花生AhPEPC1基因抑制表达的转基因后代转录组分析
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摘要
磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvatecarboxylase,PEPC)是控制油料作物种子中蛋白质/油脂含量比率的一个关键酶。本研究检测了花生AhPEPC1基因抑制表达的转基因株系种子含油量,与非转基因花生相比,转基因花生种子含油量提高了5.7%～10.3%。利用转录组测序(RNA-Seq)技术分析花生中AhPEPC1基因的抑制表达是否影响其他基因的功能。结果表明,转录组分析筛选到110个基因差异表达,其中25个基因上调表达,85个基因表达下调。对110个差异表达基因进行了KEGG富集分析,其中有34个基因成功获得了KEGG注释,发现氨基酸的生物合成途径中有2个基因(Aradu.M0JX8,Aradu.FE0Z7)下调表达。利用荧光定量PCR分析了15个DEG(differential expressed gene)在非转基因对照和转基因花生种子中的表达情况,发现其趋势与转录组测序结果基本一致。研究结果可在一定程度上解析AhPEPC1基因调控花生种子含油量的分子机制。
Phosphoenolpyruvate carboxylase is considered as a key enzyme to control the ratio of protein to lipid of oilseeds. In this study, the antisense expression of the peanut PEPC isoform 1(AhPEPC1) gene increased the lipid content by 5.7%–10.3%compared with the non-transgenic control. The high-throughput sequencing technology — RNA-Seq was used to analyze whether the inhibitory expression of AhPEPC1 gene in peanut affected the function of other genes. The results showed that 110 genes were differentially expressed, of which 25 genes were up-regulated and 85 genes were down-regulated. KEGG enrichment analysis was performed on 110 differentially expressed genes, among which 34 genes were successfully obtained KEGG annotation, and two genes(Aradu.M0 JX8 and Aradu.FE0 Z7) were down-regulated in the biosynthesis pathway of amino acids. Fifteen DEGs between non-transgenic control and transgenic peanut seeds were selected to analyze the gene expression levels using qRT-PCR. The results of qRT-PCR agreed well with most findings from RNA-seq analysis. This research might resolve to some extent the molecular mechanisms of AhPEPC1 gene regulating oil content of peanut seeds.
Phosphoenolpyruvate carboxylase is considered as a key enzyme to control the ratio of protein to lipid of oilseeds. In this study, the antisense expression of the peanut PEPC isoform 1(AhPEPC1) gene increased the lipid content by 5.7%–10.3%compared with the non-transgenic control. The high-throughput sequencing technology — RNA-Seq was used to analyze whether the inhibitory expression of AhPEPC1 gene in peanut affected the function of other genes. The results showed that 110 genes were differentially expressed, of which 25 genes were up-regulated and 85 genes were down-regulated. KEGG enrichment analysis was performed on 110 differentially expressed genes, among which 34 genes were successfully obtained KEGG annotation, and two genes(Aradu.M0 JX8 and Aradu.FE0 Z7) were down-regulated in the biosynthesis pathway of amino acids. Fifteen DEGs between non-transgenic control and transgenic peanut seeds were selected to analyze the gene expression levels using qRT-PCR. The results of qRT-PCR agreed well with most findings from RNA-seq analysis. This research might resolve to some extent the molecular mechanisms of AhPEPC1 gene regulating oil content of peanut seeds.
引文
[1]万书波.花生产业形势与对策.山东农业科学,2014,46(10):128-132.Wan S B.Situation and developing strategies of peanut industry.Shandong Agric Sci,2014,46(10):128-132(in Chinese with English abstract).
