Li Y H,Wang H.Breeding technologies and germplasm innovation on extrahigh-oil content in Brassica napus.Acta Agric Boreali-occident Sin,2011,20(12):83-87(in Chinese with English abstract)
[4]张永霞,赵锋,张红玲.中国油菜产业发展现状、问题及对策分析.世界农业,2015,(4):96-99Zhang Y X,Zhao F,Zhang H L.Analysis on the development status,problems and countermeasures of Chinese rapeseed industry.World Agric,2015,(4):96-99(in Chinese with English abstract)
[5]王汉中,殷艳.我国油料产业形势分析与发展对策建议.中国油料作物学报,2014,36:414-421Wang H Z,Yin Y.Analysis and strategy for oil crop industry in China.Chin J Oil Crop Sci,2014,36:414-421(in Chinese with English abstract)
[6]熊秋芳,张效明,文静,李兴华,傅廷栋,沈金雄.菜籽油与不同食用植物油营养品质的比较--兼论油菜品质的遗传改良.中国粮油学报,2014,29:122-128Xiong Q F,Zhang X M,Wen J,Li X H,Fu T D,Shen J X.Comparison of nutritive quality between rapeseed oil and different edible vegetable oil-on the genetic improvement of rapeseed quality.J Chin Cereals Oils Assoc,2014,29:122-128(in Chinese with English abstract)
[7]熊源.植物油的种类与营养价值.中国粮食经济,2014,(6):72Xiong Y.Types and nutritional value of vegetable oil.Chin Food Economy,2014,(6):72(in Chinese with English abstract)
[8]Saha S,Enugutti B,Rajakumari S.Cytosolic triacylglycerol biosynthetic pathway in oilseeds.Molecular cloning and expression of peanut cytosolic.diacylglycerol acyltransferase.Plant Physiol,2006,141:1533-1543
[9]Thelen J J,Ohlrogge J B.Metabolic engineering of fatty acid biosynthesis in Plants.Metab Eng,2002,4:12-21
[10]Dahlqvist A,Stahl U,Lenman M.Phospholipid:diacylglycerol acyltransferase:An enzyme that catalyzes the Acyl-CoA-Independent formation of triacylglycerol in yeast and plants.Proc Natl Acad Sci USA,2000,97:6487-6492
[11]周奕华,陈正华.植物种子中脂肪酸代谢途径的遗传调控与基因工程.植物学通报,1998,15(5):16-23Zhou Y H,Chen Z H.Genetic manipulation and gene engineering of fatty acid metabolism in plant seeds.Chin Bull Bot,1998,15(5):16-23(in Chinese with English abstract)
[12]周丹,赵江哲,柏杨,张群,井文,章文华.植物油脂合成代谢及调控的研究进展.南京农业大学学报,2012,35(5):81-90Zhou D,Zhao J Z,Bai Y,Zhang Q,Jing W,Zhang W H.Research advance in triacylglycerol synthesis,metabolism and regulation in plants.2012,35(5):81-90(in Chinese with English abstract)
[13]Bates P D,Stymne S,Ohlrogge J B.Biochemical pathways in seed oil synthesis.Curr Opin Plant Biol,2013,16:358-364
[14]Horn P J,Korte A R,Neogi P B,Love E,Fuchs J,Strupat K,Borisjuk L,Shulaev V,Lee Y J,Chapman K D.Spatial mapping of lipids at cellular resolution in embryos of cotton.Plant Cell,2012,24:622-636
[15]Horn P J,Silva J E,Anderson D,Fuchs J,Borisjuk L,Nazarenus T J,Shulaev V,Cahoon E B,Chapman K D.Imaging heterogeneity of membrane and storage lipids in transgenic Camelina sativa seeds with altered fatty acid profiles.Plant J,2013,76:138-150
[16]Sturtevant D,Due?as M E,Lee Y J,Chapman K D.Threedimensional visualization of membrane phospholipid distributions in Arabidopsis thaliana seeds:a spatial perspective of molecular heterogeneity.Biochim Biophys Acta,2017,1862:268-281
[17]Woodfield H K,Sturtevant D,Borisjuk L,Munz E,Guschina I A,Chapman K,Harwood J L.Spatial and temporal mapping of key lipid species in Brassica napus seeds.Plant Physiol,1998,173:1998-2009
[18]Sturtevant D,Lee Y J,Chapman K D.Matrix assisted laser desorption/ionization-mass spectrometry imaging(MALDI-MSI)for direct visualization of plant metabolites in situ.Curr Opin Biotech,2015,37:53-60
[19]Lu X,Chen D,Shu D.The differential transcription network between embryo and endosperm in the early developing maize seed.Plant Physiol,2013,162:440-455
[20]He R,Salvato F,Park J J.A systems-wide comparison of red rice(Oryza longistaminata)tissues identifies rhizome specific genes and proteins that are targets for cultivated rice improvement.BMC Plant Biol,2014,14:46-66
[21]Schuster S C.Next-generation sequencing transforms today’s biology.Nat Methods,2008,5:16-18
[22]Metzker M L.Sequencing technologies:the next generation.Nat Rev Genet,2010,11:31-46
