摘要
启动子在基因表达调控中起关键性作用,它在很大程度上决定目的基因表达的时间、空间和强度。当前商业化应用的植物基因工程产品大多使用的是组成型启动子,它们驱动外源基因在植物各种组织和所有发育阶段表达,这无疑增加了植物的代谢负担导致物质和能量的浪费,另外可能引起影响植物生长发育的植物形态改变,并且组成型表达的筛选标记基因不利于转基因安全。因此克隆组织特异表达启动子对于提高转基因效率及增加转基因安全性有着重要的意义。油菜中目前研究最多的是种子表达启动子,已经克隆了多个种子特异启动子,而对油菜营养器官启动子研究较少。本研究从油菜基因组中克隆茎部特异性表达基因启动子pSH4,并与GFP报告基因融合,转化拟南芥后对其进行功能的初步验证,该研究的结果可以为油菜茎的基因特异表达提供有价值的启动子。本研究的主要研究结果包括以下几个方面:
1、通过筛选拟南芥组织特异表达基因,筛选获得6个茎特异表达基因。
2、用甘蓝型油菜中双9号为材料,在盛花期取其根、茎、叶、花器官等,提取RNA进行启动子的各器官表达相对强度进行分析。结果显示候选基因SH4呈在茎中特异性表达。
3、检索白菜基因组测序序列,获得油菜SH4启动子的序列,设计引物克隆了油菜茎特异性启动子pSH4,长度1779bp。生物信息学分析显示其具有多种顺式作用元件,包括参与茎组织特异性表达的元件如CCA1ATLHCB1、DOFCOREZM等,激素诱导相关元件ASF1MOTIFCAMV、LTRECOREATCOR15等,以及物理因素诱导的相关元件GT1CORE、GT1CONSENSUS等。
4、为进一步验证SH4的功能,成功构建2个植物表达载体:pBI121-SH4-GFP,携带pSH4启动子驱动GFP基因的表达盒,由农杆菌介导转化拟南芥,获得转基因植株,观察T1代植株的GFP的表达情况,结果表明pSH4启动子具有维管束组织表达特异性,是理想的茎表达启动子。
本研究对pSH4启动子的分离和组织特异性及其序列特征的生物信息学、表达分布等方面的分析,为深入研究和利用该启动子提供了试验依据和理论基础。
另外,本研究构建一种转基因作物通用阳性质粒分子,为转基因生物筛查检测和安全监管服务。通过分析全球商业化种植的转基因作物分子特征,选取常用的调控元件启动子CaMV 35S、FMV35S、nos,终止子NOS 3'、T-35S、g7 3'、E9 3',和标记基因bar、NPT II、hpt II、pmi作为筛查靶标序列,通过重组PCR技术将这些元件克隆到双元载体pBI121上,构建成包含11个转基因元件的通用阳性质粒分子pBI121-ELEMENTS。质粒序列信息已提交至GeneBank并获得序列号HM047294。该质粒分子对目前国内外批准的5类主要转基因作物(大豆、油菜、玉米、棉花和水稻)理论筛查覆盖率达到91.5%,对我国批准进口的27种转基因作物的理论筛查覆盖率达100%。以pBI121-ELEMENTS为阳性对照,筛查7个转基因品系,结果显示该分子能够有效用于转基因作物定性筛查。
Promoter plays a key role in the regulation of gene expression. It is important to achieve temporal and tissue-specific expression of the foreign gene(s) through promoter control. Some constitutive promoters such as the cauliflower mosaic virus (CaMV) 35S promoter and maize ubiquitin are widely used in the current commercial genetic engineering products, and usually result in high and constitutive expression of the genes in host plants. However, constitutive expression of the foreign gene(s) could be harmful to the growth and development of plants because overexpression can lead to metabolic stress. Therefore, application of tissue-specific promoter may increase the safety of genetically modified crops and has an important significance to expression efficiency of target gene. Several seed-specific promoters have been cloned from rape, but few promoters with vegetative organs specificity have been well characterized. In this study, a promoter was cloned from the upstream of the stem-specific expression gene SH4 in rapeseed, and integrated with the GFP reporter gene. A preliminary functional analysis of the pSH4 was made after transformed into Arabidopsis thaliana. The results would provide a valuable promoter for genetic engineering of rapeseed. The results were obtained as follows:
1. By screening tissue-specific gene from Arabidopsis thaliana, 6 stem-specific genes were selected as candidates.
2. From the rapeseed material Zhongshuang No.9 (in full bloom), RNA was extracted from 8 organs (root, stem, leaf, flower and so on) and quantitatively analysed for the 6 candidate genes. The candidate gene SH4 was shown to express in a stem-specific way.
3. According to the known genome sequence (GenBank: AC189510.1) of B.rapa c.v.pekinensis, a 1779bp pSH4 or the 5’flanking sequence of SH4 was isolated. Bioinformatic analysis shows that pSH4 has a variety of cis-acting elements, including the stem tissue-specific elements CCA1ATLHCB1, DOFCOREZM, the hormones-inducable elements ASF1MOTIFCAMV, LTRECOREATCOR15 , physical induced elements GT1CORE、GT1CONSENSUS.
4. To characterize the expression features of pSH4, plant expression vectors pBI121-pSH4-GFP was successfully constructed and used to transform Arabidopsis thaliana through the Agrobacterium-mediated method and with the pBI121-GFP as control. We observed the expression of GFP in T1 progeny, indicating that pSH4 is a vascular tissue-specific promoter.
This study involved in the isolation and signal scan analysis of pSH4, verification of its tissue-specificity. All these data will give valuable materials and theoretical guidance to study and applification of pSH4 promoter.
Genetically modified organisms (GMO) detection is essential for biosafety management but lack of positive control, which become a challenge and a bottleneck of GMO detection. In order to provide a general positive control for GMO screening, plasmid pBI121-ELEMENTS was constructed and confirmed. The pBI121-ELEMENTS contained 11 target elements including promoters (nos, CaMV 35S, FMV35S), terminators (NOS 3', T-35S, g7 3', E9 3') and selective markers (bar, NPT II, hpt II and pmi).The plasmid sequence was submitted to GeneBank and Accession number HM047294 received. These elements covered 91.5% of the worldwide approved transgenic events of major crops including soybean, oilseed rape, maize, cotton and rice. They also covered 100% of transgenic events that China approved. The positive plasmid was used to detect seven related elements and the results showed its effectiveness for GMO screening.
引文
[1] Breamthnach R, Chambon P. Organization and expression of eukaryotic split genes coding for proteins. Ann.Rev. Biochem, 1981, 50:349-383
[2] Lagrange T, Gauvin S, Mache R, et al. S2F, a leaf-specific trans-acting factor, binds to a novel cis-acting element and differentially activates the RPL21 gene. Plant cell, 1997,9:1469-1479
[3] Joshi C P. An inspection of the domain between putative TATA-box and translation start site in 79 plant genes.Nucleic.Acid.Res, 1987, 15:6643-6653
[4] Weis L,Reinberg D.Transcription by RNA polymeraseII:Initiator-directed formation of transcription-competent complexes. FASEB J, 1992, 6:3300-3309
[5]张春晓,王文棋,蒋湘宁,陈雪梅,植物基因启动子研究进展.遗传学报,2004,31(12):1455-1464
[6] Sun L.Cai H.Xu W.Hu Y, Lin Z CaMV 35S promoter directs beta- glucuronidase expression in Ganoderma lucidum and Pleurotus citrinopileatus. Mol Biotechnol, 2002,20(3):239-244
[7] Odell J T, Nagy F, Chua N H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter [J]. Nature, 1985, 313(6005):810-812
[8] Sandhu J S, Webster C I, Gray J C. A/T-rich sequences act as quantitative enhances of gene expression in transgenic tobacco and potato plants [J]. Plant Mol Bio, 1998,37(5):885–896