[2]Chen X,Li H,Pandey M K,Yang Q,Wang X,Garg V,Li H,Chi X,Doddamani D,Hong Y,Upadhyaya H,Guo H,Khan A W,Zhu F,Zhang X,Pan L,Pierce G J,Zhou G,Krishnamohan K A,Chen M,Zhong N,Agarwal G,Li S,Chitikineni A,Zhang G Q,Sharma S,Chen N,Liu H,Janila P,Li S,Wang M,Wang T,Sun J,Li X,Li C,Wang M,Yu L,Wen S,Singh S,Yang Z,Zhao J,Zhang C,Yu Y,Bi J,Zhang X,Liu Z J,Paterson A H,Wang S,Liang X,Varshney R K,Yu S.Draft genome of the peanut A-genome progenitor(Arachis duranensis)provides insights into geocarpy,oilbiosynthesis,and allergens.Proc Natl Acad Sci USA,2016,113:6785-6790.
[3]Bertioli D J,Cannon S B,Froenicke L,Huang G,Farmer A D,Cannon E K S,Liu X,Gao D,Clevenger J,Dash S,Ren L,Moretzsohn M C,Shirasawa K,Huang W,Vidigal B,Abernathy B,Chu Y,Niederhuth C E,Umale P,Araujo A C G,Kozik A,Do Kim K,Burow M D,Varshney R K,Wang X,Zhang X,Barkley N,Guimaraes P M,Isobe S,Guo B,Liao B,Stalker H T,Schmitz R J,Scheffler B E,Leal-Bertioli S C M,Xun X,Jackson S A,Michelmore R,Ozias-Akins P.The genome sequences of Arachis duranensis and Arachis ipaensis,the diploid ancestors of cultivated peanut.Nat Genet,2016,48:438-446.
[4]Rolletschek H,Borisjuk L,Radchuk R,Miranda M,Heim U,Wobus U,Weber H.Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy.Plant Biotechnol J,2004,2:211-219.
[5]Song D,Fu J,Shi D.Exploitation of oil-bearing microalgae for biodiesel.Chin J Biotechnol,2008,24:341-348.
[6]陈锦清,郎春秀,胡张华,刘智宏,黄锐之.反义PEP基因调控油菜籽粒蛋白质/油脂含量比率的研究.农业生物技术学报,1999,7:316-320.Chen J Q,Lang C X,Hu Z H,Liu Z H,Huang R Z.Antisense PEP gene regulates to ratio of protein and lipid content in Brassica napus seeds.J Agric Biotechnol,1999,7:316-320(in Chinese with English abstract).
[7]Sugimoto T,Kawasaki T,Kato T,Whittier R F,Shibata D,Kawamura Y.cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean.Plant Mol Biol,1992,20:743-747.
[8]Pan L J,Zhang J C,Chi X Y,Chen N,Chen M N,Wang M,Wang T,Yang Z,Zhang Z M,Wan Y S,Yu S L,Liu F Z.The antisense expression of AhPEPC1 increases seed oil production in peanuts(Arachis hypogaea L.).Grasas Y Aceites,2016,67(4):e164.
[9]Sharma N,Anderson M,Kumar A,Zhang Y,Giblin E M,Abrams S R,Zaharia L I,Taylor D C,Fobert P R.Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts.BMC Genomics,2008,9:619,doi:10.1186/1471-2164-9-619.
[10]Liu J,Hua W,Yang H L,Zhan G M,Li R J,Deng L B,Wang X F,Liu G H,Wang H Z.The BnGRF2 gene(GRF2-like gene from Brassica napus)enhances seed oil production through regulating cell number and plant photosynthesis.J Exp Bot,2012,63:3727-3740.
[11]Trapnell C,Williams B A,Pertea G,Mortazavi A,Kwan G,van Baren M J,Salzberg S L,Wold B J,Pachter L.Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation.Nat Biotechnol,2010,28:511-515.
[12]Ruuska S A,Girke T,Benning C,Ohlrogge J B.Contrapuntal networks of gene expression during Arabidopsis seed filling.Plant Cell,2002,14:1191-1206.
[13]Uhrig R G,O’Leary B,Spang H E,MacDonald J A,She Y M,Plaxton W C.Coimmunopurification of phosphorylated bacterialand plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds.Plant Physiol,2008,146:1346-1357.
