兰花转CiDREB1、PeDREB2、CyMV-CP基因研究
|
摘要
兰科(Orchidaceae)是有花植物中最大的一个科,具极高的观赏价值。在全世界花卉业中,兰花具有重要的经济价值。兰花是整个兰科植物的总称,主要分布于热带、亚热带,性喜温暖潮湿,耐寒性、抗旱性能力较差。近年来兰花广泛受到建兰花叶病毒(Cymbidium Mosaic Virus ,CyMV)等病毒的侵染,导致观赏价值下降。目前国际上还未有防治植物病毒病的有效药剂,而随着世界上兰花贸易的不断扩大,兰花病毒病也开始在不同植株中蔓延,严重影响多种兰花的栽培和生产。因此,培育耐寒、抗旱、抗病毒的兰花新品种已成为兰花新品种培育的主要方向。
本研究以8个种类具有重要价值的兰花(大花蕙兰、中国兰属附生种类等)为材料,应用农杆菌和基因枪两种转化方法进行了转基因研究,将美花兰抗寒基因CiDREB1和毛竹抗旱基因PeDREB2分别转化大花蕙兰、中国兰花等种类兰花;采用实时荧光定量PCR方法对转CyMV-CP基因兰花进行表达量的检测。本研究旨在利用基因工程手段进行兰花抗逆性品质改良,为最终培育耐寒、抗旱、抗病毒兰花新品种奠定理论和实践基础。
1.研究确定了2类兰花的最佳抗生素筛选浓度。表达载体中有新霉素磷酸转移酶基因(NptⅡ)作为筛选标记基因,利用卡那霉素对转化体进行筛选。美花兰( Cymbidium insigne ) MH的卡那霉素最佳筛选浓度为50 mg/L ;树兰(Epidendrum)SL的卡那霉素最佳筛选浓度为15 mg/L。
2.采用农杆菌侵染和基因枪介导的方法,以美花兰抗寒基因CiDREB1基因和毛竹抗旱基因PeDREB2基因为外源目的基因,对美花兰MH,大花蕙兰(Cymbidium)PB、PC,兰属(Cymbidium)兰花F5、F7、C2,树兰SL,卡特兰(Cattleya)K1 8种兰花进行遗传转化,初步得到了抗生素水平的阳性苗,并对转化率分别进行了统计。结果表明,农杆菌侵染法出苗率处于3.13-15.6%之间;基因枪介导的转化出苗率处于1.7-92.2%之间。
3.基因克隆获得了兰花PB、PC肌动蛋白Actin基因部分序列,设计合成了适合于实时荧光定量PCR试验进行基因相对表达量分析的特异性引物(Actin-S/A, CyMV-S/A)。溶解曲线和标准曲线分析结果表明,Actin-S/A引物扩增Actin基因标准曲线相关系数为r2 = 0.994,线性方程为y =-3.271X+19.008,扩增效率E=102.2%;CyMV-S/A引物扩增CyMV-CP基因标准曲线相关系数为r2 = 0.997,线性方程为y =-3.731X+11.993,扩增效率E=85.4%。表明所设计的CyMV-CP基因引物能够用于目的基因相对表达量的检测。
4.利用实时荧光定量PCR方法,以两个种类转CyMV-CP基因大花蕙兰PB、PC PCR检测阳性植株为材料,对CyMV-CP基因在转基因兰花植株体内的表达量进行分析。结果表明,外源基因CyMV-CP在转基因大花蕙兰PB、PC植株体内得到较高水平的表达,表达量明显高于未转基因兰花植株,相对表达量分别是对照植株的14.8倍、100倍。
Orchidaceae is one of the largest families in the flowering plants with high ornamental value. As one of important ornamental plants, orchids have an important commercial importance in world flower industry, and distribute mainly in the tropical, subtropical, liking warm and humid, with poor ability to cold and drought tolerance. In recent years, orchids were seriously infected by Cymbidium Mosaic Virus, leading
to declining of ornamental value. Simultaneously, with the world trade widely expanding, kinds of virus diseases spread in plants, and seriously affect the cultivation and production of orchids.Therefore, antiviral, drought and cold resistant new orchid varieties have become the main direction of orchids breeding.
In this research, Kanamycin was the antibiotic selection of orchids. Eight species of orchids (Cymbidium hybridium, Cattleya, Chinese Cymbidium, etc.) were transformed by bombardment or Agrobacterium mediated transformation, and protocorm-like bodies (PLBs) were used as target explants, with CiDREB1 gene from Cymbidium insigne and PeDREB2 gene from Phyllostachys edulis. Results showed that the germinat rates of transgenic PLBs were between 1.7-92.2% under the antibiotic selection in the MS culture medium with appropriate Kanamycin concentration.
The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was applied to quantitative detect transgenic Cymbidium with CyMV-CP gene. By using RT-PCR method, partial sequence of Actin gene was cloned from Cymbidium. On the base of Actin gene sequence, the primers of Actin-S/A and CyMV-S/A were synthesized, which were suitable for quantitative real-time fluorescent PCR. The plasmid containing the target sequence was constructed to prepare for the standard curve and detect the sensitivity.The melt curve and standard curve analysis results showed that the regression equation of Actin-S/A primers was y = - 3.271X+19.008, and related coefficient: R2= 0.994, ampliative efficiency E=102.2%; and the regression equation of CyMV-S/A primers was y = -3.731X+11.993,and related coefficient: R2= 0.997, ampliative efficiency E=85.4%.It indicated that the primers can be used for detecting the expression of CyMV-CP gene in the transgenic Cymbidium.
The assay showed that the exogenous CyMV-CP genes in transgenic Cymbidium PB, PC plants had higher levels of expression, and the expression level obviously higher than control plants, and the separate relatively expression were 14.8 times and 100 times comparing to the negative Cymbidium.
