四种木本油料内参基因筛选及Actin基因时空表达分析
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摘要
以油茶、沙棘、文冠果和油用牡丹的成熟种子为材料,利用实时荧光定量PCR分析了7个内参候选基因GAPDH、Actin、TUB、eEF1α、RPL4、UBQ和e IF4A在四种木本油料中的表达稳定性,并进一步验证了Actin基因在沙棘不同发育期种子和果肉中的表达。结果表明,6个内参候选基因GAPDH、TUB、eEF1α、RPL4、UBQ和e IF4A的表达水平在四种本油料种子间存在明显差异(p<0.05),内参候选Actin基因在四种木本油料种子中的表达较稳定(p>0.05)。Actin基因在沙棘种子和果实的不同发育期表达稳定(p=0.380和p=0.603),且在果肉和种子间在各个时期不存在明显差异(p>0.05)。这不仅可为沙棘果实不同器官基因表达差异研究提供内参基因,而且可为四种木本油料基因表达研究内参基因的选择提供科学参考。
The mature seeds of four woody-oil trees(C amellia oleifera,Hippophae rhamnoides,Xanthoceras sorbifolia and Paeonia suffruticosa) were selected as materials,the expression stability of seven candidate reference genes GAPDH,Actin,TUB,eEF1α,RPL4,UBQ and eIF4A were analyzed by qRT-PCR.We also further tested the expression of Actin gene in seeds and pulp of different developmental stages of sea buckthorn(Hippophae rhamnoides).The results showed that there were significant differences(p<0.05) in the expressions of GAPDH,TUB,eEF1α,RPL4,UBQ and e IF4A among seeds of four woody-oil trees,but the expressions of Actin gene were relatively stable(p>0.05) in four seeds.The expressions of Actin gene were stable in different developmental stages of seeds and pulp of sea buckthorn(p=0.380 and p=0.603,respectively),and there was no significant difference in expressions between pulp and seeds at different development stages(p>0.05).These data not only could provide the reference gene for the analysis of different gene expressions in different organs of sea buckthorn fruits,but also provide scientific reference for the selection of reference genes in studying the gene expression of four woody-oil trees.
The mature seeds of four woody-oil trees(C amellia oleifera,Hippophae rhamnoides,Xanthoceras sorbifolia and Paeonia suffruticosa) were selected as materials,the expression stability of seven candidate reference genes GAPDH,Actin,TUB,eEF1α,RPL4,UBQ and eIF4A were analyzed by qRT-PCR.We also further tested the expression of Actin gene in seeds and pulp of different developmental stages of sea buckthorn(Hippophae rhamnoides).The results showed that there were significant differences(p<0.05) in the expressions of GAPDH,TUB,eEF1α,RPL4,UBQ and e IF4A among seeds of four woody-oil trees,but the expressions of Actin gene were relatively stable(p>0.05) in four seeds.The expressions of Actin gene were stable in different developmental stages of seeds and pulp of sea buckthorn(p=0.380 and p=0.603,respectively),and there was no significant difference in expressions between pulp and seeds at different development stages(p>0.05).These data not only could provide the reference gene for the analysis of different gene expressions in different organs of sea buckthorn fruits,but also provide scientific reference for the selection of reference genes in studying the gene expression of four woody-oil trees.