[23]Louisa F L.RNA-Seq:a revolutionary tool for transcriptomics.Nat Rev Genet,2008,9:568-574
[24]Troncoso-Ponce M A,Kilaru A,Cao X,Durrett T P,Fan J,Jensen J K,Thrower N A,Pauly M,Wilkerson C,Ohlrogge J B.Comparative deep transcriptional profiling of four developing oil seeds.Plant J,2011,68:1014-1027
[25]Dussert S,Morcillo F.Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition.Plant Physiol,2013,162:1337-1358
[26]Lu S P,Sturtevant D,Aziz M,Jin C,Li Q,Chapman K D,Guo L.Spatial analysis of lipid metabolites and expressed genes reveals tissue-specific heterogeneity of lipid metabolism in high-and low-oil Brassica napus L.seeds.Plant J,2018,94:915-932
[27]Trapnell C,Roberts A,Goff L,Pertea G,Kim D,Kelley D R,Pimentel H,Salzberg S L,Rinn J L,Pachter L.Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.Nat Protoc,2012,7:562-578
[28]Anders S,Pyl P T,Huber W.HTSeq:a Python framework to work with high-throughput sequencing data.Bioinformatics,2015,31:166-169
[29]Wang L,Feng Z,Wang X,Wang X,Zhang X.DEGseq:an Rpackage for identifying differentially expressed genes from RNA-seq data.Bioinformatics,2010,26:136-138
[30]Love M I,Huber W,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol,2014,15:550-575
[31]Baud S,Lepiniec L.Physiological and developmental regulation of seed oil production.Prog Lipid Res,2010,49:235-249
[32]Borisjuk L,Neuberger T,Schwender J,Heinzel N,Sunderhaus S,Fuchs J,Hay J O,Tschiersch H,Braun H P,Denolf P,Lambert B,Jakob P M,Rolletschek H.Seed architecture shapes embryo metabolism in oilseed rape.Plant Cell,2013,25:113-128
[33]Napier J A,Haslam R P,Beaudoin F.Understanding and manipulating plant lipid composition:Metabolic engineering leads the way.Curr Opin Plant Biol,2014,19:68-75
[2]沈琼.中国油菜产业竞争优势与劣势分析.农业产品加工,2008,(8):57-59Shen Q.Analysis on competitive advantages and disadvantages of Chinese rapeseed industry.Acad Period Farm Products Proc,2008,(8):57-59(in Chinese with English abstract)
[3]李殿荣,田建华,陈文杰,张文学,李永红,王灏.甘蓝型油菜特高含油量育种技术与资源创新.西北农业学报,2011,20(12):83-87Li D R,Tian J H,Chen W J,Zhang W X,Li Y H,Wang H.Breeding technologies and germplasm innovation on extrahigh-oil content in Brassica napus.Acta Agric Boreali-occident Sin,2011,20(12):83-87(in Chinese with English abstract)
[4]张永霞,赵锋,张红玲.中国油菜产业发展现状、问题及对策分析.世界农业,2015,(4):96-99Zhang Y X,Zhao F,Zhang H L.Analysis on the development status,problems and countermeasures of Chinese rapeseed industry.World Agric,2015,(4):96-99(in Chinese with English abstract)
[5]王汉中,殷艳.我国油料产业形势分析与发展对策建议.中国油料作物学报,2014,36:414-421Wang H Z,Yin Y.Analysis and strategy for oil crop industry in China.Chin J Oil Crop Sci,2014,36:414-421(in Chinese with English abstract)
[6]熊秋芳,张效明,文静,李兴华,傅廷栋,沈金雄.菜籽油与不同食用植物油营养品质的比较--兼论油菜品质的遗传改良.中国粮油学报,2014,29:122-128Xiong Q F,Zhang X M,Wen J,Li X H,Fu T D,Shen J X.Comparison of nutritive quality between rapeseed oil and different edible vegetable oil-on the genetic improvement of rapeseed quality.J Chin Cereals Oils Assoc,2014,29:122-128(in Chinese with English abstract)
[7]熊源.植物油的种类与营养价值.中国粮食经济,2014,(6):72Xiong Y.Types and nutritional value of vegetable oil.Chin Food Economy,2014,(6):72(in Chinese with English abstract)
[8]Saha S,Enugutti B,Rajakumari S.Cytosolic triacylglycerol biosynthetic pathway in oilseeds.Molecular cloning and expression of peanut cytosolic.diacylglycerol acyltransferase.Plant Physiol,2006,141:1533-1543
[9]Thelen J J,Ohlrogge J B.Metabolic engineering of fatty acid biosynthesis in Plants.Metab Eng,2002,4:12-21
[10]Dahlqvist A,Stahl U,Lenman M.Phospholipid:diacylglycerol acyltransferase:An enzyme that catalyzes the Acyl-CoA-Independent formation of triacylglycerol in yeast and plants.Proc Natl Acad Sci USA,2000,97:6487-6492
[11]周奕华,陈正华.植物种子中脂肪酸代谢途径的遗传调控与基因工程.植物学通报,1998,15(5):16-23Zhou Y H,Chen Z H.Genetic manipulation and gene engineering of fatty acid metabolism in plant seeds.Chin Bull Bot,1998,15(5):16-23(in Chinese with English abstract)
