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基因枪法和农杆菌介导法转化的外源DNA整合到植物梁色上是随机进行的,因此,它们可能会产生不同的转基因拷贝数,得到不同的基因表达盒完整率,这反过来会影响基因的表达,为证实这一假说,作首先将同一质粒pAcPG-CAM分别用基因枪法和农杆菌介导法转化到水稻(Oryza sativa L.cv.TNG67)愈伤组织,为了揭示不同质粒是否也出现类似结果,也用农杆菌介导法,基因枪法分别将pTOK233和pJPM44导入水稻愈伤组织,并获得一批转基因植株,R0代值转基因表达的分析用GUS组织化学染色法,用质粒上的单切点酶酶切基因组DNA后的Southern杂交结果确定转基因拷贝数,总DNA髟双切点酶(分别位于表达盒两侧)酶切后的Southern杂交结果确定了完整转基因表达盒数目,结果表明,农杆菌介导转化法的转基因植株的转基因拷贝数相对少一些(平均为2.1和2.3),而基因枪法转化产生的转基因植株的转基因撬贝数相对多一些(平均为4.2和5.6),并且农杆菌介导转化法的转基因植株的基因表达盒DNA重排概率低于由基因枪法转化产生的转基因植株的基因表达盒DNA重排概率-农杆菌介导转化法的DNA重排概率为0.07和0.106,由基因枪法转化的DNA重排概率为0.57和0.66。研究还分析了基因表达情况与转基因的拷贝数或完整表达盒数之间的关系,GUS定量分析结果表明,为了准确揭示转基因的表达情况与转基因之间的关系,用完整基因表达盒数而不是转基因DNA拷贝数更准确,并于用不同转基因方法将同种质粒导入植物体分析基因表达盒DNA重排概率为首次报道。 相似文献
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Transgeni根癌农杆菌介导的小麦转基因植株再生(英文) 总被引:1,自引:0,他引:1
根癌农杆菌菌株Agl Ⅰ的Ti 质粒pUNN-2 带有Ubi1 启动子驱动的npt Ⅱ基因。7 种基因型小麦幼胚或胚性愈伤组织用于农杆菌介导的转化实验。经过不同浓度巴龙霉素的筛选,3 种基因型小麦产生抗性愈伤组织并再生植株。再生植株经PCR 和Southern 杂交鉴定为转基因植株,转化频率( 再生转基因植株的小麦愈伤组织数/ 用于转化实验的愈伤组织数) 为3.7% ~5 .9% 。小麦基因型及转化材料的起始生理状态是影响TDNA转移的重要因素。 相似文献
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根癌农杆菌介导的小麦转基因植株再生 总被引:19,自引:0,他引:19
根癌农杆菌菌株Agl Ⅰ的Ti质粒p^UNN-2带有Ubi1启动子驱动的nptⅡ基因。7种基因型小麦幼胚或胚性愈伤组织用于农杆菌介导的转化实验。经过不同家度巴龙霉素的筛选,3种基因型小麦产生抗性愈伤组织并再生植株。再生植株经PCR和Southern杂交鉴定为转基因植株,转化频率为3.7%-5.9%。小麦基因型及转化材料的起始生理状态的影响T-DNA转移的重要因素。 相似文献
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以叶盘为外植体,通过农杆菌菌株C58Cl(pPMP90)介导,开展了用植物表达载体pK2-35S-GFP(含GFP基因)转化洋桔梗(Eustoma russellianum)的研究。洋桔梗叶盘用农杆菌浸染15-20min,在共培养基上与农杆菌共培养2d后,转移到分化培养基上诱导愈伤组织。10d后选取部分叶盘在紫外灯下观察GFP的荧光亮点,结果80%以上的叶盘都有GFP的荧光亮点,说明农杆菌对叶盘的感染效率很高。在分化培养基上红花洋桔梗叶盘不定芽分化频率约为30%,把分化培养基上形成的小苗转移到含有Km(20mg/L)的生根培养基上,进行生根诱导筛选具有Km抗性的转基因植株,统计结果说明红花洋桔梗的转化效率为2.4%。随机选取4株具有Km抗性的洋桔梗植株进行PCR检测,结果均为阳性,证明GFP基因已整合到洋桔梗基因组中,选取经PCR检测为阳性的植株叶片,用激光共聚焦显微镜观察,结果发现有GFP的绿色荧光出现在叶片细胞质中,说明叶片中有GFP蛋白的表达,转化试验成功。 相似文献
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农杆菌介导法与基因枪轰击法结合在植物遗传转化上的应用 总被引:10,自引:1,他引:9