本研究以8个种类具有重要价值的兰花(大花蕙兰、中国兰属附生种类等)为材料,应用农杆菌和基因枪两种转化方法进行了转基因研究,将美花兰抗寒基因CiDREB1和毛竹抗旱基因PeDREB2分别转化大花蕙兰、中国兰花等种类兰花;采用实时荧光定量PCR方法对转CyMV-CP基因兰花进行表达量的检测。本研究旨在利用基因工程手段进行兰花抗逆性品质改良,为最终培育耐寒、抗旱、抗病毒兰花新品种奠定理论和实践基础。
1.研究确定了2类兰花的最佳抗生素筛选浓度。表达载体中有新霉素磷酸转移酶基因(NptⅡ)作为筛选标记基因,利用卡那霉素对转化体进行筛选。美花兰( Cymbidium insigne ) MH的卡那霉素最佳筛选浓度为50 mg/L ;树兰(Epidendrum)SL的卡那霉素最佳筛选浓度为15 mg/L。
2.采用农杆菌侵染和基因枪介导的方法,以美花兰抗寒基因CiDREB1基因和毛竹抗旱基因PeDREB2基因为外源目的基因,对美花兰MH,大花蕙兰(Cymbidium)PB、PC,兰属(Cymbidium)兰花F5、F7、C2,树兰SL,卡特兰(Cattleya)K1 8种兰花进行遗传转化,初步得到了抗生素水平的阳性苗,并对转化率分别进行了统计。结果表明,农杆菌侵染法出苗率处于3.13-15.6%之间;基因枪介导的转化出苗率处于1.7-92.2%之间。
3.基因克隆获得了兰花PB、PC肌动蛋白Actin基因部分序列,设计合成了适合于实时荧光定量PCR试验进行基因相对表达量分析的特异性引物(Actin-S/A, CyMV-S/A)。溶解曲线和标准曲线分析结果表明,Actin-S/A引物扩增Actin基因标准曲线相关系数为r2 = 0.994,线性方程为y =-3.271X+19.008,扩增效率E=102.2%;CyMV-S/A引物扩增CyMV-CP基因标准曲线相关系数为r2 = 0.997,线性方程为y =-3.731X+11.993,扩增效率E=85.4%。表明所设计的CyMV-CP基因引物能够用于目的基因相对表达量的检测。
4.利用实时荧光定量PCR方法,以两个种类转CyMV-CP基因大花蕙兰PB、PC PCR检测阳性植株为材料,对CyMV-CP基因在转基因兰花植株体内的表达量进行分析。结果表明,外源基因CyMV-CP在转基因大花蕙兰PB、PC植株体内得到较高水平的表达,表达量明显高于未转基因兰花植株,相对表达量分别是对照植株的14.8倍、100倍。
Orchidaceae is one of the largest families in the flowering plants with high ornamental value. As one of important ornamental plants, orchids have an important commercial importance in world flower industry, and distribute mainly in the tropical, subtropical, liking warm and humid, with poor ability to cold and drought tolerance. In recent years, orchids were seriously infected by Cymbidium Mosaic Virus, leading
to declining of ornamental value. Simultaneously, with the world trade widely expanding, kinds of virus diseases spread in plants, and seriously affect the cultivation and production of orchids.Therefore, antiviral, drought and cold resistant new orchid varieties have become the main direction of orchids breeding.
In this research, Kanamycin was the antibiotic selection of orchids. Eight species of orchids (Cymbidium hybridium, Cattleya, Chinese Cymbidium, etc.) were transformed by bombardment or Agrobacterium mediated transformation, and protocorm-like bodies (PLBs) were used as target explants, with CiDREB1 gene from Cymbidium insigne and PeDREB2 gene from Phyllostachys edulis. Results showed that the germinat rates of transgenic PLBs were between 1.7-92.2% under the antibiotic selection in the MS culture medium with appropriate Kanamycin concentration.
The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was applied to quantitative detect transgenic Cymbidium with CyMV-CP gene. By using RT-PCR method, partial sequence of Actin gene was cloned from Cymbidium. On the base of Actin gene sequence, the primers of Actin-S/A and CyMV-S/A were synthesized, which were suitable for quantitative real-time fluorescent PCR. The plasmid containing the target sequence was constructed to prepare for the standard curve and detect the sensitivity.The melt curve and standard curve analysis results showed that the regression equation of Actin-S/A primers was y = - 3.271X+19.008, and related coefficient: R2= 0.994, ampliative efficiency E=102.2%; and the regression equation of CyMV-S/A primers was y = -3.731X+11.993,and related coefficient: R2= 0.997, ampliative efficiency E=85.4%.It indicated that the primers can be used for detecting the expression of CyMV-CP gene in the transgenic Cymbidium.
The assay showed that the exogenous CyMV-CP genes in transgenic Cymbidium PB, PC plants had higher levels of expression, and the expression level obviously higher than control plants, and the separate relatively expression were 14.8 times and 100 times comparing to the negative Cymbidium.
引文
[1]Wang W, Vinocur B , Altman A. Plant responses to drought , salinity and extreme temperatures : towards genetic engineering for stress tolerance [J].Planta , 2003 , 218 :1 -14.
[2]倪志勇,徐兆师,李连城,等.DREB转录因子在植物抗逆胁迫中的作用机理及应用研究进展[J].麦类作物学报,2008 ,28 (6) :1100-1106.
[3]刘强,张贵友,陈受宜.植物转录因子的结构与调控作用[J].中国科学,2000 ,45 :1465-1474.
[4]Riechmann J L, Heard J. Arabidopsis Transcription Factors: Genome-Wide Comparative Analysis Among Eukaryotes [J].Science,2000,290 :2105-2110.
[5]孙啸.大豆抗逆相关转录因子基因GmDREB3的功能分析及启动子的克隆[J].新疆农业大学,2007,5:1-92.
[6]Jofuku K D, Denboer B G W, Vanmontagu M, et al. Control of Arabidopsis Flower and Seed Development by the Homeotic Gene APETALA2[J]. Plant Cell, 1994, 6(9):1211-1225.
[7]Ohme-Takagi, Shinshi H. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element [J].Plant Cell, 1995, 7(2):173-182.
[8]Weigel D. The APETALA2 domain is related to a novel type of DNA binding domain [J].Plant Cell, 1995, 7(4):388-389.
[9]Moose S P, Sisco P H. Glossy15, an APETALA2-like gene from maize that regulates leaf epidermal cell identity[J]. Genes & Development, 1996, 10(23):3018-3027
[10]Gu Y Q, Wildermuth M C, Chakravarthy S, et al. Tomato transcription factors Pti4, Pti5 and Pti6 activate defense responses when expressed in Arabidopsis[J].Plant Cell, 2002,14(4): 817-831.
[11]Riechmann J L, Meyerowitz E M. The AP2/EREBP family of plant transcription factors[J]. Biol. Chem, 1998, 379:633-646.
[12]Krizek B A. AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain[J]. Nucleic Acids Res, 2003, 31: 1859-1868.
[13]Wuitschick J D, Lindstrom P R, Meyer A E, et al. Homing endonucleases encoded by germ line-limited genes in Tetrahymena thermophilahave APETALA2 DNA binding domains[J]. Eukaryot.Cell, 2004, 3:685-694.
