油菜种子发育过程中MATH结构域基因的干扰载体构建与转基因研究
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摘要
在对油菜脂肪酸合成的研究中,对其相关分子调控机理研究较少,本研究通过分析甘蓝型油菜种子发育过程SSH文库的EST序列,筛选得到了一个与拟南芥At3g20370有高度相似性的具MATH结构域蛋白的基因,命名为BnaMT-1。本研究利用半定量RT-PCR方法分析了甘蓝、白菜、甘蓝型油菜种子不同发育时期BnaMT-1基因的表达谱,发现该基因在三种植物中的表达存在明显差异。同时,为了研究该基因在油菜种子发育过程中的功能,克隆了BnaMT-1基因片段,长度为327bp,并插入至含种子特异性表达NapinA启动子的RNA干扰载体,构建成种子特异表达的RNA干扰载体,用于沉默该基因。利用根癌农杆菌介导转化法将干扰载体转至甘蓝型油菜湘油15号中,转化子在除草剂PPT的选择压下进行培养。目前已获得一株稳定的转基因苗。这为进一步研究BnaMT-1基因在甘蓝型油菜中的功能打下了基础。
Molecular regulation mechanism to synthesis of fatty acid was researched less in Brassica napus. A gene including MATH domain screened from EST of SSH Library about seed development in Brassica napus. Alignment showed that it had a high degree of similarity with Arabidopsis thaliana At3g20370, named BnaMT-1.Semiquantitative RT-PCR showed BnaMT-1 gene had different expression in Brassica oleracea, Brassica pekinensis and Brassica napus.The BnaMT-1 gene fragment with 327bp cloned was inserted to plasmid, with a seed specific expression NapinA promoter, to construct a BnaMT-1 gene RNA interference vector which was used to interfere the gene expression. The RNA interference vector was transferred into Brassica napus XY15 by Agrobacterium-mediated transfrormation.Transformants were screened by PPT resistance and PCR amplification.One positive transformation of seedlings were detected,which paved the way for further study on the function of BnaMT-1 gene in Brassica napus.
Molecular regulation mechanism to synthesis of fatty acid was researched less in Brassica napus. A gene including MATH domain screened from EST of SSH Library about seed development in Brassica napus. Alignment showed that it had a high degree of similarity with Arabidopsis thaliana At3g20370, named BnaMT-1.Semiquantitative RT-PCR showed BnaMT-1 gene had different expression in Brassica oleracea, Brassica pekinensis and Brassica napus.The BnaMT-1 gene fragment with 327bp cloned was inserted to plasmid, with a seed specific expression NapinA promoter, to construct a BnaMT-1 gene RNA interference vector which was used to interfere the gene expression. The RNA interference vector was transferred into Brassica napus XY15 by Agrobacterium-mediated transfrormation.Transformants were screened by PPT resistance and PCR amplification.One positive transformation of seedlings were detected,which paved the way for further study on the function of BnaMT-1 gene in Brassica napus.
引文
[1]Ohlrogge JB,Kuhn DN, Stumpf PK. Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea [J]. Proc Natl Acad Sci USA,1979,76 (3):1194-1198
[2]Ohlrogge JB, Browse JA. Lipid biosynthesis[J]. Plant Cell,1995,7:957-970
[3]Thelen JJ, Ohlrogge JB.Metabolic engineering of fatty acid biosynthesis in plants [J]. Metab Eng,2002,4(1):12-21
[4]Pollard MR, Anderson L, Fan C, et al. Aspecific acyl-ACP thioesterase implicated in medium chain fatty acid production immature cotyledons of Umbellularia California [J]. Archives of biochemistry and biophysics,1991,284:306-412
[5]Knutzon DS, Lardizabal KD, Nelsen JS, Bleibaum JL, Davies HM, Metz JG. Cloning of a coconut endosperm cDNA encoding al-acyl-sn-glycerol-3-phosphate acyltransfera-se that accepts medium-chain-length substractes[J]. Plant Physiol,1995,109(3):999-1006
[6]Voelker TA, Hayes TR, Cranmer AM, Turner JC, Davies HM. Genetic engineering of a quantitative trait:metabolic and genetic parameters influencing the accumulation of laurate in rapeseed[J]. Plant J,1996,9:229-241