[9] Benfey P N, Ren L, Chua N H. Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J, 1990,9(6):1677-1684
[10] Mitsuhara I, Ugaki M, Hirochika H, et al. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants[J]. Plant Cell Physiol, 1996, 37(1):49-59
[11] Verdaguer B, de Kochko A, Fux C I, et al. Functional organization of the cassava vein mosaic virus (CsVMV) promoter. Plant Mol Biol, 1998, 37(6):1055-1067
[12] Mcelroy D, Zhang W G, Cao J. Isolation of an efficient actin promoter for use in rice transformation. Plant cell, 1990, 2:163-171
[13] Streatfield S J, Beifuss K K, Brooks C A, et al. Analysis of the maizepolyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics. Transgenic Res, 2004, 13(4):299-312
[14] Leisner, S.M., and Gelvin, S.B. Structure of the octopine synthase upstream activator sequence. Proc. Natl. Acad. Sci. USA 1988,85:2553-2557
[15] Leisner, S.M., and Gelvin, S.B. Multiple domains exist within the upstream activator sequence of the octopine synthase gene.Plant Cell, 1989,1:925-936
[16] Bouchez, D., Tokuhisa, J.G., et al. The ocs-element is a component of the promoters of severa1 T-DNA and plant vira1 genes. EMBO J, 1989, 8:4197-4204
[17] Yu D.,Chen C.,and Chen Z.Evidence for an important role of WRKY DNA binding proteins in theregulation of NPR1 gene expression.Plant Cell, 2001,13:1527-1540
[18] Rushton P.J.,Reinstadler A.,Lipka V.,Lippok B.,and Somssich I.E.Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen-and wound-induced signaling.Plant Cell 2002,14:749-762
[19] Manavella P.A.,Dezar C.A.,Ariel F.D.,and Chan R.L.Two ABREs,two redundant root-specific and one W-box cis-acting elements are functional in the sunflower HAHB4 promoter.Plant Physiol Biochem 2008,46:860-867
[20] Menke F.L.,Champion A.,Kijne J.W.,and Memelink J.A novel jasmonate-and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate and elicitor-inducible AP2-domain transcription factor,ORCA2.EMBO J 1999,18:4455-4463
[21] Boter M.,Ruiz-Rivero O.,Abdeen A.,and Prat S.Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis thaliana.Genes Dev 2004,18:1577-1591
[22] Xu B.,and Timko M. Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements.Plant Mol Biol 2004, 55:743-761
[23] Vom Endt D.,Soares e Silva M.,Kijne J.W.,Pasquali G.,and Memelink J.Identification of a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3transcription factor gene that interacts specifically with AT-Hook DNA-binding proteins.PlantPhysiol 2007,144:1680-1689
[24] Liu Z.B.,Ulmasov T.,Shi X.,Hagen G.,and Guilfoyle T.J.Soybean GH3 promoter contains multiple auxin-inducible elements. Plant Cell ,1994,6:645-657
[25] Guo H.,and Ecker J.R. The ethylene signaling pathway:new insights. Curr Opin Plant Biol, 2004,7:40-49
[26] Seki M.,Narusaka M.,et al. Monitoring the expression profiles of 7000 Arabidopsis thaliana genesunder drought,cold and high-salinity stresses using a full-length cDNA microarray. Plant J, 2002,31:279-292
[27] Shen Q.J.,Casaretto J.A.,Zhang P.,and Ho T.H. Functional definition of ABA-response complexes:the promoter units necessary and sufficient for ABA induction of gene expression in barley(Hordeum vulgare L.).Plant Mol Biol, 2004,54:111-124
[28] Hobo T.,Asada M.,Kowyama Y.,and Hattori T.ACGT-containing abscisic acid response element(ABRE) and coupling element 3(CE3) are functionally equivalent.Plant J ,1999,19:679-689