[14]Magnani E, Sj lander K, Hake S. From endonucleases to transcription factors: evolution of the AP2 DNA binding domain in plants[J].Plant Cell,2004,16:2265-2277.
[15]Chang C, Shockey J A. The ethylene-response pathway: signal perception to gene regulation[J]. Current Opinion in Plant Biology, 1999, 2:352-358.
[16]Klucher K M, Chow H, Reiser L, et al. The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floal homeotic gene APETA-LA2[J]. Plant Cell, 1996, 8(2):137-153.
[17]Liu Q, Kasuga M, Sakuma Y, et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression in Arabidopsis[J].Plant Cell,1998, 10(8):1391-1406.
[18]Nole-Wilson Staci, Krizek B A. DNA binding properties of the Arabidopsis floral develop-ent protein AINTEGUMENTA [J]. Nucleic Acids Research, 2000, 28(21):4076-4082.
[19]Ohme-Takagi, Masaru, Hideaki Shishi. Ethylene inducible DNA binding proteins that interact with an ethylene responsive element[J].The Plant Cell,1995,(7):173-l82.
[20]葛宝宇,林轶,侯和胜.ERF类转录因子的结构与功能[J].安徽农学通报,2007,13(20):32-36.
[21]Toshitsugu Nakano, Kaoru Suzuki, Tatsuhito Fujimura, et al. Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice[J].Plan t Physiology, 2006,140(2):411-432.
[22]Yamaguchi-ShinoZaki K, Shinozaki K. Characteriaztion of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plnats[J]. Mol Gen Genet, 1993, 236:331-340.
[23]Yamaguchi-ShinoZaki K., Shinozaki K. 1994.A novel cis acting element in an Arabidopsis gene is involved in responsiveness to draught, low-temperature or high-salt stress[J].Plant Cell,6:251-264.
[24]Martin ML, Busconi L. A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature [J].Plant Physiol., 2001, 125: 1442-1449.
[25]Saijo Y, Hata S, Kyozuka J, et al. Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants[J].Plant J., 2000, 23: 319-327.
[26]Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis 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 low temperature and water deficit[J]. Proceeding of the National Academy of Sciences of the USA, 1997,94:1035-1040.
[27]王少峡,王振英,彭永康.DREB转录因子及其在植物抗逆中的作用[J].植物生理学通讯,2004, 2, 40(1):7-13.
[28]Gilmour SJ, Zarka DG, Stockinger EJ, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression [J]. Plant J, 1998,16(4):433-442.
[29]Chinnusamy V, Ohta M, Kanrar S, et al. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis[J]. Gene Dev, 2003, 17(8):1043-1054.
[30]Dong CH, Agarwal M, Zhang Y, et al. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[J].Proc Natl Acad Sci USA,2006,103(21):8281-8286.
[31]Nakashima K, Shinwari Z K, Sakuma Y, et al. Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression [J]. Plant Molecular Biology, 2000, 42: 657-665
[32]Haake V, Cook D, Riehmann J L, et al. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis [J].Plant Physiology,2002: 130,639-648
[33]Kizis D and Pages M. Maize DRE-binding proteins DBFI and DBF2 are involved in tsbl7regulation through the drought-responsive element in an ABA-dependent pathway[J].The Plant Journal, 2002, 30: 679-689
[34]Mohamed Badawi, Yedulla Venkat Reddy, Zahra Agharbaoui, et al. Structure and Functional Analysis of Wheat ICE (Inducer of CBF Expression) Genes[J].Plant Cell Physiol, 2008 ,49(8): 1237-1249.
[35]Agarwal P, Agarwal P K, Nair S , et al. Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity[J].Molecular Genetics and Genomics,2007,277:189-198.
[36]Lifeng Zhao, Yibing Hu, Kang Chong, et al.ARAG1, an ABA-responsive DREB gene, plays a role in seed germination and drought tolerance of rice[J].Annals of Botany, 2010,105(3):401-409 .
[37]Feng Qin, Yoh Sakuma, Jie Li, et al. Cloning and Functional Analysis of a Novel DREB1/CBF Transcription Factor Involved in Cold-Responsive Gene Expression in Zea mays L[J].Plant and Cell Physiology, 2004 ,45(8):1042-1052 .
[38]Yoshihisa Kasukabe, Lixiong He, Kazuyoshi Nada, et al. Over expression of Spermidine Synthase Enhances Tolerance to Multiple Environmental Stresses and Up-Regulates the Expression of Various Stress-Regulated Genes in Transgenic Arabidopsis thaliana [J]. Plant and Cell Physiology, 2004, 45(6):712-722.
[39]高峰,熊爱生,彭日荷,等.抗逆相关DREB转录因子的研究进展及应用[J].上海农业学报,2008, 24(1):118-123
[40]罗赛男,杨国顺,石雪晖.转录因子在植物抗逆性上的应用研究[J].湖南农业大学学报(自然科学版),2005,4,31(2):220-224.
[41]刘强,赵南明,Yamaguch-Shinozaki K,等.DREB转录因子在提高植物抗逆性中的作用[J].科学通报,2000,45(1):ll-l6.
[42]郝晓燕,陈明,徐惠君,等.GH-DREB基因转化小麦及转基因植株后代的抗旱生理指标鉴定[J].西南农业学报, 2005 ,18(5):616-620.
[43]李峰,曹刚强,梁秋霞,等.农杆菌介导Prd29A:DREB1A基因转化番茄的研究[J].江苏农业科学, 2008, 4:61-65.
[44]Wanmei Jin, Jing Dong , Yuanlei Hu, et al. Improved Cold-resistant Performance in Transgenic Grape (Vitis vinifera L.) Over expressing Cold-inducible Transcription Factors AtDREB1b[J]. Hortscience, 2009,44(1):35-39.
[45]Yusuke Ito, Koji Katsura, Kyonoshin Maruyama, et al. Functional Analysis of Rice DREB1/C- BF-type Transcription Factors Involved in Cold-responsive Gene Expression in Transgenic Rice [J].Plant Cell Physiol,2006 ,47(1):141-53.
[46]Ji-Ren Chen, Jing-Jing Lu, Rong Liu, et al.DREB1C from Medicago truncatula enhances freezing tolerance in transgenic M. truncatula and China Rose (Rosa chinensis Jacq.) [J]. Plant Growth Regul (2010) 60:199–211.