[12]周丹,赵江哲,柏杨,张群,井文,章文华.植物油脂合成代谢及调控的研究进展.南京农业大学学报,2012,35(5):81-90Zhou D,Zhao J Z,Bai Y,Zhang Q,Jing W,Zhang W H.Research advance in triacylglycerol synthesis,metabolism and regulation in plants.2012,35(5):81-90(in Chinese with English abstract)
[13]Bates P D,Stymne S,Ohlrogge J B.Biochemical pathways in seed oil synthesis.Curr Opin Plant Biol,2013,16:358-364
[14]Horn P J,Korte A R,Neogi P B,Love E,Fuchs J,Strupat K,Borisjuk L,Shulaev V,Lee Y J,Chapman K D.Spatial mapping of lipids at cellular resolution in embryos of cotton.Plant Cell,2012,24:622-636
[15]Horn P J,Silva J E,Anderson D,Fuchs J,Borisjuk L,Nazarenus T J,Shulaev V,Cahoon E B,Chapman K D.Imaging heterogeneity of membrane and storage lipids in transgenic Camelina sativa seeds with altered fatty acid profiles.Plant J,2013,76:138-150
[16]Sturtevant D,Due?as M E,Lee Y J,Chapman K D.Threedimensional visualization of membrane phospholipid distributions in Arabidopsis thaliana seeds:a spatial perspective of molecular heterogeneity.Biochim Biophys Acta,2017,1862:268-281
[17]Woodfield H K,Sturtevant D,Borisjuk L,Munz E,Guschina I A,Chapman K,Harwood J L.Spatial and temporal mapping of key lipid species in Brassica napus seeds.Plant Physiol,1998,173:1998-2009
[18]Sturtevant D,Lee Y J,Chapman K D.Matrix assisted laser desorption/ionization-mass spectrometry imaging(MALDI-MSI)for direct visualization of plant metabolites in situ.Curr Opin Biotech,2015,37:53-60
[19]Lu X,Chen D,Shu D.The differential transcription network between embryo and endosperm in the early developing maize seed.Plant Physiol,2013,162:440-455
[20]He R,Salvato F,Park J J.A systems-wide comparison of red rice(Oryza longistaminata)tissues identifies rhizome specific genes and proteins that are targets for cultivated rice improvement.BMC Plant Biol,2014,14:46-66
[21]Schuster S C.Next-generation sequencing transforms today’s biology.Nat Methods,2008,5:16-18
[22]Metzker M L.Sequencing technologies:the next generation.Nat Rev Genet,2010,11:31-46
[23]Louisa F L.RNA-Seq:a revolutionary tool for transcriptomics.Nat Rev Genet,2008,9:568-574
[24]Troncoso-Ponce M A,Kilaru A,Cao X,Durrett T P,Fan J,Jensen J K,Thrower N A,Pauly M,Wilkerson C,Ohlrogge J B.Comparative deep transcriptional profiling of four developing oil seeds.Plant J,2011,68:1014-1027
[25]Dussert S,Morcillo F.Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition.Plant Physiol,2013,162:1337-1358
[26]Lu S P,Sturtevant D,Aziz M,Jin C,Li Q,Chapman K D,Guo L.Spatial analysis of lipid metabolites and expressed genes reveals tissue-specific heterogeneity of lipid metabolism in high-and low-oil Brassica napus L.seeds.Plant J,2018,94:915-932
[27]Trapnell C,Roberts A,Goff L,Pertea G,Kim D,Kelley D R,Pimentel H,Salzberg S L,Rinn J L,Pachter L.Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.Nat Protoc,2012,7:562-578
[28]Anders S,Pyl P T,Huber W.HTSeq:a Python framework to work with high-throughput sequencing data.Bioinformatics,2015,31:166-169
[29]Wang L,Feng Z,Wang X,Wang X,Zhang X.DEGseq:an Rpackage for identifying differentially expressed genes from RNA-seq data.Bioinformatics,2010,26:136-138
[30]Love M I,Huber W,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol,2014,15:550-575
[31]Baud S,Lepiniec L.Physiological and developmental regulation of seed oil production.Prog Lipid Res,2010,49:235-249
[32]Borisjuk L,Neuberger T,Schwender J,Heinzel N,Sunderhaus S,Fuchs J,Hay J O,Tschiersch H,Braun H P,Denolf P,Lambert B,Jakob P M,Rolletschek H.Seed architecture shapes embryo metabolism in oilseed rape.Plant Cell,2013,25:113-128
[33]Napier J A,Haslam R P,Beaudoin F.Understanding and manipulating plant lipid composition:Metabolic engineering leads the way.Curr Opin Plant Biol,2014,19:68-75