根癌农杆菌介导法(Agrohacterium mediated transformation)和基因枪轰击法( particle bombardment transformation)是植物遗传转化的主要方法。两种方法各有优缺点.农杆菌介导法是一种天然的植物遗传转化系统,外源基因在转基因植物中的拷贝数低,遗传稳定性好;基因枪转化法不受材料基因型的限制。通过结合两种方法的优点,发展了3种农杆菌介导和基因枪轰击法相结合的遗传转化方法,分别为农杆枪法、基因枪轰击/农杆菌感染法、金粉或钨粉包裹菌体细胞作为微弹轰击法。对3种结合转化方法的技术途径、原理、转化受体及研究进展等方面进行了综述。 相似文献
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通过根癌农杆菌介导法获得菊花转基因植株 总被引:26,自引:0,他引:26
以带叶茎段为外植体,通过根癌农杆菌介导法,将兔防御NP-1基因导入菊花品种“001”中。经梯度卡那霉素(kanamycin,Km)筛选,获得了大量Km抗性植株,其中部分Km抗性植株经Southern杂交鉴定为转基因植株。从而成功地建立了菊花遗传转化系统,为菊花分子育种奠定了基础。 相似文献
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抗烟青虫转基因烟草的培育 总被引:3,自引:0,他引:3
烟青虫属鳞翅且(Lpidoptera)昆虫。前人研究表明,苏云金杆菌(Bacillus thuringiensis)中的Bt毒蛋白对其具有很强的毒杀作用。设法将Bt基因导入到烟草中,是防治这类虫害的一种有效途径。80年代后期以来,为了提高Bt基因在植物中的表达水平,在编码区密码子的优化、编码顺序的改进和高效启动子的选配方面.都有了很快的发展。有些经过部分改进或人工全合成的Bt基因,还被先后导入到番茄〔1.2〕、烟草〔2-4〕、棉花〔5.6〕、玉米〔7〕、水稻〔8〕等重要作物中。迄1995年止.在美国获准可供商业用的Bt毒蛋白转基因作物已有槔花、玉米和马铃薯等〔9〕。在烟草中迄今未见有Bt毒蛋白转基因株达到生产实用水平的报道。本实验试图通过农杆菌介导法,将密码子经过优化的Bt基因cryIA(b)及cryIA?〔10〕叫导入离体培养的烟草叶片,然后使之再生,形成转基因植株,再从其后代中选育出既保留原品种优良农艺性状,又具有对烟青虫稳定抗性的转基因株,以期提高该品种的稳产性,降低杀虫农药成本。保护烟田的良好生态环境。 相似文献
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D. S. Choffnes R. Philip L. O. Vodkin 《In vitro cellular & developmental biology. Plant》2001,37(6):756-762
Summary We have examined transgene inheritance in over 300 progeny of a line of soybean (Glycine max) transformed by particle bombardment with a construct containing bovine β-casein under the control of the soybean lectin
5′ and 3′ regulatory elements. Four copies of the casein transgene, located at a single locus, exhibit a high frequency of
recombination that resulted in novel patterns in approximately 16% of the progeny in both the T1 and T2 generations. Characterization of the transgene locus using restriction enzymes that do not cut the transformation plasmid
showed that all four transgene copies are at a single locus no larger than approximately 40 kb in size. Therefore, the recombination
events resulting in the loss of transgene DNA are taking place within a limited physical distance on the host chromosome.