引文
Ao Y.,Duan J.,Yu H.Y.,Jiang C.Y.,and Ma L.Y.,2012,Research progress on Xanthoceras sorbifolia Bunge,Zhongguo Nongye Daxue Xuebao(Journal of China Agricultural University),17(6):197-203(敖妍,段劼,于海燕,姜常玉,马履一,2012,文冠果研究进展,中国农业大学学报,17(6):197-203)
Cai J.L.,Zhu Y.L.,Xie S.P.,Chen L.,Ou Y.L.,Liu F.,Zhang Y.L.,Liu X.D.,Tong Q.Z.,and Yi G.Q.,2016,Screening of reference genes in Lonicera macranthoides and spatio-temporal expression analysis of Lm AGL15 in Mads-box family,Zhongcaoyao(Chinese Traditional and Herbal Drugs),47(15):2727-2733(蔡嘉洛,朱贻霖,谢舒平,陈林,欧阳琳,刘峰,张亚丽,刘湘丹,童巧珍,易刚强,2016,灰毡毛忍冬内参基因筛选和MADS-box家族基因AGL15的时空表达分析,中草药,47(15):2727-2733)
Dheda K.,Huggett J.F.,Bustin S.A.,Johnson M.A.,Rook G.,and Zumla A.,2004,Validation of housekeeping genes for normalizing RNA expression in real-time PCR,Biotechniques,37(1):112-119
Ding J.,Ruan C.J.,Guan Y.,Wu L.R.,and Shan J.Y.,2017,Coordinated regulation mechanism of multigenes involved in high accumulation of C18 unsaturated fatty acids in sea buckthorn seed,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(6):1080-1089(丁健,阮成江,关莹,吴立仁,单金友,2017,沙棘种子高积累碳十八不饱和脂肪酸的多基因协同调控机制,西北植物学报,37(6):1080-1089)
Ding J.,Ruan C.J.,Shan J.Y.,and Guan Y.,2016,Expression of key gene involved in lipid biosynthesis and accumulation during seeds formation and development in Hippophae rhamnoides,Xibei Zhiwu Xuebao(Acta Botanica BorealiOccidentalia Sinica),36(8):1642-1647(丁健,阮成江,单金友,关莹,2016,沙棘种子形成发育过程中油脂合成积累关键基因的表达分析,西北植物学报,36(8):1642-1647)
Meng T.,Ruan C.J.,Ding J.,and Tang D.H.,2017,Oil content in germplasm and composition of oil of different species of Camellia oleifera in Yuping,Zhongguo Youzhi(China Oils and Fats),42(6):129-133(孟婷,阮成江,丁健,唐东慧,2017,玉屏不同油茶种质含油率及油中脂肪酸组成成分分析,中国油脂,42(6):129-133)
Nicot N.,Hausman J.F.,Hoffmann L.,and Evers D.,2005,House-keeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress,J.Exp.Bot.,56(421):2907-2914
Quackenbush J.,2002,Microarray data normalization and transformation,Nat.Genet.,32(S):496-501
Ruan C.J.,Rumpunen K.,and Nybom H.,2013,Advances in improvement of quality and resistance in a multipurpose crop:sea buckthorn,Crit.Rev.Biotechnol.,33(2):126-144
Schmittgen T.D.,and Liveak K.J.,2008,Analyzing real-time PCR data by the comparative CTmethod,Nat.Protoc.,3(6):1101-1108
Song Z.B.,2014,The selection reference gene and expression analysis of oil content enzyme gene in Camellia oleifera,Thesis for M.S.,Central South University of Forestry and Technology,Supervisor:Zhang L.,pp.1-2(宋志波,2014,油茶内参基因的筛选与油脂合成相关酶基因的表达特征分析,硕士学位论文,中南林业科技大学,导师:张琳,pp.1-2)
Tang D.H.,Ruan C.J.,Meng T.,and Ding J.,2017,Oil contents and fatty acid composition in different germplasm of Xanthoceras sorbifolia Bunge,Zhongguo Youzhi(China Oils and Fats),42(3):77-81(唐东慧,阮成江,孟婷,丁健,2017,不同种质文冠果含油量及油中脂肪酸组成成分分析,中国油脂,42(3):77-81)
Wang H.,2011,The research on the extraction and purification technology of tea oil and high purity tea saponin from the camellia cakes,Thesis for M.S.,Lanzhou University of Technology,Supervisor:Shi G.F.,pp.1-2(汪虎,2011,油茶茶枯中茶油及高纯度茶皂素的提取纯化工艺研究,硕士学位论文,兰州理工大学,导师:史高峰,pp.1-2)
Zhang T.,Gao T.S.,Bai R.Y.,Huang X.L.,Guo N.,Lu J.X.,and Luo A.C.,2015,Utilization and research progress of oil tree peony,Chongqing Shifan Daxue Xuebao(Journal of Chongqing Normal University),32(2):143-149(张涛,高天姝,白瑞英,黄兴琳,郭楠,陆俊杏,罗安才,2015,油用牡丹利用与研究进展,重庆师范大学学报,32(2):143-149)