[29] Ballas N.,Wong L.M.,Ke M.,and Theologis,A.Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.Proc Natl Acad Sci USA ,1995,92:3483-3487
[30] Ulmasov T.,Liu Z.B.,Hagen G.,and Guilfoyle T.J.Composite structure of auxin response elements.Plant Cell,1995,7:1611-1623
[31] Qin X.F.,Holuigue L.,Horvath D.M.,and Chua N.H. Immediate early transcription activation by salicylic acid via the cauliflower mosaic virus as-1 element. Plant Cell,1994,6:863-874
[32] Fukuda Y.,and Shinshi H.Characterization of a novel cis-acting element that is responsive to afungal elicitor in the promoter of a tobacco class I chitinase gene.Plant Mol Biol 1994,24: 485-493
[33] Rushton P.J.,Torres J.T.,Parniske M.,Wernert P.,Hahlbrock K.,and Somssich I.E. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.EMBO J,1996,15:5690-5700
[34] Warner S.A.,Gill A.,and Draper J.The developmental expression of the asparagus intracellular PR protein(AoPR1)gene correlates with sites of phenylpropanoid biosynthesis.Plant J,1994,6:31-43
[35] Despres C.,Subramaniam R.,Matton D.P.,and Brisson N.The activation of the potato PR-10a gene requires the phosphorylation of the nuclear factor PBF-1.Plant Cell 1995,7:589-598
[36] Raventos D.,Jensen A.B.,Rask M.B.,Casacuberta J.M.,Mundy J.,and San Segundo B.A 20 bp cis-acting element is both necessary and sufficient tomediate elicitor response of a maize PRms gene.Plant J,1995,7:147-155
[37] Strompen G.,Gruner R.,and Pfitzner U.M.An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobacco.Plant Mol Biol,1998,37:871-883
[38] Toquin V.,Grausem B.,Geoffroy P., Legrand M.Structure of the tobacco caffeic acid O-methyltransferase(COMT)II gene:identification of promoter sequences involved in gene inducibility by various stimuli. Plant Mol Biol ,2003,52:495-509
[39] Grimmig B., Matern U. Structure of the parsley caffeoyl-CoA O-methyltransferase gene, harbouring a novel elicitor responsive cis-acting element.Plant Mol Biol,1997,33:323-341
[40] Niu X, Bressan RA, Hasegawa PM et al. Ion homeostasis in NaCl stress environments. Plant Physiol, 1995,109:735-742
[41] Flowers TJ, Troke PF, Yeo AR. The mechanism of salt tolerance in halophytes. Annual Review of Plant Physiology,1977,28: 89-121
[42] Greenway H., Munns R. .Mechanisms of salt tolerance in non-halophytes, Ann. Rev. Plant Physiol., 1980, 31: 149-190
[43] H. Giese and H.E. Hopp, Influence of nitrogen nutrition on the amount of hordein, protein Z andβ-amylase messenger RNA in developing endosperms of barley, Carlsberg Research Communication,1984, 49:365–383
[44] Singletary GW, Doehlert DC, Wilson CM, Muhitch MJ ,Below FE .Response of enzymes and storage proteins of maize endosperm to nitrogen supply. Plant Physiol. 1990, 94: 858–864
[45] Park HC, Shin J, Appel B. Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling. Development , 2004,131:5959–5969
[46] Qi, X., Zhang, Y. and Chai, T. Characterization of a novel plant promoter specifically induced by heavy metal and identification of the promoter regions conferring heavy metal responsiveness.Plant Physiol. 2007,143:50-59
[47] Müller M, Knudsen S. The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box.Plant J,1993,6:343-355
[48] Boaz Kaplan,Olga Davydov, Heather Knight,et al . Rapid Transcriptome Changes Induced by Cytosolic Ca2+ Transients Reveal ABRE-Related Sequences as Ca2+-Responsive cis Elements in Arabidopsis thaliana.The Plant Cell , 2006,18:2733-2748