[7]Lassner MW, Levering CK, Davies HM, Knutzon DS. Lysophosphatidic acid acyltrans-ferase from meadowfoam mediates insertion of erucic acid at the sn-2 positon of tracy-lglycerol in transgenic raeseed oil [J]. Plant Physiol,1995,109:1389-1394
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[9]Wang YP, Sonntag K, Rudloff E, Han J. Production of fertile transgenic Brassica napus by Agrobacetium-mediated transformation of protoplasts [J]. Plant Breed,2005, 124:1-4
[10]Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, Heinz E. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constri-nts on their accumulation [J].Plant Cell,2004,16:2734-2748
[11]Qi B, Fraser T, Mugford S, Dobson G, Sayanova O, Butler J, Napier JA, Stobart AK, Lazarus CM. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants [J]. Nat Biotechnol,2004,22:739-745
[12]Bession F, Koo AJ, Ruuska S, Schwender J, Pollard M, Thelen JJ, Paddock T, Salas JJ, et al. Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database [J].Plant Physiol,2003,132(2):681-697
[13]彭琦,胡燕,杜培粉,谢青轩,阮颖,刘春林.甘蓝型油菜种子不同发育时期SSH文库的构建.作物学报,2009,35(9)：1-8
[14]Chung JY,Park YC,Ye H,et al.All TRAFs are not created equal:Common and distinct molecular mechanisms of TRAF-mediated signal transduction[J]. J Cell Sci,2002, 115:679-688
[15]Zapata JM.TNF-receptor-associated factors as targets for drug development [J]. Ex-pert Opin Ther Targets,2003,7(3):411-425
[16]Bradley JR,Pober JS.Tumor necrosis factor receptor-associated factor(TRAFs) [J]. Oncogene,2001,20:6482-6491
[17]Biship GA.The multifaceted roles of TRAFs in the regulation of B-cell function [J]. Nat Rev Immunol,2004,4(10):775-786
[18]Uren AG,Vaux DL.TRAF proteins and meprins share a conserved domain[J]. Tren-ds Biochem Sci,1996,21(7):244-245
[19]Sunnerhagen M,Pursglove S,Fladvad M.The new MATH:Homology suggests shared binding surfaces in meprin tetramers and TRAF trimers[J]. FEBS Lett,2002,530(1-3):1-3
[20]Yang P,Wiser JL, Peairs JJ, et al. Human RPE expression of cell survival factors[J]. Invest Ophthalmol Vis Sci,2005,46(5):1755-1764
[21]王承兴,邓锡云,李晓艳等.TRAF1在鼻咽癌及癌旁组织中的表达[J].癌症,2000,19(9)：857-859
[22]Siegler G, Kremmer E, Gonnella R,et al.Epstein-Barr virus encoded latent mem-brane protein 1(LMP1) and TNF receptor associated factors(TRAF):colocalisation of LMP1 and TRAF1 in primary EBV infection and in EBV associated Hodgkin lymph-oma[J]. Mol Pathol,2003,56 (3):156-161
[23]Jeannie QH, Suriya KS, Jason RK, Brian Z, Genhong C. Specificity of TRAF3 in its negative regulation of the noncanonical NF-κB pathway[J]. J Biol Chem,2007,282 (6):3688-3694
[24]Krajewska M, Krajewski S, Zapata JM, et al. TRAF-4 expression in epithelial proge-nitor cells. Analysis in normal adult, fetal, and tumor tissue[J]. Am J Pathol.,1998, 152(6):1549-61
[25]Abell AN, Johnson GL. MEKK4 is an effector of the embryonic TRAF4 for JNK activation [J]. J Biol Chem,2005,280(43):35793-35796
[26]Esparza EM, Lindsten T, Stockhausen JM, et al. Tumor necrosis factor recapto r (TNFR) associated factor5 is a critical intermediated of costimulatory signali-ng pathways triggered by glucocoticoid-indeced TNFR in cells[J]. J Biol Chem,2006, 281(13):8559-8564
[27]Betty L, Alejandro DC, William KW, et al. The RING Domain and First Zinc Finger of TRAF6 Coordinate Signaling by Interleukin-1, Lipopolysaccharide, and R-ANKL [J]. J Biol Chem,2008,283(36):24871-24880
