Comprehensive transcriptomic analysis of developing seed in Cerasus humilis for lipid related gene discovery
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摘要
Cerasus humilis is a woody shrub with increasing economic importance. Due to its high oil content in seed with desired fatty acid(FA) composition, C. humilis has been considered as a potential woody oilseed crop in China. However, FAs accumulation and related molecular mechanisms of FA biosynthesis in C. humilis seed have not been elucidated well. In this study, oil contents and FA compositions of developing C. humilis seed were analyzed. The total oil content in mature seed reached 48.7% while unsaturated FA concentration reached a high level of 96.2%. Three c DNA libraries of C. humilis developing seed were constructed at the beginning and 2 fast oil-accumulation stages, followed by Illumina sequencing with the platform of HiSeq~TM 2000. Differentially expressed unigenes(DEGs) were identified during the respective seed development stages to investigate transcription dynamics. Among DEGs, 82 unigenes were identified as being closely involved in de novo FA and triacylglycerol(TAG) biosynthesis. Surprisingly, among DEGs involved in TAG biosynthesis, expression of unigenes encoding GPATs(glycerol-3-phosphate acyltransferase) were relatively low and unigenes encoding LPAATs(lysophosphatidic acid acyltransferase) were highly expressed, suggesting that LPAAT contributed more in storage lipids metabolism in C. humilis seed. Genes encoding DGAT2(diacylgycerol acyltransferase2) were the most highly expressed while expression of DGAT1 was very low, suggesting DGAT2 was the dominant DGAT which catalyzed TAG biosynthesis. To verify these results,10 unigenes were selected and their expression patterns were analyzed by quantitative RTPCR. These data provided comprehensive information for understanding the molecular mechanism of FA and TAG biosynthesis in C. humilis seed.
Cerasus humilis is a woody shrub with increasing economic importance. Due to its high oil content in seed with desired fatty acid(FA) composition, C. humilis has been considered as a potential woody oilseed crop in China. However, FAs accumulation and related molecular mechanisms of FA biosynthesis in C. humilis seed have not been elucidated well. In this study, oil contents and FA compositions of developing C. humilis seed were analyzed. The total oil content in mature seed reached 48.7% while unsaturated FA concentration reached a high level of 96.2%. Three c DNA libraries of C. humilis developing seed were constructed at the beginning and 2 fast oil-accumulation stages, followed by Illumina sequencing with the platform of HiSeq~TM 2000. Differentially expressed unigenes(DEGs) were identified during the respective seed development stages to investigate transcription dynamics. Among DEGs, 82 unigenes were identified as being closely involved in de novo FA and triacylglycerol(TAG) biosynthesis. Surprisingly, among DEGs involved in TAG biosynthesis, expression of unigenes encoding GPATs(glycerol-3-phosphate acyltransferase) were relatively low and unigenes encoding LPAATs(lysophosphatidic acid acyltransferase) were highly expressed, suggesting that LPAAT contributed more in storage lipids metabolism in C. humilis seed. Genes encoding DGAT2(diacylgycerol acyltransferase2) were the most highly expressed while expression of DGAT1 was very low, suggesting DGAT2 was the dominant DGAT which catalyzed TAG biosynthesis. To verify these results,10 unigenes were selected and their expression patterns were analyzed by quantitative RTPCR. These data provided comprehensive information for understanding the molecular mechanism of FA and TAG biosynthesis in C. humilis seed.
Cerasus humilis is a woody shrub with increasing economic importance. Due to its high oil content in seed with desired fatty acid(FA) composition, C. humilis has been considered as a potential woody oilseed crop in China. However, FAs accumulation and related molecular mechanisms of FA biosynthesis in C. humilis seed have not been elucidated well. In this study, oil contents and FA compositions of developing C. humilis seed were analyzed. The total oil content in mature seed reached 48.7% while unsaturated FA concentration reached a high level of 96.2%. Three c DNA libraries of C. humilis developing seed were constructed at the beginning and 2 fast oil-accumulation stages, followed by Illumina sequencing with the platform of HiSeq~TM 2000. Differentially expressed unigenes(DEGs) were identified during the respective seed development stages to investigate transcription dynamics. Among DEGs, 82 unigenes were identified as being closely involved in de novo FA and triacylglycerol(TAG) biosynthesis. Surprisingly, among DEGs involved in TAG biosynthesis, expression of unigenes encoding GPATs(glycerol-3-phosphate acyltransferase) were relatively low and unigenes encoding LPAATs(lysophosphatidic acid acyltransferase) were highly expressed, suggesting that LPAAT contributed more in storage lipids metabolism in C. humilis seed. Genes encoding DGAT2(diacylgycerol acyltransferase2) were the most highly expressed while expression of DGAT1 was very low, suggesting DGAT2 was the dominant DGAT which catalyzed TAG biosynthesis. To verify these results,10 unigenes were selected and their expression patterns were analyzed by quantitative RTPCR. These data provided comprehensive information for understanding the molecular mechanism of FA and TAG biosynthesis in C. humilis seed.