[49] Kobayashi T.,Nakayama Y.,Itai R.N.,Nakanishi H.,Yoshihara T.,Mori S., Nishizawa N.K.Identification of novel cis-acting elements,IDE1 and IDE2,of the barley IDS2 gene promoter conferring iron-deficiency-inducible, root-specific expression in heterogeneous tobacco plants. Plant J,2003,36:780-793
[50] Logemann E.,Parniske M.,and Hahlbrock K.Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley.Proc Natl Acad Sci USA,1995,92:5905-5909
[51] Loake G.J., Faktor O., Lamb C.J., Dixon R.A. Combination of H-box [CCTACC(N)7CT] and G-box(CACGTG)cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid.Proc Natl Acad Sci USA,1992,89:9230-9234
[52] Takeda S., Sugimoto K.,Otsuki H. ,and Hirochika H. A 13-bp cis-regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture,wounding,methyl jasmonate and fungal elicitors.Plant J,1999,18:383-393
[53] Sugimoto K.,Takeda S.,and Hirochika H.Transcriptional activation mediated by binding of a plant GATA-type zinc finger protein AGP1 to the AG-motif(AGATCCAA)of the wound-inducible Myb gene NtMyb2. Plant J 2003,36:550-564
[54] Sugimoto K.,Takeda S., Hirochika H. MYB-related transcription factor NtMYB2 induced by wounding and elicitors is a regulator of the tobacco retrotransposon Tto1 and defense-related genes.Plant Cell,2000,12:2511-2528
[55] Asao H., Yoshida K., Nishi Y., Shinmyo A. Wound-responsive cis-element in the 5'-upstream region of cucumber ascorbate oxidase gene.Biosci Biotechnol Biochem,2003,67:271-277
[56] Sasaki K., Ito H., Mitsuhara I., Hiraga S.,Seo S., Matsui H., Ohashi Y. A novel wound-responsive cis-element, VWRE, of the vascular system-specific expression of a tobacco peroxidase gene,tpoxN1. Plant Mol Biol , 2006,62:753-768
[57] Nishiuchi T., Shinshi H., Suzuki K. Rapid and transient activation of transcription of the ERF3 gene by wounding in tobacco leaves: possible involvement of NtWRKYs and auto repression. J Biol Chem, 2004,279:55355-55361
[58] Takumi S,Koike A,Nakata M,et al. Cold-specific and light-stimulated expression of a wheat (Triticum aestivum L.) Cor gene Wcor15 encoding a chloroplast-targeted protein. Journal of Experimental Botany, 2003,54:2265-2274
[59] Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana thaliana CBF1 encodes an AP2 domain–containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to temperature and water deficit. Proc. Natl. Acad. Sci. USA, 1997,94:1035-1040
[60]张海利,吕淑霞,田颍川.韧皮部特异性启动子研究概述.中国生物工程杂志.2003,23(11):11-15
[61] Yin Y, Chen L ,Beachy R. Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice. Plant J ,1997 ,12 5:1179-1188
[62] Hehn A , Rohde W. Characterization of cis2acting elements affecting strength and phloem specificity of the coconut foliar decay virus promoter. J Gen Virol ,1998 ,79 (6) :1495-1499
[63] Medberry S L , Olszewski N E. Identification of cis elements involved in Commelina yellow mottle virus promoter activity. Plant J ,1993 ,3 (4) :619-626
[64] Yang, N. S. Russell, D. Maize sucrose synthase-1 promoter directs phloem cell-specific expression of GUS gene in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA , 1990,87: 4144-4148
[65] Keller, B., Heierli, D.. Vascular expression of the GRP1.8-promoter is controlled by 3 specific regulatory elements and one unspecific activating sequence. Plant Mol. Biol. 1994,26:747-756