[28]Xu L G, Li L Y and Shu H B. TRAF7 potentiates MEKK3-induced API and CH-OP activation and induces apoptosis [J]. J Biol Chem,2004,279(17):17278-17282
[29]Hiroki Y, Hirofunmi J, Hirofumi K, et al. The tumor suppressor cylinderomatosi-s(CYLD) acts as a negative regulator for tool-like receptor 2 signaling via negative cross-talk with TRAF6 and TRAF7[J]. J Biol Chem,2005,280(49):41111-41121
[30]He L, Wu X, Siegel R, et al. TRAF6 regulates cell fate decisions by inducing cas-pase 8-dependent apoptosis and the activation of NF-kappaB[J]. J Biol Chem,2006, 281(16):11235-11249
[31]Henriette W, Anne B, Monika D, Perdita H, et al. Arabidopsis AtCUL3a and AtCUL-3b from complexes with members of the BTB/POZ-MATH protein family[J]. Plant Physiology,2005,137:83-93
[32]Tatjana P, Bationa S, Pham HG, et al. Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant-micro-be interactions and involes early plant protein modifications in the endoplasmic reti-culum and at the plasma memebrane[J]. Physiologia Plantarum,2004,122:465-477
[33]Ralf O, Tatjana P, Bationa S, et al. MATH domain proteins represent a novel protein family in Arabidopsis thaliana, and at least one member is modified in roots during the course of a plant-microbe interaction[J]. Physiologia Plantarum,2005,124:152-166
[34]Jane G, Sally WR, Mike D, Paul D, John CR. A unique family of proteins associated with internalized membranes in protein storage vacuoles of the Brassi-caceae[J]. The Plant Journal,2005,41:429-441
[35]Guo S,Kemphues KJ.Par-l,a gene required for establishing polarity in C.elehans embryos, enoodes a putative Ser/Thr kinase that is asymmetrically distributed [J]. Cell,1995,81:611-620
[36]Fire A, Xu SQ, Montgomery MK, et al. Potent and specific enetic interference by dou-ble-stranded RNA in Caenorhabditiselegans[J]. Nature,1998,391 (6669):806-811
[37]Senthil S, Madge YG, Qliver Y, Terrence L. RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and enhan-ced susceptibility to phytophthora sojace[J]. Plant Physiology,2005,137 (4):1345-1353
[38]Jens H, Sayda ME, et al. Identification of essential gene in cultured mammalian cells using small interfering RNAi[J]. Cell Science,2001,114(24):14557-4565
[39]Liu L, Sun BZ, Liang YM, et al.The effect of telomerase anti-sense RNA on telomere-ase activity and cell proliferation of HL-60. Cancer Research on prevention and treatment,2001,28 (1):4-6
[40]Willians RW, Rubin GW. Argonarte 1 is required for efficient RNA interference in colon cancer [J]. PNAS,2003,99(5):6889-6894
[41]Stege A, Priebsch A, Nietht C, et al. Stable and complete overcoming of MDR1/P-gly coprote-in-mediated multi drug resistance in human gastric carcinomal cells by RNA interference cancer [J]. Gene Therapy,2004, 11(11):699-706
[42]Giladi H, Ketzinel, Gilad M, Rivkinl. Small interfering RNA inhibits virus replication in mice [J]. Molecular Therapy,2003,85(1):769
[43]Yang D, Sun B, Zhang Z, et al. Interference of hepatitis B virus expression and replication by RNA interference[J]. Surgers,2003,114(2):257
[44]Deng J, Yang YL, Fu SH, et al. Inhibition of XJ-160 virus replication by RNAi[J]. Chin J Microbio Immunol,2005,25(6):496-500
[45]Tenllado F, Diaz-Ruiz JR. Double-stranded RNAi mediated interference with plant virus infection [J]. J Virol,2001,75:12288-12297
[46]Andika IB, Kondo H, Tamada T. Evidence that RNA silencing-mediated resistance to beet necrotic yellowvein virus is less effective in roots than in leaves[J]. MPMI,2005, 18(3):194-204