[47]高世庆,徐惠君,程宪国,等.转大豆GmDREB基因增强小麦的耐旱及耐盐性[J].科学通报,2005, 50(23):2617-2625.
[48]Oh SJ, Song SI, Kim YS, et al. Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth [J]. Plant Physiol, 2005, 138:341-351.
[49]杨帆,苗灵凤,胥晓,等.植物对干旱胁迫的响应研究进展[J].应用与环境生物学报,2007, 13 (4) : 586-591.
[50]杜洪伟,陈芬,肖国樱.提高作物耐旱性的DREB转录因子研究进展[J].生物技术通报,2008,6:1-6.
[51]徐莎,胡军,陈宇红,等.DREB转录因子研究进展[J].农业生物技术学报,2008,16(4): 706-713.
[52]Powell Abel P, Nelson R S, De B, et al. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene [J].Science, 1986, 232:738-743.
[53]Rubino L, Capriotti G. Resistance to Cymbidium ringspot tombusvirus infection in transgenic Nicotiana benthamiana plants expressing the virus coat protein gene [J]. Plant Mol Biol, 1993, 2 ,21 (4) :665 - 672.
[54]潘俊松,刘志昕,郑学勤.建兰花叶病毒的分离、鉴定及检测研究[J].热带作物学报,1997, 18(1): 63-70.
[55]Yang J, Lee H J, Shin D H, et a1. Genetic transformation of Cymbidium orchid by particle bombardment[J ]. Plant Cell Rep., 1999, 18: 978-984.
[56]赵学敏.植物抗病毒基因工程研究进展[J].现代农业科技,2008 ,16 :317-318.
[57]Golemboski D B, Lomonossoff G P, Zaitlin M,et al. Plants transformed with tobacco mosaic virus nonstructural gene sequence are resistant to the virus[J].Proc Natl Acad Sci USA,1990,87:6311-6315.
[58]程英豪,王继伟.植物抗病毒基因工程[J].生物学通报,1998,33(5): 5-6.
[59]刘志昕,吴豪,潘俊松,等.建兰花叶病毒运动蛋白基因克隆及序列分析[J].中国病毒学,2001, 16(1):51-54
[60]Obsuwan Kullanart, Borth Wayne, Hu John, et a1.Development of transgenic Dendrobium orchid resistant to CyMV[EB/OL].http://abstracts.aspb.org/pb2003/public/p49/0748.html.
[61]Ajjikuttira P, Loh CS, Wong SM. Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein [J]. J Gen Virol, 2005, 86 (5):1543 - 53.
[62]徐莺,施农农,王慧中.兰花病毒病分子生物学及抗病毒基因工程研究进展[J].浙江农业学报, 2007,19 (1) :65-69 .
[63]田波.植物基因工程[M].山东泰山科技出版社,1996.
[64]Hoopes B C, McClure W R, A cl1-dependent promoter is located within the Q gene of baterioph-age [J].Proc Natl Acad Sci USA, 1985, 82: 3134-3138.
[65]Nelson A, Roth K A, ohnson J D. Tobacco mosaic virus infection of transgenic Nicotiana tabacum plants is inhibited by antisense constructs directed at the 5’region of viral RNA[J].Gene, 1993, 127 (2): 227-232.
[66]Bendahmane M, Gronenborn B. Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA[J].Plant Mol Bio ,1997 , 33 : 351-357
[67]Szittya G, Burgyan J. Cymbidium ringspot tombusvirus coat protein coding sequence acts as anavirulent RNA[J ].Virol , 2001 ,75 (5) :2411 - 2420.
[68]黄凤兰,孟凡娟,牛红云,等.共抑制技术在花卉植物中的应用[J].内蒙古民族大学学报(自然科学版),2008,7,23(4):409-413.
[69]Tanzer M M ,Thompson W F ,Law M D ,et al. Characterization of post-transcriptionally suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco [J].Plant Cell , 1997 ,9 :1411 - 1423.
[70]Jacobs John JMR , Litiere K, van Dijk V ,et al. Post-transcriptionalβ-1,3-glucanase gene silencing involves increased transcript turnover that is translation independent[J].Plant ,1997,12 (4):885- 893.
[71]张恭,刘立峰,马峙英.RNA干扰及其植物抗病毒应用[J].中国农学通报, 2007,23(1):42-45.
[72]Napoli C, Lemieux C, Jorgensen R. Introduction of a chalcone synthase gene into petunia results in reversible co-suppression of homologous gene in transplant[J].Cell,1990,2:279-289.
[73]Fire A., Xu S., Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391:806-811.
[74]Wang M.B., Abbott D.C., Waterhouse P.M.A single copy of virus derived transgene encoding hairpin RNA give immunity to Barley yellow dwarf virus[J].Molecular Plant Pathology,2000, 1: 347-356.
[75]Lawson, C. et al. Engineering resistance to mixed virus infection in a commercial potato cultivar: resistace to potato virus X and potato virus Y in transgenic Russet Burbank[J].Bio Technolgy, 1990 ,8 :127-134
[76]Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants[J].Nature, 1989, 342 (6245): 76-78.
[77]Clegg R M. Fluorescence resonance energy transfer [J].Curr Opin Biotechnol, 1995, 6 (1): 103-110.
[78]Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression [J]. Methods, 2001, 25(4):386-401.
[79]Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR[J].Genome Res,1996, 6(10):986-994
[80]袁亚男,刘文忠.实时荧光定量PCR技术的类型、特点与应用[J].中国畜牧兽,2008,35(3):27-30.
[81]蔡霞.定量PCR技术及其应用现状[J].现代诊断与治疗,2005, 16 (2): 112-115.
[82]Hardegger D, Nadal D, Bossart W, et al. Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR[J]. Journal of Microbiological Methods, 2000, 41 (1): 45-51.
[83]蒋春燕,王泰健,王琴,等.实时荧光定量PCR技术[J].动物医学进展, 2005, 26 (12): 97-101.
[84]敖金霞,高学军,仇有文,等.实时荧光定量PCR技术在转基因检测中的应用[J].东北农业大学学报,2009,40(6):141-144.
[85]Marta H, David R L, Teresa E, et al. Real-time quantitative polymerase chain reaction methods for multiplex genetically modified organism with melting curve method[J]. Analytical Biochemistry, 2003, 323: 164-170.
[86]Alary R, Serin A, Maury D. Comparison of simplex and duplex real-time PCR for the quantifica-tion of GMO in maize and soybean[J]. Food Control, 2002, 13: 235-244.