This is the first report extensively documenting transgene instability at the DNA level in a plant transformed via particle
bombardment. As this report indicates, a seemingly simple phenotype (presence of the foreign protein) may conceal inherent
genetic instability at the DNA level. 相似文献
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Molecular Characteristics of Transgenic Wheat and the Effect on Transgene Expression 总被引:20,自引:0,他引:20
A population of R0 transgenic wheat plants, generated by particle bombardment, was analyzed to define molecular, genetic and phenotypic properties resulting from transformation with a cointegrate vector, or cotransformation with two separate plasmids. By evaluating the progeny of 70 independently-derived transgenic plants, we also identified rare events such as chimerism and transgene elimination, which provide valuable information concerning the development of transgenic cereal plants following bombardment experiments. The frequency of chimerism in our transgenic wheat plants was very low. Furthermore, while transgene elimination did occur, this was also a very rare event. We determined the copy numbers of integrated transgenes and the levels of transgene expression. Comparisons to transgenic rice plants generated in the same manner demonstrated some similarities, but also important differences in transgene behavior. Whereas in rice there is no evidence for any direct relationship between transgene copy number and transgene expression or stability, multicopy populations in wheat demonstrated a bias towards higher levels of expression for the two genes and the maize ubiquitin promoter evaluated in the present study. 相似文献
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Constitutive versus seed specific expression in transgenic wheat: temporal and spatial control 总被引:5,自引:0,他引:5
Transgenic wheat plants from specific cultivars can now be routinely engineered in many laboratories. However, our understanding of the factors controlling transgene expression and stability in wheat compared to other cereals is rather limited. Only a few promoters have been tested in transgenic wheat, and relatively little is known of their relative activities and expression parameters. In the present study, the spatial and temporal properties of one heterologous constitutive promoter and one seedspecific wheat promoter were investigated. We generated constructs with the reporter gene gusA (glucuronidase) driven by: (a) the constitutive maize ubiquitin1 (ubi1) promoter, and (b) two differentsized fragments of the seedspecific low molecular weight glutenin (LMWG1D1) promoter from wheat. The activities of all three promoter constructs were comparable in endosperm tissue. A detailed analysis of spatial and temporal properties of the promoters is described. Heat shock treatment of transgenic plants carrying the ubi1: gusA construct resulted in a significant elevation in the levels of GUS activity. The inheritance of transgene expression levels and stability was evaluated over four generations, as a function of transgene integration patterns and copy number. 相似文献
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Analysis of a large number of independent transgenic rice plants produced by the biolistic method 总被引:2,自引:0,他引:2
Rongda Qu Alexandre de Kochko Lanying Zhang Philippe Marmey Liangcai Li Wenzhong Tian Shiping Zhang Claude M. Fauquet Roger N. Beachy 《In vitro cellular & developmental biology. Plant》1996,32(4):233-240
Summary Over 500 independent transgenic rice plants have been obtained by the biolistic method with an average transformation frequency
of 9.7% for japonica variety Taipei 309. A tight selection procedure using 50 mg/l of hygromycin B successfully prevented
the growth of nontransformed tissues. Analysis of the T0 transgenic rice plants revealed that more than 97% of the transgenic
plants were morphologically normal and more than 80% were at least partially fertile. The hygr trait was inherited as a dominant trait in a Mendelian manner in 8 out of 11 transgenic events assayed. Thirty-seven out
of fifty transgenic plants were estimated to contain no more than five copies of the transgenes. In six out of seven transformation
events, unlinked, co-transformed genes co-segregated in the T1 generation. The hygr trait has been stably inherited to the T4 generation. No chimerical transgenic plant has been found in an intensive search.