[66] Ringli C, Keller B. Specific interaction of the tomato bZIP transcription factor VSF-1 with a non-palindromic DNA sequence that controls vascular gene expression. Plant Molecular Biology ,1998.37, 977–988
[67] Liu Yuhui, Wang Zhixing, Jia Shirong. Effect of 5’-deletion of Arabidopsis thaliana profilin2 promoter on its vascular specific expression. Chinese Science Bulletin, 2001,46 (19):1627-1630
[68] Vicente-Carbajosa J, Moose S P, Parsons R L,et al. A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.Proc Natl Acad Sci U S A,1997,94(14):7685-7690
[69] Yoshihara T, Washida H, Takaiwa F. A 45-bp proximal region containing AACA and GCN4 motif is sufficient to confer endosperm-specific expression of the rice storage protein glutelin gene, GluA-3. FEBS Lett, 1996, 383(3) :213-218
[70] Fiedler U, Filistein R, Wobus U, et al. A complex ensemble of cis-regulatory elements controls the expression of a Vicia faba non-storage seed protein gene. Plant Mol Biol, 1993,22:669-679
[71] De Pater S, Pham K, Chua N H, et al. A 22-bp fragment of the pea lectin promoter containing essential TGAC-like motifs confers seed-specific gene expression. Plant Cell, 1993,5(8):877-886
[72]王莹,韩烈保,曾会明.高等植物启动子克隆方法的研究进展.生物技术通报,2007,3:97-100
[73]刘次桃.利用水稻启动子捕获系筛选启动子的研究.广州,华南师范大学, 2007
[74] De Raffaele M, Montini E, Santoni de Sio F R, et al. Promoter Trapping Reveals Significant Differences in Integration Site Selection Between MLV and HIV Vectors in Primary Hematopoietic Cells. Blood, 2005, 105 (6) : 2307-2315.
[75] Markus Kiechle, PalaniyandiManivasakam1, Friederike Eckardt- Schupp, et al. Promoter - trapping in Saccharomyces cerevisiae by Radiation - Assisted Fragment Insertion. Nucleic Acids Research, 2002, 30 (24) : 1-6.
[76] Hudson ME, Quail PH. Identification of promoter motifs involved in the network of phytochrome A-regulated gene expression by combined analysis of genomic sequence and microarray data. Plant Physiol,2003,133: 1605-1616
[77] AWAZUHARA Motoko, et al.A 235-bp region from a nutritionally reglated soybean seed-specific gene promoter can confer its sulfur and nitrogen response to a constitutive promoter in aerial tissues of Arabidopsis thaliana traliana. Plant Science ,2002, 163(1):75-82
[78] Li JJ, Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 1993;262:1870-1873
[79] Sergiy Lopato, Natalia Bazanova, et al. Isolation of plant transcription factors using a modified yeast one-hybrid system. Plant Methods. 2006; doi: 10.1186/1746-4811-2-3.
[80] David A. Christopher, Yanxin Shen, et al. Expression of a higher-plant chloroplast psbD promoter in a cyanobacterium (Synechococcus sp. strainPCC7942) reveals a conserved cis-element, designated PGT, that differentially interacts with sequence-specific binding factors during leaf development. Current Genetics, 1999,35: 657–666
[81] Kuo-Chih Lin, David Shiuan. A simple method for DNaseI footprinting analysis. Journal of Biochemical and Biophysical Methods, 1995, 30:85-89
[82]官春云,李枸.转基因油菜应用研究.细胞生物学杂志,1997,19(1):18-22
[83]王贵春,杨光圣.油菜高含油量育种研究进展.安徽农业科学, 2007, 35(18):5373-537
[84]杨仲毅,王兰岚,宋桂英,等.利用微束激光穿刺法将菜豆几丁质酶基因导入油菜的研究[J].光生物学报,1999,8:1-5
[85]谢莲萍.转基因植物的研究进展[J].安徽农业科学, 2009, 37(22): 10 384-10 386,10 412.
[86] Clive James. 2008年全球生物技术/转基因作物商业化发展态势[J].中国生物工程杂志,2009,29(2):1-10.
[87] Matsuoka T, Kuribara H, Akiyama H, et al. A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize [J]. Shokuhin Eiseigaku Zasshi, 2001, 42(1):24-32.
[88]王广印,韩世栋,陈碧华,等.转基因食品的安全性与标识管理[J].食品科学, 2008, 29(11): 667-673.