[87]Kim J H, Song H S, Kim D H, et al. Quantification of genetically modified canola GT73 using TaqMan real-time PCR[J]. Journal of Microbiology and Biotechnology, 2006, 16: 1778-1783.
[88]Peanoa R C, Bordoni M, Gulli A, et al. Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods[J]. Analytical Biochemistry, 2005, 346(1): 90-100.
[89]Shindo Y, Kuribara H, Matsuoka T, et al. Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules[J]. AOAC Int, 2002, 85: 1119-1126.
[90]Vollenhofer S, Burg K, Schmidt J, et al.Genetically modified organisms in food -screening and specific detection by polymerase chain reaction [J]. Journal of Agriculture and Food Chemistry, 2004, 47(12): 5038-5043.
[91]La Paz J L, Garcia-Muniz N, Nadal A, et al. Inter laboratory transfer of a real-time polymerase chain reaction assay for quantitative detection of genetically modified maize event TC-1507[J]. Journal of AOAC International, 2006, 89(5): 1347-1352.
[92]Permingeat H R, Reggiardo M I, Vallejos R H. Detection and quantification of transgenes in grains by multiplex and real-time PCR [J]. J Agric Food Chem, 2002, 50(16): 4431-4436.
[93]Hird H, Powell J, Johnson M L, et al. Determination of percentage of roundup ready soya in soya flour using real-time polymerase chain reaction: inter laboratory study [J]. AOAC Int, 2003, 86(1):66-71.
[94]Song P, Cai C Q, Skokut M, et al.Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS(tm)-derived transgenic maize [J]. Plant Cell Reports, 2004, 20: 948-954.
[95]Ding J, Jia J,Yang L, et al. Validation of a rice-specific gene, sucrose-phosphate synthase used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of Transgenes [J]. J Agric Food Chem, 2004, 52(11): 3372-3377.
[96]Shi J, Thomas C J, King L A, et al. The expression of a baculovirus derived chitinase gene increased resistance of tobacco cultivars to brown spot [J]. Ann Appl Biol, 2000, 136: 1-8.
[97]Arne H, Sissel B R, Astrid L, et al. Berdal PCR technology for screening and quantification of genetically modified organisms(GMOs)[J]. Anal Bioanal Chem, 2003, 375: 985-993.
[98]Schmittgen T D. Real-time quantitative PCR [J]. Methods, 2006, 25(4): 383-385.
[99]Ke L D, Chen Z, Yung W K. A reliability test of standard-based quantitative PCR: exogenous VS endogenous standards [J]. Mol Cell Probes, 2004, 14 (2): 127-135.
[100]Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures [J]. Physiol Plant, 1962, 15: 473-497.
[101]张兰.几个种类兰花的转基因抗病毒新品种培育研究[D].河北保定:河北大学生命科学学院,2009.
[102]Kuehnle A R, JonesW E. Transformation of Dendrobium orchid using particle bombardment of protocorms[J ]. Plant Cell Rep., 1992, 11: 484-488.
[103]满若君,李杨瑞,卜朝阳.蝴蝶兰的组织培养和遗传转化体系的研究进展[J].广西农业科学, 2007,38 (1): 6-10.
[104]Blarmino M M, Chomczynski P. Agrobacterium-mediated genetic transformation of a phalaeno-psis orchid [J]. Plant Cell Rep, 2000, 19: 435-442.
[105]Yu H, et al. Agrobaeterium-mediated transformation of a Dendrobium orchid with the class knox gene DOHI[J].Plant Cell Report,2001,20:30-305.
[106]柴明良,汪炳良.若干花卉转基因研究的现状和前景[J].园艺学报, 2002,29(增刊): 664-670.
[107]柴明良,金斗焕.农杆菌介导的蝴蝶兰基因转化系统的建立[J].园艺学报,2004,31(4): 537-539.
[108]Men S, Ming X, Wang Y, et al. Genetic transformation of two species of orchid by biolistic bombardment[J]. Plant Cell Reports, 2003, 21: 592-598.
[109]Anzai H, Ishii Y, Shichinohe M, et al. Transformation of Phalaenopsis by particle bombardment [J]. Plant Tiss Cult Lett, 1996, 13: 265-271.
[110]Chia T F, Chan Y S, Chuan H.Genetic engineering of tolerance to Cymbidium mosaic virus[C]. Proc 13thWorld Orchi Conf, 1990, 284.
[111]Chia T F, Chan Y S, Chuan H.The firefly lucifera gene as a non-invasive reporter for Dendrobium transformation[J]. Plant, 1995, 6: 441-446.
[112]Knapp J. Transformation of three genera of orchid using the bar gene as a selectable maker[J]. Plant Cell Report, 2000, 19: 893-898.
[113]Maton L, Browse J. Facile transformation of Arabidopsis[J].Plant cell Rep ,1991 ,10 :235-239.
[114]Renckens S, De Greve H, Van Montagu M, et al . Petunia plants escape from negative selective against a transgene by silencing the foreign DNA via methylation[J].Mol Gen Genet, 1992 , 233 : 53-64.
[115]Koncz C, Schell J, Redei GP. T-DNA transformation and insertion mutagenesis .In: Koncz C, Chua N H, Schell J (eds) Methods in Arabidopsis research[J].Singapore:World Scientific, 1992 , P224-273.
[116]王紫萱,易自力.卡那霉素在植物转基因中的应用及其抗性基因的生物安全性评价[J].中国生物工程杂志,2003,23 (6 ): 9-13.
[2]倪志勇,徐兆师,李连城,等.DREB转录因子在植物抗逆胁迫中的作用机理及应用研究进展[J].麦类作物学报,2008 ,28 (6) :1100-1106.
[3]刘强,张贵友,陈受宜.植物转录因子的结构与调控作用[J].中国科学,2000 ,45 :1465-1474.
[4]Riechmann J L, Heard J. Arabidopsis Transcription Factors: Genome-Wide Comparative Analysis Among Eukaryotes [J].Science,2000,290 :2105-2110.
[5]孙啸.大豆抗逆相关转录因子基因GmDREB3的功能分析及启动子的克隆[J].新疆农业大学,2007,5:1-92.
[6]Jofuku K D, Denboer B G W, Vanmontagu M, et al. Control of Arabidopsis Flower and Seed Development by the Homeotic Gene APETALA2[J]. Plant Cell, 1994, 6(9):1211-1225.
[7]Ohme-Takagi, Shinshi H. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element [J].Plant Cell, 1995, 7(2):173-182.