[14]Sugimoto T,Tanaka K,Monma M,Kawamura Y,Saio K.Phosphoenolpyruvate carboxylase level in soybean seed highly correlates to its contents of protein and lipid.Agric Biol Chem,1989,53:885-887.
[15]Vazquez-Tello A,Whittier R P,Kawasaki T,Sugimoto T,Kawamura Y,Shibata D.Sequence of a soybean(Glycine max L.)phosphoenolpyruvate carboxylase cDNA.Plant Physiol,1993,103:1025-1026.
[16]张占琴,王金梅,王学军,汪凯华,袁春新,麻浩.油菜籽粒发育过程中PEPCase活性与油脂,蛋白质及亚基积累的特点.中国油料作物学报,2009,31:14-18.Zhang Z Q,Wang J M,Wang X J,Wang K H,Yuan C X,Ma H.The characteristics of PEPCase activity and accumulation of oil,protein and major protein subunits during seed development of rape(Brassica napus).Chin J Oil Crop Sci,2009,31:14-18(in Chinese with English abstract).
[17]Ward J K,Tissue D T,Thomas R B,Strain B R.Comparative responses of model C3 and C4 plants to drought in low and elevated CO2.Global Change Biol,1999,5:857-867.
[18]Nayyar H,Gupta D.Differential sensitivity of C3 and C4 plants to water deficit stress:association with oxidative stress and antioxidants.Environ Exp Bot,2006,58:106-113.
[19]Brown A P,Kroon J T,Swarbreck D,Febrer M,Larson T R,Graham I A,Caccamo M,Slabas A R.Tissue-specific whole transcriptome sequencing in castor,directed at understanding triacylglycerol lipid biosynthetic pathways.PLoS One,2012,7:e301
[2]Chen X,Li H,Pandey M K,Yang Q,Wang X,Garg V,Li H,Chi X,Doddamani D,Hong Y,Upadhyaya H,Guo H,Khan A W,Zhu F,Zhang X,Pan L,Pierce G J,Zhou G,Krishnamohan K A,Chen M,Zhong N,Agarwal G,Li S,Chitikineni A,Zhang G Q,Sharma S,Chen N,Liu H,Janila P,Li S,Wang M,Wang T,Sun J,Li X,Li C,Wang M,Yu L,Wen S,Singh S,Yang Z,Zhao J,Zhang C,Yu Y,Bi J,Zhang X,Liu Z J,Paterson A H,Wang S,Liang X,Varshney R K,Yu S.Draft genome of the peanut A-genome progenitor(Arachis duranensis)provides insights into geocarpy,oilbiosynthesis,and allergens.Proc Natl Acad Sci USA,2016,113:6785-6790.
[3]Bertioli D J,Cannon S B,Froenicke L,Huang G,Farmer A D,Cannon E K S,Liu X,Gao D,Clevenger J,Dash S,Ren L,Moretzsohn M C,Shirasawa K,Huang W,Vidigal B,Abernathy B,Chu Y,Niederhuth C E,Umale P,Araujo A C G,Kozik A,Do Kim K,Burow M D,Varshney R K,Wang X,Zhang X,Barkley N,Guimaraes P M,Isobe S,Guo B,Liao B,Stalker H T,Schmitz R J,Scheffler B E,Leal-Bertioli S C M,Xun X,Jackson S A,Michelmore R,Ozias-Akins P.The genome sequences of Arachis duranensis and Arachis ipaensis,the diploid ancestors of cultivated peanut.Nat Genet,2016,48:438-446.
[4]Rolletschek H,Borisjuk L,Radchuk R,Miranda M,Heim U,Wobus U,Weber H.Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy.Plant Biotechnol J,2004,2:211-219.
[5]Song D,Fu J,Shi D.Exploitation of oil-bearing microalgae for biodiesel.Chin J Biotechnol,2008,24:341-348.