[89] Holst-Jensen A. Testing for genetically modified organisms (GMOs): Past, present and future perspectives [J].Biotechnology Advances, 2009, 27(6):1 071-1 082.
[90]林清,彭于发.转基因作物及产品检测技术研究进展[J].西南农业学报, 2009, 22(2):513-517.
[91] Hubner P, Waiblinger H U, Pietsch K, et al. Validation of PCR methods for quantitation of genetically modified plants in food[J].J. AOAC Int., 2001, 84(6):1855-1864.
[92] Miraglia M, Berdal K G, Brera C. Detection and traceability of genetically modified organisms in the food production chain[J].Food and Chemical Toxicology, 2004,42:1157-1180.
[93]段武德.转基因植物检测[M].北京:中国农业出版社,2009:12-32
[94] Hardegger M, Brodmann P, Herrmann A. Quantitative detection of the 35S promoter and the Nos terminator using quantitative competitive PCR [J]. Eur Food Res Technol , 1999, 209:83-87.
[95] Kuribara H, Shindo Y, Matsuoka T, et al. Novel reference molecules forquantitation of genetically modified maize and soybean[J]. J AOAC Int. 2002, 85(5):1077-1089.
[96] Corbisier P, Broeders S, Charels D, et al. Certification of plasmidic DNA containing MON 810 maize DNA fragments, ERM?-AD413[R]. EC certification report, 2007,EUR 22948, ISBN 978-92-79-07139-3.
[97] Wolf C, Scherzinger M, Wurz A, et al. Detection of cauliflower mosaic virus by the polymerase chain reaction: testing of food components for false-positive 35S-promoter screening results [J]. Eur Food Res Technol, 2000, 210:367-372.
[98]刘丹,刘贵华,王汉中.油菜抗性相关基因的分离及其基因工程研究进展.中国农业科技导报,2008,10(3):6-11
[99]双桑,阮颖,彭冬平,刘春林.油菜菌核病及抗病育种研究进展.作物研究,2006,5:552-556
[100] Takeshi Obayashi,1 Takashi Okegawa,et al. Distinctive Features of Plant Organs Characterized by Global Analysis of Gene Expression in Arabidopsis thaliana. DNA Research,2004,11:11-25
[101] Rachel A. Burton, David M. Gibeaut. et al. Virus-Induced Silencing of a Plant Cellulose Synthase Gene. Plant Cell, 2000,12:691-706
[102] BAO W,O MALLEYDM,WHETTEN R, et al. A laccase associated with lifgnification in loblolly pine xylem. Science, 1993, 260: 672-674
[103] Ranocha, P.; Chabannes, M.; Chamayou, S, et al. Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar. Plant Physiol, 2002, 129: 145-155
[104]巩万奎.阿拉伯半乳糖蛋白与植物细胞分化.植物生理学通讯,2000,36(4):362-367
[105]赖秋安,胡建军,孙久荣.腺苷酸激酶与细胞凋亡.生物化学与生物物理进展.2001,28(4):444-446
[106] Manzara T, Gruissem W. Organization and expression of the genes encoding ribulose-1,5-bisphosphate carboxylase in higher plants. Photosynth Res ,1988,16:117-139
[107] Donald RGK, Cashmore AR. Mutation of either G box or l box sequences profoundly affects expression from the Arabidopsis thaliana rbcS-1A promoter. EMBO J,1990,9:1717-1726
[108] Wanner LA.Gruissem W Expression dynamics of the tomato rbcS gene family during development The Plant cell 1991;3(12):1289-303
[109] Yanagisawa S, Schmidt RJ.Diversity and similarity among recognition sequences of Dof transcription factors,Plant J, 1999,17:209-214
[110] Yanagisawa S.Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize.Plant J , 2000,21:281-288
[111] Abe M, Takahashi T, Komeda Y. Identification of a cis-regulatory element for L1 layer-specific gene expression, which is targeted by an L1-specific homeodomain protein.Plant J ,2001,26: 487-494