[8]Weigel D. The APETALA2 domain is related to a novel type of DNA binding domain [J].Plant Cell, 1995, 7(4):388-389.
[9]Moose S P, Sisco P H. Glossy15, an APETALA2-like gene from maize that regulates leaf epidermal cell identity[J]. Genes & Development, 1996, 10(23):3018-3027
[10]Gu Y Q, Wildermuth M C, Chakravarthy S, et al. Tomato transcription factors Pti4, Pti5 and Pti6 activate defense responses when expressed in Arabidopsis[J].Plant Cell, 2002,14(4): 817-831.
[11]Riechmann J L, Meyerowitz E M. The AP2/EREBP family of plant transcription factors[J]. Biol. Chem, 1998, 379:633-646.
[12]Krizek B A. AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain[J]. Nucleic Acids Res, 2003, 31: 1859-1868.
[13]Wuitschick J D, Lindstrom P R, Meyer A E, et al. Homing endonucleases encoded by germ line-limited genes in Tetrahymena thermophilahave APETALA2 DNA binding domains[J]. Eukaryot.Cell, 2004, 3:685-694.
[14]Magnani E, Sj lander K, Hake S. From endonucleases to transcription factors: evolution of the AP2 DNA binding domain in plants[J].Plant Cell,2004,16:2265-2277.
[15]Chang C, Shockey J A. The ethylene-response pathway: signal perception to gene regulation[J]. Current Opinion in Plant Biology, 1999, 2:352-358.
[16]Klucher K M, Chow H, Reiser L, et al. The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floal homeotic gene APETA-LA2[J]. Plant Cell, 1996, 8(2):137-153.
[17]Liu Q, Kasuga M, Sakuma Y, et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression in Arabidopsis[J].Plant Cell,1998, 10(8):1391-1406.
[18]Nole-Wilson Staci, Krizek B A. DNA binding properties of the Arabidopsis floral develop-ent protein AINTEGUMENTA [J]. Nucleic Acids Research, 2000, 28(21):4076-4082.
[19]Ohme-Takagi, Masaru, Hideaki Shishi. Ethylene inducible DNA binding proteins that interact with an ethylene responsive element[J].The Plant Cell,1995,(7):173-l82.
[20]葛宝宇,林轶,侯和胜.ERF类转录因子的结构与功能[J].安徽农学通报,2007,13(20):32-36.
[21]Toshitsugu Nakano, Kaoru Suzuki, Tatsuhito Fujimura, et al. Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice[J].Plan t Physiology, 2006,140(2):411-432.
[22]Yamaguchi-ShinoZaki K, Shinozaki K. Characteriaztion of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plnats[J]. Mol Gen Genet, 1993, 236:331-340.
[23]Yamaguchi-ShinoZaki K., Shinozaki K. 1994.A novel cis acting element in an Arabidopsis gene is involved in responsiveness to draught, low-temperature or high-salt stress[J].Plant Cell,6:251-264.
[24]Martin ML, Busconi L. A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature [J].Plant Physiol., 2001, 125: 1442-1449.
[25]Saijo Y, Hata S, Kyozuka J, et al. Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants[J].Plant J., 2000, 23: 319-327.
[26]Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis 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 low temperature and water deficit[J]. Proceeding of the National Academy of Sciences of the USA, 1997,94:1035-1040.
[27]王少峡,王振英,彭永康.DREB转录因子及其在植物抗逆中的作用[J].植物生理学通讯,2004, 2, 40(1):7-13.
[28]Gilmour SJ, Zarka DG, Stockinger EJ, et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression [J]. Plant J, 1998,16(4):433-442.
[29]Chinnusamy V, Ohta M, Kanrar S, et al. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis[J]. Gene Dev, 2003, 17(8):1043-1054.
[30]Dong CH, Agarwal M, Zhang Y, et al. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[J].Proc Natl Acad Sci USA,2006,103(21):8281-8286.
[31]Nakashima K, Shinwari Z K, Sakuma Y, et al. Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression [J]. Plant Molecular Biology, 2000, 42: 657-665
[32]Haake V, Cook D, Riehmann J L, et al. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis [J].Plant Physiology,2002: 130,639-648
[33]Kizis D and Pages M. Maize DRE-binding proteins DBFI and DBF2 are involved in tsbl7regulation through the drought-responsive element in an ABA-dependent pathway[J].The Plant Journal, 2002, 30: 679-689
[34]Mohamed Badawi, Yedulla Venkat Reddy, Zahra Agharbaoui, et al. Structure and Functional Analysis of Wheat ICE (Inducer of CBF Expression) Genes[J].Plant Cell Physiol, 2008 ,49(8): 1237-1249.
[35]Agarwal P, Agarwal P K, Nair S , et al. Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity[J].Molecular Genetics and Genomics,2007,277:189-198.
[36]Lifeng Zhao, Yibing Hu, Kang Chong, et al.ARAG1, an ABA-responsive DREB gene, plays a role in seed germination and drought tolerance of rice[J].Annals of Botany, 2010,105(3):401-409 .
[37]Feng Qin, Yoh Sakuma, Jie Li, et al. Cloning and Functional Analysis of a Novel DREB1/CBF Transcription Factor Involved in Cold-Responsive Gene Expression in Zea mays L[J].Plant and Cell Physiology, 2004 ,45(8):1042-1052 .
[38]Yoshihisa Kasukabe, Lixiong He, Kazuyoshi Nada, et al. Over expression of Spermidine Synthase Enhances Tolerance to Multiple Environmental Stresses and Up-Regulates the Expression of Various Stress-Regulated Genes in Transgenic Arabidopsis thaliana [J]. Plant and Cell Physiology, 2004, 45(6):712-722.
[39]高峰,熊爱生,彭日荷,等.抗逆相关DREB转录因子的研究进展及应用[J].上海农业学报,2008, 24(1):118-123
[40]罗赛男,杨国顺,石雪晖.转录因子在植物抗逆性上的应用研究[J].湖南农业大学学报(自然科学版),2005,4,31(2):220-224.
[41]刘强,赵南明,Yamaguch-Shinozaki K,等.DREB转录因子在提高植物抗逆性中的作用[J].科学通报,2000,45(1):ll-l6.
[42]郝晓燕,陈明,徐惠君,等.GH-DREB基因转化小麦及转基因植株后代的抗旱生理指标鉴定[J].西南农业学报, 2005 ,18(5):616-620.
[43]李峰,曹刚强,梁秋霞,等.农杆菌介导Prd29A:DREB1A基因转化番茄的研究[J].江苏农业科学, 2008, 4:61-65.