[6]陈锦清,郎春秀,胡张华,刘智宏,黄锐之.反义PEP基因调控油菜籽粒蛋白质/油脂含量比率的研究.农业生物技术学报,1999,7:316-320.Chen J Q,Lang C X,Hu Z H,Liu Z H,Huang R Z.Antisense PEP gene regulates to ratio of protein and lipid content in Brassica napus seeds.J Agric Biotechnol,1999,7:316-320(in Chinese with English abstract).
[7]Sugimoto T,Kawasaki T,Kato T,Whittier R F,Shibata D,Kawamura Y.cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean.Plant Mol Biol,1992,20:743-747.
[8]Pan L J,Zhang J C,Chi X Y,Chen N,Chen M N,Wang M,Wang T,Yang Z,Zhang Z M,Wan Y S,Yu S L,Liu F Z.The antisense expression of AhPEPC1 increases seed oil production in peanuts(Arachis hypogaea L.).Grasas Y Aceites,2016,67(4):e164.
[9]Sharma N,Anderson M,Kumar A,Zhang Y,Giblin E M,Abrams S R,Zaharia L I,Taylor D C,Fobert P R.Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts.BMC Genomics,2008,9:619,doi:10.1186/1471-2164-9-619.
[10]Liu J,Hua W,Yang H L,Zhan G M,Li R J,Deng L B,Wang X F,Liu G H,Wang H Z.The BnGRF2 gene(GRF2-like gene from Brassica napus)enhances seed oil production through regulating cell number and plant photosynthesis.J Exp Bot,2012,63:3727-3740.
[11]Trapnell C,Williams B A,Pertea G,Mortazavi A,Kwan G,van Baren M J,Salzberg S L,Wold B J,Pachter L.Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation.Nat Biotechnol,2010,28:511-515.
[12]Ruuska S A,Girke T,Benning C,Ohlrogge J B.Contrapuntal networks of gene expression during Arabidopsis seed filling.Plant Cell,2002,14:1191-1206.
[13]Uhrig R G,O’Leary B,Spang H E,MacDonald J A,She Y M,Plaxton W C.Coimmunopurification of phosphorylated bacterialand plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds.Plant Physiol,2008,146:1346-1357.
[14]Sugimoto T,Tanaka K,Monma M,Kawamura Y,Saio K.Phosphoenolpyruvate carboxylase level in soybean seed highly correlates to its contents of protein and lipid.Agric Biol Chem,1989,53:885-887.
[15]Vazquez-Tello A,Whittier R P,Kawasaki T,Sugimoto T,Kawamura Y,Shibata D.Sequence of a soybean(Glycine max L.)phosphoenolpyruvate carboxylase cDNA.Plant Physiol,1993,103:1025-1026.
[16]张占琴,王金梅,王学军,汪凯华,袁春新,麻浩.油菜籽粒发育过程中PEPCase活性与油脂,蛋白质及亚基积累的特点.中国油料作物学报,2009,31:14-18.Zhang Z Q,Wang J M,Wang X J,Wang K H,Yuan C X,Ma H.The characteristics of PEPCase activity and accumulation of oil,protein and major protein subunits during seed development of rape(Brassica napus).Chin J Oil Crop Sci,2009,31:14-18(in Chinese with English abstract).
[17]Ward J K,Tissue D T,Thomas R B,Strain B R.Comparative responses of model C3 and C4 plants to drought in low and elevated CO2.Global Change Biol,1999,5:857-867.
[18]Nayyar H,Gupta D.Differential sensitivity of C3 and C4 plants to water deficit stress:association with oxidative stress and antioxidants.Environ Exp Bot,2006,58:106-113.
[19]Brown A P,Kroon J T,Swarbreck D,Febrer M,Larson T R,Graham I A,Caccamo M,Slabas A R.Tissue-specific whole transcriptome sequencing in castor,directed at understanding triacylglycerol lipid biosynthetic pathways.PLoS One,2012,7:e301
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