Cai J.L.,Zhu Y.L.,Xie S.P.,Chen L.,Ou Y.L.,Liu F.,Zhang Y.L.,Liu X.D.,Tong Q.Z.,and Yi G.Q.,2016,Screening of reference genes in Lonicera macranthoides and spatio-temporal expression analysis of Lm AGL15 in Mads-box family,Zhongcaoyao(Chinese Traditional and Herbal Drugs),47(15):2727-2733(蔡嘉洛,朱贻霖,谢舒平,陈林,欧阳琳,刘峰,张亚丽,刘湘丹,童巧珍,易刚强,2016,灰毡毛忍冬内参基因筛选和MADS-box家族基因AGL15的时空表达分析,中草药,47(15):2727-2733)
Dheda K.,Huggett J.F.,Bustin S.A.,Johnson M.A.,Rook G.,and Zumla A.,2004,Validation of housekeeping genes for normalizing RNA expression in real-time PCR,Biotechniques,37(1):112-119
Ding J.,Ruan C.J.,Guan Y.,Wu L.R.,and Shan J.Y.,2017,Coordinated regulation mechanism of multigenes involved in high accumulation of C18 unsaturated fatty acids in sea buckthorn seed,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(6):1080-1089(丁健,阮成江,关莹,吴立仁,单金友,2017,沙棘种子高积累碳十八不饱和脂肪酸的多基因协同调控机制,西北植物学报,37(6):1080-1089)
Ding J.,Ruan C.J.,Shan J.Y.,and Guan Y.,2016,Expression of key gene involved in lipid biosynthesis and accumulation during seeds formation and development in Hippophae rhamnoides,Xibei Zhiwu Xuebao(Acta Botanica BorealiOccidentalia Sinica),36(8):1642-1647(丁健,阮成江,单金友,关莹,2016,沙棘种子形成发育过程中油脂合成积累关键基因的表达分析,西北植物学报,36(8):1642-1647)
Meng T.,Ruan C.J.,Ding J.,and Tang D.H.,2017,Oil content in germplasm and composition of oil of different species of Camellia oleifera in Yuping,Zhongguo Youzhi(China Oils and Fats),42(6):129-133(孟婷,阮成江,丁健,唐东慧,2017,玉屏不同油茶种质含油率及油中脂肪酸组成成分分析,中国油脂,42(6):129-133)
Nicot N.,Hausman J.F.,Hoffmann L.,and Evers D.,2005,House-keeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress,J.Exp.Bot.,56(421):2907-2914
Quackenbush J.,2002,Microarray data normalization and transformation,Nat.Genet.,32(S):496-501
Ruan C.J.,Rumpunen K.,and Nybom H.,2013,Advances in improvement of quality and resistance in a multipurpose crop:sea buckthorn,Crit.Rev.Biotechnol.,33(2):126-144
Schmittgen T.D.,and Liveak K.J.,2008,Analyzing real-time PCR data by the comparative CTmethod,Nat.Protoc.,3(6):1101-1108
Song Z.B.,2014,The selection reference gene and expression analysis of oil content enzyme gene in Camellia oleifera,Thesis for M.S.,Central South University of Forestry and Technology,Supervisor:Zhang L.,pp.1-2(宋志波,2014,油茶内参基因的筛选与油脂合成相关酶基因的表达特征分析,硕士学位论文,中南林业科技大学,导师:张琳,pp.1-2)
Tang D.H.,Ruan C.J.,Meng T.,and Ding J.,2017,Oil contents and fatty acid composition in different germplasm of Xanthoceras sorbifolia Bunge,Zhongguo Youzhi(China Oils and Fats),42(3):77-81(唐东慧,阮成江,孟婷,丁健,2017,不同种质文冠果含油量及油中脂肪酸组成成分分析,中国油脂,42(3):77-81)
Wang H.,2011,The research on the extraction and purification technology of tea oil and high purity tea saponin from the camellia cakes,Thesis for M.S.,Lanzhou University of Technology,Supervisor:Shi G.F.,pp.1-2(汪虎,2011,油茶茶枯中茶油及高纯度茶皂素的提取纯化工艺研究,硕士学位论文,兰州理工大学,导师:史高峰,pp.1-2)
Zhang T.,Gao T.S.,Bai R.Y.,Huang X.L.,Guo N.,Lu J.X.,and Luo A.C.,2015,Utilization and research progress of oil tree peony,Chongqing Shifan Daxue Xuebao(Journal of Chongqing Normal University),32(2):143-149(张涛,高天姝,白瑞英,黄兴琳,郭楠,陆俊杏,罗安才,2015,油用牡丹利用与研究进展,重庆师范大学学报,32(2):143-149)