引文
Abdullah H.M.,Akbari P.,Paulose B.,Schnell D.,Qi W.P.,Park Y.,Pareek A.,Dhankher O.P.2016.Transcrip‐tome profiling of Camelinasativa to identify genes in‐volved in triacylglycerol biosynthesis and accumula‐tion in the developing seeds.Biotechnol.Biofuels.9:e136.doi:10.1186/s13068-016-0555-5.
Anders S.,Huber W.2010.Differential expression analysis for sequence count data.Genome Biol.11:1-12.doi:10.1186/gb-2010-11-10-r106.
Anders S.,Huber W.2010.Differential expression analysis for sequence count data.Genome Biol.11(10):R106.doi:10.1186/gb-2010-11-10-r106.
Bates P.D.,Durrett T.P.,Ohlrogge J.B.,Pollard M.2009.Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean em‐bryos.Plant Physiol.150(1):55-72.doi:10.1104/pp.109.137737.
Benjamini Y,Yekutieli D.2001.The control of the false discovery rate in multiple testing under dependency.Ann.Statist.29(4):1165-1188.doi:10.1214/aos/1013699998.
Brown A.P.,Kroon J.T.M.,Swarbreck D.,Febrer M.,Lar‐son T.R.,Graham I.A.,Caccamo M.,Slabas A.R.2012.Tissue-specific whole transcriptome sequenc‐ing in Castor directed at understanding triacylglycerol lipid biosynthetic pathways.Plos One.7(2):e30100.doi:10.1371/journal.pone.0030100.
Costa G.G.,Cardoso K.C.,del Bem L.E.,Lima A.C.,Cunha M.A.,de Campos-Leite L.,Vicentini R.,Papes F.,Moreira R.C.,Yunes J.A.,et al.2010.Transcrip‐tome analysis of the oil-rich seed of the bioenergy crop Jatrophacurcas L.BMC Genom.11(1):462-471.doi:10.1186/1471-2164-11-462.
Dong S.B.,Liu Y.L.,Niu J.,Ning Y.,Lin S.Z.,Zhang Z.X.2014.De novo transcriptome analysis of the Siberian apricot(Prunussibirica L.)and search for potential SSR markers by 454 pyrosequencing.Gene.544(2):220-227.doi:10.1016/j.gene.2014.04.031.
Fan S.Q.,Liang T.,Yu H.Y.,Bi Q.X.,Li G.T.,Wang L.B.2016.Kernel characteristics,oil contents,fatty acid compositions and biodiesel properties in developing Siberian apricot(Prunussibirica L.)seeds.Ind.Crop.Prod.89:195-199.doi:10.1016/j.indcrop.2016.05.012.
Fatima T.,Snyder C.L.,Schroeder W.R.,Cram D.,Datla R.,Wishart D.,Weselake R.J.,Krishna P.2012.Fatty acid composition of developing sea buckthorn(Hip‐pophaerhamnoides L.)berry and the transcriptome of the mature seed.Plos One.7(4):e34099.doi:10.1371/journal.pone.0034099.
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thomp‐son D.A.,Amit I.,Adiconis X.,Fan L.,Raychowd‐hury R.,Zeng Q.D.,et al.2011.Full-length transcrip‐tome assembly from RNA-Seq data without a refer‐ence genome.Nat.Biotechnol.29(7):644-652.doi:10.1038/nbt.1883.
Gupta K.,Kayam G.,Faigenboim-Doron A.,Clevenger J.,Ozias-Akins P.,Hovav R.2016.Gene expression pro‐filing during seed-filling process in peanut with em‐phasis on oil biosynthesis networks.Plant Sci.248:116-127.doi:10.1016/j.plantsci.2016.04.014.
Kroon J.T.M.,Wei W.X.,Simon W.J.,Slabas A.R.2006.Identification and functional expression of a type 2acyl-CoA:diacylglycerol acyltransferase(DGAT2)in developing Castor bean seeds which has high homolo‐gy to the major triglyceride biosynthetic enzyme of fungi and animals.Phytochemistry.67(23):2541-2549.doi:10.1016/j.phytochem.2006.09.020.