[47]Ogita S, Uefuji H, Yamaguchi Y, et al. Producing decaffeinated coffee plants [J]. Na-ture,2003,424(6949):823
[48]Shimada T, Otanim, Hamad T, et al. Increase of amylase content of sweetpotato by RNA interference of the starch branching enzyme Ⅱ gene(IbSBE Ⅱ)[J]. Plant Biotechnol,2006,23:85-90
[49]Liu YS, Roof S, Ye ZB,et al.Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. PNAS,2004,101:387-397
[50]Qi P, Yan H, Ran W, Yuan Z, Chunyun G, Ying R, Chunlin L. Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds[J]. Plant Cell Rep,2010,29:317-325
[51]Inoue H, Nojima H, and Okayama H. High efficiency transformation of Escherichia coli with plasmids [J]. Gene,1990,96:23-28
[52]Sambrook J, and Russell D. Molecular Cloning---A laboratory manual, third edition [M]. Cool Harbor Laboratory Press,2001.《分子克隆实验指南》第三版,科学出版社,2002
[53]P.马利加,D.F.克莱森,W.格瑞森姆等著.刘进元,吴庆余译.植物分子生物学实验指南[M].科学出版社,2000
[54]彭琦.Fad2和Fae1基因RNA双干扰载体的构建与转化甘蓝型油菜的研究,2007,硕士学位论文,湖南农业大学农学院,导师：刘春林
[55]刘青,金岩,吴织芬,刘源,邢向辉.TRAF-6真核表达载体的构建和转染牙龈上皮细胞[J].牙体牙髓牙病学杂志,2005,15(6)：305-308
[56]王妮丹,王晶,潘玉玲,秦娜林,庞艳,朱建华,刘涛,张述,李卓娅.TRAF1-DsRed真核表达载体的构建及其在cos-7细胞中的定位[J].免疫学杂志,2007,23(5)：491-493
[2]Ohlrogge JB, Browse JA. Lipid biosynthesis[J]. Plant Cell,1995,7:957-970
[3]Thelen JJ, Ohlrogge JB.Metabolic engineering of fatty acid biosynthesis in plants [J]. Metab Eng,2002,4(1):12-21
[4]Pollard MR, Anderson L, Fan C, et al. Aspecific acyl-ACP thioesterase implicated in medium chain fatty acid production immature cotyledons of Umbellularia California [J]. Archives of biochemistry and biophysics,1991,284:306-412
[5]Knutzon DS, Lardizabal KD, Nelsen JS, Bleibaum JL, Davies HM, Metz JG. Cloning of a coconut endosperm cDNA encoding al-acyl-sn-glycerol-3-phosphate acyltransfera-se that accepts medium-chain-length substractes[J]. Plant Physiol,1995,109(3):999-1006
[6]Voelker TA, Hayes TR, Cranmer AM, Turner JC, Davies HM. Genetic engineering of a quantitative trait:metabolic and genetic parameters influencing the accumulation of laurate in rapeseed[J]. Plant J,1996,9:229-241
[7]Lassner MW, Levering CK, Davies HM, Knutzon DS. Lysophosphatidic acid acyltrans-ferase from meadowfoam mediates insertion of erucic acid at the sn-2 positon of tracy-lglycerol in transgenic raeseed oil [J]. Plant Physiol,1995,109:1389-1394
[8]Knutzon DS, Hayes TR, Wyrick A, Xiong H, Maelor DH, Voelker TA. Lysophosphati-dic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels [J]. Plant Physiol,1999,120(3):739-746
[9]Wang YP, Sonntag K, Rudloff E, Han J. Production of fertile transgenic Brassica napus by Agrobacetium-mediated transformation of protoplasts [J]. Plant Breed,2005, 124:1-4
[10]Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, Heinz E. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constri-nts on their accumulation [J].Plant Cell,2004,16:2734-2748
[11]Qi B, Fraser T, Mugford S, Dobson G, Sayanova O, Butler J, Napier JA, Stobart AK, Lazarus CM. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants [J]. Nat Biotechnol,2004,22:739-745
[12]Bession F, Koo AJ, Ruuska S, Schwender J, Pollard M, Thelen JJ, Paddock T, Salas JJ, et al. Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database [J].Plant Physiol,2003,132(2):681-697
[13]彭琦,胡燕,杜培粉,谢青轩,阮颖,刘春林.甘蓝型油菜种子不同发育时期SSH文库的构建.作物学报,2009,35(9)：1-8