[44]Wanmei Jin, Jing Dong , Yuanlei Hu, et al. Improved Cold-resistant Performance in Transgenic Grape (Vitis vinifera L.) Over expressing Cold-inducible Transcription Factors AtDREB1b[J]. Hortscience, 2009,44(1):35-39.
[45]Yusuke Ito, Koji Katsura, Kyonoshin Maruyama, et al. Functional Analysis of Rice DREB1/C- BF-type Transcription Factors Involved in Cold-responsive Gene Expression in Transgenic Rice [J].Plant Cell Physiol,2006 ,47(1):141-53.
[46]Ji-Ren Chen, Jing-Jing Lu, Rong Liu, et al.DREB1C from Medicago truncatula enhances freezing tolerance in transgenic M. truncatula and China Rose (Rosa chinensis Jacq.) [J]. Plant Growth Regul (2010) 60:199–211.
[47]高世庆,徐惠君,程宪国,等.转大豆GmDREB基因增强小麦的耐旱及耐盐性[J].科学通报,2005, 50(23):2617-2625.
[48]Oh SJ, Song SI, Kim YS, et al. Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth [J]. Plant Physiol, 2005, 138:341-351.
[49]杨帆,苗灵凤,胥晓,等.植物对干旱胁迫的响应研究进展[J].应用与环境生物学报,2007, 13 (4) : 586-591.
[50]杜洪伟,陈芬,肖国樱.提高作物耐旱性的DREB转录因子研究进展[J].生物技术通报,2008,6:1-6.
[51]徐莎,胡军,陈宇红,等.DREB转录因子研究进展[J].农业生物技术学报,2008,16(4): 706-713.
[52]Powell Abel P, Nelson R S, De B, et al. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene [J].Science, 1986, 232:738-743.
[53]Rubino L, Capriotti G. Resistance to Cymbidium ringspot tombusvirus infection in transgenic Nicotiana benthamiana plants expressing the virus coat protein gene [J]. Plant Mol Biol, 1993, 2 ,21 (4) :665 - 672.
[54]潘俊松,刘志昕,郑学勤.建兰花叶病毒的分离、鉴定及检测研究[J].热带作物学报,1997, 18(1): 63-70.
[55]Yang J, Lee H J, Shin D H, et a1. Genetic transformation of Cymbidium orchid by particle bombardment[J ]. Plant Cell Rep., 1999, 18: 978-984.
[56]赵学敏.植物抗病毒基因工程研究进展[J].现代农业科技,2008 ,16 :317-318.
[57]Golemboski D B, Lomonossoff G P, Zaitlin M,et al. Plants transformed with tobacco mosaic virus nonstructural gene sequence are resistant to the virus[J].Proc Natl Acad Sci USA,1990,87:6311-6315.
[58]程英豪,王继伟.植物抗病毒基因工程[J].生物学通报,1998,33(5): 5-6.
[59]刘志昕,吴豪,潘俊松,等.建兰花叶病毒运动蛋白基因克隆及序列分析[J].中国病毒学,2001, 16(1):51-54
[60]Obsuwan Kullanart, Borth Wayne, Hu John, et a1.Development of transgenic Dendrobium orchid resistant to CyMV[EB/OL].http://abstracts.aspb.org/pb2003/public/p49/0748.html.
[61]Ajjikuttira P, Loh CS, Wong SM. Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein [J]. J Gen Virol, 2005, 86 (5):1543 - 53.
[62]徐莺,施农农,王慧中.兰花病毒病分子生物学及抗病毒基因工程研究进展[J].浙江农业学报, 2007,19 (1) :65-69 .
[63]田波.植物基因工程[M].山东泰山科技出版社,1996.
[64]Hoopes B C, McClure W R, A cl1-dependent promoter is located within the Q gene of baterioph-age [J].Proc Natl Acad Sci USA, 1985, 82: 3134-3138.
[65]Nelson A, Roth K A, ohnson J D. Tobacco mosaic virus infection of transgenic Nicotiana tabacum plants is inhibited by antisense constructs directed at the 5’region of viral RNA[J].Gene, 1993, 127 (2): 227-232.
[66]Bendahmane M, Gronenborn B. Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA[J].Plant Mol Bio ,1997 , 33 : 351-357
[67]Szittya G, Burgyan J. Cymbidium ringspot tombusvirus coat protein coding sequence acts as anavirulent RNA[J ].Virol , 2001 ,75 (5) :2411 - 2420.
[68]黄凤兰,孟凡娟,牛红云,等.共抑制技术在花卉植物中的应用[J].内蒙古民族大学学报(自然科学版),2008,7,23(4):409-413.
[69]Tanzer M M ,Thompson W F ,Law M D ,et al. Characterization of post-transcriptionally suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco [J].Plant Cell , 1997 ,9 :1411 - 1423.
[70]Jacobs John JMR , Litiere K, van Dijk V ,et al. Post-transcriptionalβ-1,3-glucanase gene silencing involves increased transcript turnover that is translation independent[J].Plant ,1997,12 (4):885- 893.
[71]张恭,刘立峰,马峙英.RNA干扰及其植物抗病毒应用[J].中国农学通报, 2007,23(1):42-45.
[72]Napoli C, Lemieux C, Jorgensen R. Introduction of a chalcone synthase gene into petunia results in reversible co-suppression of homologous gene in transplant[J].Cell,1990,2:279-289.
[73]Fire A., Xu S., Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391:806-811.
[74]Wang M.B., Abbott D.C., Waterhouse P.M.A single copy of virus derived transgene encoding hairpin RNA give immunity to Barley yellow dwarf virus[J].Molecular Plant Pathology,2000, 1: 347-356.
[75]Lawson, C. et al. Engineering resistance to mixed virus infection in a commercial potato cultivar: resistace to potato virus X and potato virus Y in transgenic Russet Burbank[J].Bio Technolgy, 1990 ,8 :127-134
[76]Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants[J].Nature, 1989, 342 (6245): 76-78.
[77]Clegg R M. Fluorescence resonance energy transfer [J].Curr Opin Biotechnol, 1995, 6 (1): 103-110.
[78]Giulietti A, Overbergh L, Valckx D, et al. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression [J]. Methods, 2001, 25(4):386-401.
[79]Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR[J].Genome Res,1996, 6(10):986-994
[80]袁亚男,刘文忠.实时荧光定量PCR技术的类型、特点与应用[J].中国畜牧兽,2008,35(3):27-30.