Kunst L.,Taylor D.C.,Underhill E.W.1992.Fatty acid elongation in developing seeds of Arabidopsis thali‐ana.Plant Physiol.Biochem.30:425-434.
Li H.Y.,Dong Y.Y.,Yang J.,Liu X.M.,Wang Y.F.,Yao N.,Guan L.L.,Wang N.,Wu J.Y.,Li X.K.2012.De novo transcriptome of safflower and the identification of putative genes for oleosin and the biosynthesis of fla‐vonoids.Plos One.7(2):e30987.doi:10.1371/jour‐nal.pone.0030987.
Li S.S.,Wang L.S.,Shu Q.Y.,Wu J.,Chen L.G.,Shao S.,Yin D.D.2015.Fatty acid composition of developing tree peony(Paeoniasection Moutan DC.)seeds and transcriptome analysis during seed development.BMC Genom.e16:208.doi:10.1186/s12864-015-1429-0.
Li W.D.,Li O.,Zhang A.R.,Li L.,Hao J.H.,Jin J.S.,Yin S.J.2014.Genotypic diversity of phenolic compounds and antioxidant capacity of Chinese dwarf cherry[Cerasushumilis(Bge.)Sok.]in China.Sci.Hortic.175:208-213.doi:10.1016/j.scienta.2014.06.015.
Li X.,Cao Y.,Hu Y.,Xiao L.,Wu Y.,Wu G.,Lu C.2009.Fatty acid accumulation pattern in developing seeds of peanut.Chin.J.Oil Crop.Sci.31(2):157-162.doi:10.3321/j.issn:1007-9084.2009.02.009
Lu C.F.,Fulda M.,Wallis J.G.,Browse J.2006.A highthroughput screen for genes from Castor that boost hydroxy fatty acid accumulation in seed oils of trans‐genic Arabidopsis.Plant J.45(5):847-856.doi:10.1111/j.1365-313x.2005.02636.x.
Lung S.C.,Weselake R.J.2006.Diacylglycerol acyltrans‐ferase:A key mediator of plant triacylglycerol synthe‐sis.Lipids.41(12):1073-1088.doi:10.1007/s11745-006-5057-y.
Mu X.P.,Aryal N.,Du J.M.,Du J.J.2015.Oil content and fatty acid composition of the kernels of 31 genotypes of Chinese dwarf cherry[Cerasushumilis(Bge.)Sok.].J.Hortic.Sci.Biotechnol.90(5):525-529.doi:10.1080/14620316.2015.11668709.
Mu X.P.,Liu M.,Wang P.F.,Shou J.P.,Du J.J.2016.Agro‐bacterium-mediated transformation and plant regener‐ation in Chinese dwarf cherry[Cerasushumilis(Bge.)Sok].J.Hortic.Sci.Biotechnol.91(1):71-78.doi:10.1080/14620316.2015.1110994.
Ohlrogge J.,Browse J.1995.Lipid biosynthesis.Plant Cell.7(7):957-970.doi:10.1105/tpc.7.7.957.
Shockey J.M.,Gidda S.K.,Chapital D.C.,Kuan J.C.,Dha‐noa P.K.,Bland J.M.,Rothstein S.J.,Mullen R.T.,Dy‐er J.M.2006.Tung tree DGAT1 and DGAT2 have non-redundant functions in triacylglycerol biosynthe‐sis and are localized to different subdomains of the endoplasmic reticulum.Plant Cell.18(9):2294-2313.doi:10.1105/tpc.106.043695.
Song X.S.,Shang Z.W.,Yin Z.P.,Ren J.,Sun M.C.,Ma X.L.2011.Mechanism of xanthophyll-cycle-mediated photoprotection in Cerasushumilis seedlings under water stress and subsequent recovery.Photosyntheti‐ca.49(4):523-530.doi:10.1007/s11099-011-0065-4.
Wang P.F.,Jia L.T.,Du J.J.,Zhang J.C.,Mu X.P.,Ding W.2017.Improvement of soil quality by Chinese dwarf cherry cultivation in the Loess Plateau steep hill re‐gion.Acta Prataculturae Sin.26(3):65-74.doi:10.11686/cyxb2016233.
Wei W.L.,Qi X.Q.,Wang L.H.,Zhang Y.X.,Hua W.,Li D.H.,Lv H.,Zhang X.R.2011.Characterization of the sesame(Sesamumindicum L.)global transcriptome using Illumina paired-end sequencing and develop‐ment of EST-SSR markers.BMC Genom.12:e451.doi:10.1186/1471-2164-12-451.