[14]Chung JY,Park YC,Ye H,et al.All TRAFs are not created equal:Common and distinct molecular mechanisms of TRAF-mediated signal transduction[J]. J Cell Sci,2002, 115:679-688
[15]Zapata JM.TNF-receptor-associated factors as targets for drug development [J]. Ex-pert Opin Ther Targets,2003,7(3):411-425
[16]Bradley JR,Pober JS.Tumor necrosis factor receptor-associated factor(TRAFs) [J]. Oncogene,2001,20:6482-6491
[17]Biship GA.The multifaceted roles of TRAFs in the regulation of B-cell function [J]. Nat Rev Immunol,2004,4(10):775-786
[18]Uren AG,Vaux DL.TRAF proteins and meprins share a conserved domain[J]. Tren-ds Biochem Sci,1996,21(7):244-245
[19]Sunnerhagen M,Pursglove S,Fladvad M.The new MATH:Homology suggests shared binding surfaces in meprin tetramers and TRAF trimers[J]. FEBS Lett,2002,530(1-3):1-3
[20]Yang P,Wiser JL, Peairs JJ, et al. Human RPE expression of cell survival factors[J]. Invest Ophthalmol Vis Sci,2005,46(5):1755-1764
[21]王承兴,邓锡云,李晓艳等.TRAF1在鼻咽癌及癌旁组织中的表达[J].癌症,2000,19(9)：857-859
[22]Siegler G, Kremmer E, Gonnella R,et al.Epstein-Barr virus encoded latent mem-brane protein 1(LMP1) and TNF receptor associated factors(TRAF):colocalisation of LMP1 and TRAF1 in primary EBV infection and in EBV associated Hodgkin lymph-oma[J]. Mol Pathol,2003,56 (3):156-161
[23]Jeannie QH, Suriya KS, Jason RK, Brian Z, Genhong C. Specificity of TRAF3 in its negative regulation of the noncanonical NF-κB pathway[J]. J Biol Chem,2007,282 (6):3688-3694
[24]Krajewska M, Krajewski S, Zapata JM, et al. TRAF-4 expression in epithelial proge-nitor cells. Analysis in normal adult, fetal, and tumor tissue[J]. Am J Pathol.,1998, 152(6):1549-61
[25]Abell AN, Johnson GL. MEKK4 is an effector of the embryonic TRAF4 for JNK activation [J]. J Biol Chem,2005,280(43):35793-35796
[26]Esparza EM, Lindsten T, Stockhausen JM, et al. Tumor necrosis factor recapto r (TNFR) associated factor5 is a critical intermediated of costimulatory signali-ng pathways triggered by glucocoticoid-indeced TNFR in cells[J]. J Biol Chem,2006, 281(13):8559-8564
[27]Betty L, Alejandro DC, William KW, et al. The RING Domain and First Zinc Finger of TRAF6 Coordinate Signaling by Interleukin-1, Lipopolysaccharide, and R-ANKL [J]. J Biol Chem,2008,283(36):24871-24880
[28]Xu L G, Li L Y and Shu H B. TRAF7 potentiates MEKK3-induced API and CH-OP activation and induces apoptosis [J]. J Biol Chem,2004,279(17):17278-17282
[29]Hiroki Y, Hirofunmi J, Hirofumi K, et al. The tumor suppressor cylinderomatosi-s(CYLD) acts as a negative regulator for tool-like receptor 2 signaling via negative cross-talk with TRAF6 and TRAF7[J]. J Biol Chem,2005,280(49):41111-41121
[30]He L, Wu X, Siegel R, et al. TRAF6 regulates cell fate decisions by inducing cas-pase 8-dependent apoptosis and the activation of NF-kappaB[J]. J Biol Chem,2006, 281(16):11235-11249
[31]Henriette W, Anne B, Monika D, Perdita H, et al. Arabidopsis AtCUL3a and AtCUL-3b from complexes with members of the BTB/POZ-MATH protein family[J]. Plant Physiology,2005,137:83-93
[32]Tatjana P, Bationa S, Pham HG, et al. Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant-micro-be interactions and involes early plant protein modifications in the endoplasmic reti-culum and at the plasma memebrane[J]. Physiologia Plantarum,2004,122:465-477
[33]Ralf O, Tatjana P, Bationa S, et al. MATH domain proteins represent a novel protein family in Arabidopsis thaliana, and at least one member is modified in roots during the course of a plant-microbe interaction[J]. Physiologia Plantarum,2005,124:152-166