[81]蔡霞.定量PCR技术及其应用现状[J].现代诊断与治疗,2005, 16 (2): 112-115.
[82]Hardegger D, Nadal D, Bossart W, et al. Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR[J]. Journal of Microbiological Methods, 2000, 41 (1): 45-51.
[83]蒋春燕,王泰健,王琴,等.实时荧光定量PCR技术[J].动物医学进展, 2005, 26 (12): 97-101.
[84]敖金霞,高学军,仇有文,等.实时荧光定量PCR技术在转基因检测中的应用[J].东北农业大学学报,2009,40(6):141-144.
[85]Marta H, David R L, Teresa E, et al. Real-time quantitative polymerase chain reaction methods for multiplex genetically modified organism with melting curve method[J]. Analytical Biochemistry, 2003, 323: 164-170.
[86]Alary R, Serin A, Maury D. Comparison of simplex and duplex real-time PCR for the quantifica-tion of GMO in maize and soybean[J]. Food Control, 2002, 13: 235-244.
[87]Kim J H, Song H S, Kim D H, et al. Quantification of genetically modified canola GT73 using TaqMan real-time PCR[J]. Journal of Microbiology and Biotechnology, 2006, 16: 1778-1783.
[88]Peanoa R C, Bordoni M, Gulli A, et al. Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods[J]. Analytical Biochemistry, 2005, 346(1): 90-100.
[89]Shindo Y, Kuribara H, Matsuoka T, et al. Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules[J]. AOAC Int, 2002, 85: 1119-1126.
[90]Vollenhofer S, Burg K, Schmidt J, et al.Genetically modified organisms in food -screening and specific detection by polymerase chain reaction [J]. Journal of Agriculture and Food Chemistry, 2004, 47(12): 5038-5043.
[91]La Paz J L, Garcia-Muniz N, Nadal A, et al. Inter laboratory transfer of a real-time polymerase chain reaction assay for quantitative detection of genetically modified maize event TC-1507[J]. Journal of AOAC International, 2006, 89(5): 1347-1352.
[92]Permingeat H R, Reggiardo M I, Vallejos R H. Detection and quantification of transgenes in grains by multiplex and real-time PCR [J]. J Agric Food Chem, 2002, 50(16): 4431-4436.
[93]Hird H, Powell J, Johnson M L, et al. Determination of percentage of roundup ready soya in soya flour using real-time polymerase chain reaction: inter laboratory study [J]. AOAC Int, 2003, 86(1):66-71.
[94]Song P, Cai C Q, Skokut M, et al.Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS(tm)-derived transgenic maize [J]. Plant Cell Reports, 2004, 20: 948-954.
[95]Ding J, Jia J,Yang L, et al. Validation of a rice-specific gene, sucrose-phosphate synthase used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of Transgenes [J]. J Agric Food Chem, 2004, 52(11): 3372-3377.
[96]Shi J, Thomas C J, King L A, et al. The expression of a baculovirus derived chitinase gene increased resistance of tobacco cultivars to brown spot [J]. Ann Appl Biol, 2000, 136: 1-8.
[97]Arne H, Sissel B R, Astrid L, et al. Berdal PCR technology for screening and quantification of genetically modified organisms(GMOs)[J]. Anal Bioanal Chem, 2003, 375: 985-993.
[98]Schmittgen T D. Real-time quantitative PCR [J]. Methods, 2006, 25(4): 383-385.
[99]Ke L D, Chen Z, Yung W K. A reliability test of standard-based quantitative PCR: exogenous VS endogenous standards [J]. Mol Cell Probes, 2004, 14 (2): 127-135.
[100]Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures [J]. Physiol Plant, 1962, 15: 473-497.
[101]张兰.几个种类兰花的转基因抗病毒新品种培育研究[D].河北保定:河北大学生命科学学院,2009.
[102]Kuehnle A R, JonesW E. Transformation of Dendrobium orchid using particle bombardment of protocorms[J ]. Plant Cell Rep., 1992, 11: 484-488.
[103]满若君,李杨瑞,卜朝阳.蝴蝶兰的组织培养和遗传转化体系的研究进展[J].广西农业科学, 2007,38 (1): 6-10.
[104]Blarmino M M, Chomczynski P. Agrobacterium-mediated genetic transformation of a phalaeno-psis orchid [J]. Plant Cell Rep, 2000, 19: 435-442.
[105]Yu H, et al. Agrobaeterium-mediated transformation of a Dendrobium orchid with the class knox gene DOHI[J].Plant Cell Report,2001,20:30-305.
[106]柴明良,汪炳良.若干花卉转基因研究的现状和前景[J].园艺学报, 2002,29(增刊): 664-670.
[107]柴明良,金斗焕.农杆菌介导的蝴蝶兰基因转化系统的建立[J].园艺学报,2004,31(4): 537-539.
[108]Men S, Ming X, Wang Y, et al. Genetic transformation of two species of orchid by biolistic bombardment[J]. Plant Cell Reports, 2003, 21: 592-598.
[109]Anzai H, Ishii Y, Shichinohe M, et al. Transformation of Phalaenopsis by particle bombardment [J]. Plant Tiss Cult Lett, 1996, 13: 265-271.
[110]Chia T F, Chan Y S, Chuan H.Genetic engineering of tolerance to Cymbidium mosaic virus[C]. Proc 13thWorld Orchi Conf, 1990, 284.
[111]Chia T F, Chan Y S, Chuan H.The firefly lucifera gene as a non-invasive reporter for Dendrobium transformation[J]. Plant, 1995, 6: 441-446.
[112]Knapp J. Transformation of three genera of orchid using the bar gene as a selectable maker[J]. Plant Cell Report, 2000, 19: 893-898.
[113]Maton L, Browse J. Facile transformation of Arabidopsis[J].Plant cell Rep ,1991 ,10 :235-239.
[114]Renckens S, De Greve H, Van Montagu M, et al . Petunia plants escape from negative selective against a transgene by silencing the foreign DNA via methylation[J].Mol Gen Genet, 1992 , 233 : 53-64.
[115]Koncz C, Schell J, Redei GP. T-DNA transformation and insertion mutagenesis .In: Koncz C, Chua N H, Schell J (eds) Methods in Arabidopsis research[J].Singapore:World Scientific, 1992 , P224-273.
[116]王紫萱,易自力.卡那霉素在植物转基因中的应用及其抗性基因的生物安全性评价[J].中国生物工程杂志,2003,23 (6 ): 9-13.