Zhang J.N.,Liang S.,Duan J.L.,Wang J.,Chen S.L.,Cheng Z.S.,Zhang Q.,Liang X.Q.,Li Y.R.2012.De novo assembly and characterisation of the transcrip‐tome during seed development,and generation of genic-SSR markers in peanut(Arachishypogaea L.).BMC Genom.13(1):e90.doi:10.1186/1471-2164-13-90.
Anders S.,Huber W.2010.Differential expression analysis for sequence count data.Genome Biol.11:1-12.doi:10.1186/gb-2010-11-10-r106.
Anders S.,Huber W.2010.Differential expression analysis for sequence count data.Genome Biol.11(10):R106.doi:10.1186/gb-2010-11-10-r106.
Bates P.D.,Durrett T.P.,Ohlrogge J.B.,Pollard M.2009.Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean em‐bryos.Plant Physiol.150(1):55-72.doi:10.1104/pp.109.137737.
Benjamini Y,Yekutieli D.2001.The control of the false discovery rate in multiple testing under dependency.Ann.Statist.29(4):1165-1188.doi:10.1214/aos/1013699998.
Brown A.P.,Kroon J.T.M.,Swarbreck D.,Febrer M.,Lar‐son T.R.,Graham I.A.,Caccamo M.,Slabas A.R.2012.Tissue-specific whole transcriptome sequenc‐ing in Castor directed at understanding triacylglycerol lipid biosynthetic pathways.Plos One.7(2):e30100.doi:10.1371/journal.pone.0030100.
Costa G.G.,Cardoso K.C.,del Bem L.E.,Lima A.C.,Cunha M.A.,de Campos-Leite L.,Vicentini R.,Papes F.,Moreira R.C.,Yunes J.A.,et al.2010.Transcrip‐tome analysis of the oil-rich seed of the bioenergy crop Jatrophacurcas L.BMC Genom.11(1):462-471.doi:10.1186/1471-2164-11-462.
Dong S.B.,Liu Y.L.,Niu J.,Ning Y.,Lin S.Z.,Zhang Z.X.2014.De novo transcriptome analysis of the Siberian apricot(Prunussibirica L.)and search for potential SSR markers by 454 pyrosequencing.Gene.544(2):220-227.doi:10.1016/j.gene.2014.04.031.
Fan S.Q.,Liang T.,Yu H.Y.,Bi Q.X.,Li G.T.,Wang L.B.2016.Kernel characteristics,oil contents,fatty acid compositions and biodiesel properties in developing Siberian apricot(Prunussibirica L.)seeds.Ind.Crop.Prod.89:195-199.doi:10.1016/j.indcrop.2016.05.012.
Fatima T.,Snyder C.L.,Schroeder W.R.,Cram D.,Datla R.,Wishart D.,Weselake R.J.,Krishna P.2012.Fatty acid composition of developing sea buckthorn(Hip‐pophaerhamnoides L.)berry and the transcriptome of the mature seed.Plos One.7(4):e34099.doi:10.1371/journal.pone.0034099.
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thomp‐son D.A.,Amit I.,Adiconis X.,Fan L.,Raychowd‐hury R.,Zeng Q.D.,et al.2011.Full-length transcrip‐tome assembly from RNA-Seq data without a refer‐ence genome.Nat.Biotechnol.29(7):644-652.doi:10.1038/nbt.1883.
Gupta K.,Kayam G.,Faigenboim-Doron A.,Clevenger J.,Ozias-Akins P.,Hovav R.2016.Gene expression pro‐filing during seed-filling process in peanut with em‐phasis on oil biosynthesis networks.Plant Sci.248:116-127.doi:10.1016/j.plantsci.2016.04.014.
Kroon J.T.M.,Wei W.X.,Simon W.J.,Slabas A.R.2006.Identification and functional expression of a type 2acyl-CoA:diacylglycerol acyltransferase(DGAT2)in developing Castor bean seeds which has high homolo‐gy to the major triglyceride biosynthetic enzyme of fungi and animals.Phytochemistry.67(23):2541-2549.doi:10.1016/j.phytochem.2006.09.020.
Kunst L.,Taylor D.C.,Underhill E.W.1992.Fatty acid elongation in developing seeds of Arabidopsis thali‐ana.Plant Physiol.Biochem.30:425-434.
Li H.Y.,Dong Y.Y.,Yang J.,Liu X.M.,Wang Y.F.,Yao N.,Guan L.L.,Wang N.,Wu J.Y.,Li X.K.2012.De novo transcriptome of safflower and the identification of putative genes for oleosin and the biosynthesis of fla‐vonoids.Plos One.7(2):e30987.doi:10.1371/jour‐nal.pone.0030987.