[34]Jane G, Sally WR, Mike D, Paul D, John CR. A unique family of proteins associated with internalized membranes in protein storage vacuoles of the Brassi-caceae[J]. The Plant Journal,2005,41:429-441
[35]Guo S,Kemphues KJ.Par-l,a gene required for establishing polarity in C.elehans embryos, enoodes a putative Ser/Thr kinase that is asymmetrically distributed [J]. Cell,1995,81:611-620
[36]Fire A, Xu SQ, Montgomery MK, et al. Potent and specific enetic interference by dou-ble-stranded RNA in Caenorhabditiselegans[J]. Nature,1998,391 (6669):806-811
[37]Senthil S, Madge YG, Qliver Y, Terrence L. RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and enhan-ced susceptibility to phytophthora sojace[J]. Plant Physiology,2005,137 (4):1345-1353
[38]Jens H, Sayda ME, et al. Identification of essential gene in cultured mammalian cells using small interfering RNAi[J]. Cell Science,2001,114(24):14557-4565
[39]Liu L, Sun BZ, Liang YM, et al.The effect of telomerase anti-sense RNA on telomere-ase activity and cell proliferation of HL-60. Cancer Research on prevention and treatment,2001,28 (1):4-6
[40]Willians RW, Rubin GW. Argonarte 1 is required for efficient RNA interference in colon cancer [J]. PNAS,2003,99(5):6889-6894
[41]Stege A, Priebsch A, Nietht C, et al. Stable and complete overcoming of MDR1/P-gly coprote-in-mediated multi drug resistance in human gastric carcinomal cells by RNA interference cancer [J]. Gene Therapy,2004, 11(11):699-706
[42]Giladi H, Ketzinel, Gilad M, Rivkinl. Small interfering RNA inhibits virus replication in mice [J]. Molecular Therapy,2003,85(1):769
[43]Yang D, Sun B, Zhang Z, et al. Interference of hepatitis B virus expression and replication by RNA interference[J]. Surgers,2003,114(2):257
[44]Deng J, Yang YL, Fu SH, et al. Inhibition of XJ-160 virus replication by RNAi[J]. Chin J Microbio Immunol,2005,25(6):496-500
[45]Tenllado F, Diaz-Ruiz JR. Double-stranded RNAi mediated interference with plant virus infection [J]. J Virol,2001,75:12288-12297
[46]Andika IB, Kondo H, Tamada T. Evidence that RNA silencing-mediated resistance to beet necrotic yellowvein virus is less effective in roots than in leaves[J]. MPMI,2005, 18(3):194-204
[47]Ogita S, Uefuji H, Yamaguchi Y, et al. Producing decaffeinated coffee plants [J]. Na-ture,2003,424(6949):823
[48]Shimada T, Otanim, Hamad T, et al. Increase of amylase content of sweetpotato by RNA interference of the starch branching enzyme Ⅱ gene(IbSBE Ⅱ)[J]. Plant Biotechnol,2006,23:85-90
[49]Liu YS, Roof S, Ye ZB,et al.Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. PNAS,2004,101:387-397
[50]Qi P, Yan H, Ran W, Yuan Z, Chunyun G, Ying R, Chunlin L. Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds[J]. Plant Cell Rep,2010,29:317-325
[51]Inoue H, Nojima H, and Okayama H. High efficiency transformation of Escherichia coli with plasmids [J]. Gene,1990,96:23-28
[52]Sambrook J, and Russell D. Molecular Cloning---A laboratory manual, third edition [M]. Cool Harbor Laboratory Press,2001.《分子克隆实验指南》第三版,科学出版社,2002
[53]P.马利加,D.F.克莱森,W.格瑞森姆等著.刘进元,吴庆余译.植物分子生物学实验指南[M].科学出版社,2000
[54]彭琦.Fad2和Fae1基因RNA双干扰载体的构建与转化甘蓝型油菜的研究,2007,硕士学位论文,湖南农业大学农学院,导师：刘春林
[55]刘青,金岩,吴织芬,刘源,邢向辉.TRAF-6真核表达载体的构建和转染牙龈上皮细胞[J].牙体牙髓牙病学杂志,2005,15(6)：305-308
[56]王妮丹,王晶,潘玉玲,秦娜林,庞艳,朱建华,刘涛,张述,李卓娅.TRAF1-DsRed真核表达载体的构建及其在cos-7细胞中的定位[J].免疫学杂志,2007,23(5)：491-493