[112] Villain P, Mache R, Zhou DX. The mechanism of GT element-mediated cell type-specific transcriptional control. J Biol Chem,1996, 271:32593-32598
[113] Zhou DX.. Regulatory mechanism of plant gene transcription by GT-elements and GT-factor. Trends in Plant Science,1999,4:210-214
[114]王建林,栾运芳,大次卓嘎,张永青,等.中国栽培油菜的起源和进化.作物研究.2006,3:199-205
[115]张振桢,许煜泉,黄海.拟南芥——一把打开植物生命奥秘大门的钥匙.生命科学,2006,18(05):442-446
[116] Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 2000, 408(6814): 796-815
[117] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana thaliana. Plant J. 1998,16(6):735-743
[118] MIGUEL MARTINEZ-TRUJILLO1, VERONICA LIMONES-BRIONES. Improving Transformation Efficiency of Arabidopsis thaliana thaliana by Modifying the Floral Dip Method. Plant Molecular Biology Reporter. 2004, 22: 63-70
[119]祖元刚,孟庆焕,郭晓瑞,孙佳音,刘红梅.超声破碎拟南芥原生质体后的再生培养初报.植物研究,2005,25(4):441-443
[120]张小莉,王鹏程,宋纯鹏.基于基因枪技术的拟南芥瞬时表达转化方法的建立.河南大学学报, 2008, 38(5 ):506-509
[121]赵华;梁婉琪;杨永华;张大兵;绿色荧光蛋白及其在植物分子生物学研究中的应用.植物生理学通讯, 2003,2 :171-178
[122]王关林,方宏筠.植物基因工程[M].北京:科学出版社,744-746.
[123] Anthony J, Kinney, Kevin L, et al. Soybean event DP-305423-1 and compositions and methods for the identification and/or detection thereof [P].European Patent Application: EP20070861586, 07/15/2009.
[124] Mark A, Dizigan. High lysine maize compositions and event LY038 maize plants [P]. United States Application: US7157281 B2, 01/02/2007.
[125] SN/T1196-2003玉米中转基因成分定性PCR检测方法.
[126] NY/T675-2003转基因植物及其产品检测——大豆定性PCR方法.
[127] SN/T1197-2003油菜籽中转基因成分定性PCR检测方法.
[128] Mattarucchi, E., Weighardt, F., Barbati, C., Querci, M., & Van den Eede, G..Development and applications of real-time PCR standards for GMO quantification based on tandem-marker plasmids [J]. European Food Research and Technology, 2005,221:511-519.
[129] Zbigniew Sieradzki , Krzysztof Kwiatek. Validation of real-time pcr methods for the quantification of genetically-modified maize and soybean[J]. Bull Vet Inst Pulawy 2009,53:741-746.
[130] CHIA-CHIA HUANG, TZU-MING PAN Event-specific real-time detection and quantification of genetically modified roundup ready soybean[J].J. Agric. Food Chem. 2005, 53:3833-3839.
[131] Xiang Li, Kailin Shen, Litao Yang, Shu Wang, Liangwen Pan, Dabing Zhang Applicability of a novel reference molecule suitable for event-specific detections of maize NK603 based on both 50 and 30 flanking sequences. [J]Food Control.2010, 21:927-934
[132] L. YANG, J. GUO, A. PAN, H. ZHANG, K. ZHANG, Z. WANG, D. ZHANG. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule[J] Journal of Agricultural and Food Chemistry. 2007, 55(1): 15-24.
[133] I-Jen Lu, Chih-Hui Lin, Tzu-Ming Pan Establishment of a system based on universal multiplex-PCR for screening genetically modified crops[J] Anal Bioanal Chem.2010,396:2055-2064.
[134] Marcia J. Holden, Marci Levine, Tandace Scholdberg, Ross J. Haynes,G. Ronald Jenkins.The use of 35S and Tnos expression elements in the measurement of genetically engineered plant materials[J]Anal Bioanal Chem.2010,396:2175-2187.