Li S.S.,Wang L.S.,Shu Q.Y.,Wu J.,Chen L.G.,Shao S.,Yin D.D.2015.Fatty acid composition of developing tree peony(Paeoniasection Moutan DC.)seeds and transcriptome analysis during seed development.BMC Genom.e16:208.doi:10.1186/s12864-015-1429-0.
Li W.D.,Li O.,Zhang A.R.,Li L.,Hao J.H.,Jin J.S.,Yin S.J.2014.Genotypic diversity of phenolic compounds and antioxidant capacity of Chinese dwarf cherry[Cerasushumilis(Bge.)Sok.]in China.Sci.Hortic.175:208-213.doi:10.1016/j.scienta.2014.06.015.
Li X.,Cao Y.,Hu Y.,Xiao L.,Wu Y.,Wu G.,Lu C.2009.Fatty acid accumulation pattern in developing seeds of peanut.Chin.J.Oil Crop.Sci.31(2):157-162.doi:10.3321/j.issn:1007-9084.2009.02.009
Lu C.F.,Fulda M.,Wallis J.G.,Browse J.2006.A highthroughput screen for genes from Castor that boost hydroxy fatty acid accumulation in seed oils of trans‐genic Arabidopsis.Plant J.45(5):847-856.doi:10.1111/j.1365-313x.2005.02636.x.
Lung S.C.,Weselake R.J.2006.Diacylglycerol acyltrans‐ferase:A key mediator of plant triacylglycerol synthe‐sis.Lipids.41(12):1073-1088.doi:10.1007/s11745-006-5057-y.
Mu X.P.,Aryal N.,Du J.M.,Du J.J.2015.Oil content and fatty acid composition of the kernels of 31 genotypes of Chinese dwarf cherry[Cerasushumilis(Bge.)Sok.].J.Hortic.Sci.Biotechnol.90(5):525-529.doi:10.1080/14620316.2015.11668709.
Mu X.P.,Liu M.,Wang P.F.,Shou J.P.,Du J.J.2016.Agro‐bacterium-mediated transformation and plant regener‐ation in Chinese dwarf cherry[Cerasushumilis(Bge.)Sok].J.Hortic.Sci.Biotechnol.91(1):71-78.doi:10.1080/14620316.2015.1110994.
Ohlrogge J.,Browse J.1995.Lipid biosynthesis.Plant Cell.7(7):957-970.doi:10.1105/tpc.7.7.957.
Shockey J.M.,Gidda S.K.,Chapital D.C.,Kuan J.C.,Dha‐noa P.K.,Bland J.M.,Rothstein S.J.,Mullen R.T.,Dy‐er J.M.2006.Tung tree DGAT1 and DGAT2 have non-redundant functions in triacylglycerol biosynthe‐sis and are localized to different subdomains of the endoplasmic reticulum.Plant Cell.18(9):2294-2313.doi:10.1105/tpc.106.043695.
Song X.S.,Shang Z.W.,Yin Z.P.,Ren J.,Sun M.C.,Ma X.L.2011.Mechanism of xanthophyll-cycle-mediated photoprotection in Cerasushumilis seedlings under water stress and subsequent recovery.Photosyntheti‐ca.49(4):523-530.doi:10.1007/s11099-011-0065-4.
Wang P.F.,Jia L.T.,Du J.J.,Zhang J.C.,Mu X.P.,Ding W.2017.Improvement of soil quality by Chinese dwarf cherry cultivation in the Loess Plateau steep hill re‐gion.Acta Prataculturae Sin.26(3):65-74.doi:10.11686/cyxb2016233.
Wei W.L.,Qi X.Q.,Wang L.H.,Zhang Y.X.,Hua W.,Li D.H.,Lv H.,Zhang X.R.2011.Characterization of the sesame(Sesamumindicum L.)global transcriptome using Illumina paired-end sequencing and develop‐ment of EST-SSR markers.BMC Genom.12:e451.doi:10.1186/1471-2164-12-451.
Zhang J.N.,Liang S.,Duan J.L.,Wang J.,Chen S.L.,Cheng Z.S.,Zhang Q.,Liang X.Q.,Li Y.R.2012.De novo assembly and characterisation of the transcrip‐tome during seed development,and generation of genic-SSR markers in peanut(Arachishypogaea L.).BMC Genom.13(1):e90.doi:10.1186/1471-2164-13-90.