Novel phenomena observed in transgenic rice plants are also reported. 相似文献
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Advantages and disadvantages of using PCR techniques to characterize transgenic plants 总被引:3,自引:0,他引:3
Wassenegger M 《Molecular biotechnology》2001,17(1):73-82
The polymerase chain reaction (PCR) revolutionized molecular biology to a similar extent as the discovery of plasmids and
restriction endonucleases. However, there are some limitations to the use of PCR. Transgenic plants containing potato spindle
tuber viroid (PSTVd) cDNA constructs, demonstrated to become de novo methylated upon PSTVd infection, represent a good example to illustrate the advantages of PCR. PSTVd is a 359 nt long autonomously
replicating plant pathogenic RNA where all of its enzymatic requirements are entirely provided by the host cell. In addition,
viroids that propagate without a DNA intermediate barely tolerate nucleotide substitutions of their RNA genome without losing
infectivity. PCR is the method of choice to characterize the sequence context of genome-integrated viroid cDNA or of reverse
transcribed PSTVd RNA, and can hardly be replaced by any alternative procedure. Furthermore, the precise examination of DNA
methylation patterns (genomic sequencing) is entirely dependent on PCR. In contrast, the use of PCR is critical for the determination
of copy number and arrangement of transgene constructs. Here, the advantages and disadvantages of PCR are discussed and protocols
for PCR amplification of cDNA, genomic DNA, and bisulfite-treated DNA from transgenic plants are presented. 相似文献
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S. R. Min J. W. Woo W. J. Jeong S. K. Han Y. B. Lee J. R. Liu 《Biologia Plantarum》2006,50(1):131-134
Shoot apical meristem-derived calli were transformed with a hLF cDNA in an attempt to produce human lactoferrin (hLF) in transgenic
cell suspension cultures of sweet potato [Ipomoea batatas (L.) Lam.]. Calli were bombarded with tungsten particles coated with the binary vector pLSM1 containing a hLF cDNA under
the control of the 35S promoter and the neomycin phosphotransferase gene as a selection marker. Calli were then transferred
to Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 100 mg dm−3 kanamycin. Kanamycin-resistant calli were selected at four-week intervals and subcultured. Cell suspension cultures were
established in liquid MS medium with 4.52 μM 2,4-D. Southern and Northern blot analyses confirmed that hLF cDNA was incorporated
into the plant genome and was properly expressed in the cells. ELISA analysis showed that transgenic cells produced hLF up
to 3.2 μg mg−1 (total protein). 相似文献
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A protocol for consistent, large-scale production of fertile transgenic rice plants 总被引:10,自引:0,他引:10
A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment
of embryogenic tissues (Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines
carrying the hygromycin phosphotransferase (hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates,
when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure,
transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0
plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model
for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies.
Received: 12 March 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998 相似文献
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基因枪轰击谷子幼穗获得转基因植株 总被引:6,自引:1,他引:5
以JQ-700型国产基因枪轰击豫谷2号谷子幼穗,在150mg/L卡那霉素选择培养基上筛选到908块抗性愈伤组织,其中,绿芽块愈伤5块,共分化出16株绿苗,组织化学检测GUS表达,获得3株阳性植株,Southern杂交证明1株为阳性。以轰击总外植体计算的转基因植株频率为0.05%。 相似文献
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This work is the first to report the isolation and identification of bacteria colonizing the roots of the tropical epiphytic orchids Acampe papillosa (Lindl.) Lindl. and Dendrobium moschatum (Buch.-Ham.) Swartz. and bacteria inhabiting inner layers of the aerial and substrate roots of A. papillosa. We showed by the example of this epiphyte that associative bacteria are present in large amounts on the aerial but not the substrate roots. We isolated and identified bacteria from the substrate roots of D. Moschatum and from its growth substrate (pine bark). The structure of the intercellular matrix of the associative bacteria was studied.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 825–831.Original Russian Text Copyright © 2004 by Tsavkelova, Cherdyntseva, Netrusov. 相似文献