转基因甘蔗BtG-2的T-DNA侧翼序列分析及其转化事件特异性检测     

冯翠莲  万玥  冯小艳  王俊刚  赵婷婷  王文治  沈林波  张树珍 《热带作物学报》2021,42(9):2468-2477

转基因甘蔗BtG-2是利用农杆菌介导法把Cry1Ac-2A-gna融合抗虫基因导入‘新台糖22号’的转基因甘蔗株系,具有良好的抗虫特性和农艺性状。为了明确转基因甘蔗BtG-2的分子特征及其检测方法,推进其生物安全性评价工作,以BtG-2的T2代为研究材料,利用Southern杂交检测外源基因在转基因甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立了该转化体高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BtG-2株系;经过3次的热不对称巢式PCR扩增,获得外源基因T-DNA左边侧翼序列984 bp和右边侧翼序列705 bp;以这2个序列和相应的T-DNA的左右端序列分别设计3对检测引物对,建立了BtG-2株系的转化事件特异性PCR检测方法,扩增效率最高的引物对LS011/LA451和RS160/RA588分别扩增到440 bp和428 bp的特异片段。其中T-DNA左侧设计的LS011/LA451检测引物对扩增的灵敏度高、特异性强,能够在甘蔗BtG-2基因组DNA相对含量为0.1%的模板中检测出转基因目的成分,相当于9个单倍体基因组拷贝数。本研究完成了转基因株系BtG-2的分子特征及其转化事件特异性检测,为该转基因甘蔗及其衍生产品的检测和身份识别提供技术依据。  相似文献   

转基因动物研究进展   总被引：4，自引：0，他引：4  

胡建宏李青旺  王立强江中良于永生  解广勤 《家畜生态》2004,25(2):50-53

转基因动物是现代生物技术中一个极其重要的研究领域，目前已经有转基因小鼠、兔、绵羊、山羊、猪、牛、鸡和鱼等多种转基因动物问世。本文综述了转基因动物的制作方法、转基因动物的应用研究以及所取得的重要成就，并指出了转基因动物存在的主要问题，展望了其发展前景。  相似文献   

人血栓调节蛋白基因的表达及转基因小鼠的建立     

陈红英  苟克勉  王晓琳  陈学进  黄秀英  孙方臻 《中国兽医学报》2003,23(2):115-118

用PCR方法从人的基因组DNA中扩增了人血栓调节蛋白（hTM)基因，将其克隆到带有人EF－1α启动子的pEF-neo哺乳动物表达载体上，得到表达质粒pEF-TM。在转染pEF-TM的COS－1细胞中，hTM得到了瞬时表达，并且正确地定位到细胞膜上。用pEF-TM转染猪内皮细胞（PEC），经G418筛先得到稳定转染的克隆。凝血活性测定结果显示，表达hTM的PEC凝血时间明显延长，表明其抗凝血能力增强。通过显微注射方法，得到了1只F0代hTM转基因小鼠。Southern杂交结果表明，该转基因小鼠整合了9个拷贝的pEF-TM DNA,并能将外源DNA遗传给后代。  相似文献   

转基因植物对蜜蜂的安全性（下）     

黄少康 《蜜蜂杂志》2001,(10):5-6

转基因植物已在全球范围内大面积推广应用 ,全球 5大转基因植物中的油菜、棉花、玉米均是主要的蜜粉源植物 ,对养蜂生产有重要意义 ,其对蜜蜂的安全性问题值得关注。 2种主要的抗虫 (Bt毒蛋白、蛋白酶抑制剂 )基因或抗真菌基因的产物只有在高浓度时才可能对工蜂的寿命、消化酶活性、行为等有不良影响。从目前的研究来看 ,转基因植物对蜜蜂是安全的  相似文献   

烤烟适宜施氮量试验初报   总被引：9，自引：3，他引：6  

刘诚  王振飞 《江西农业学报》2005,17(3):95-96

试验结果表明,烤烟施氮量以90.0~112.5kg/hm2为佳,在此条件下,烤烟的产量和产值最高。  相似文献   

Influence of the Indoleacetic Acid-Lysine Synthetase Gene (iaaL) of Pseudomonas syringae subsp. savastanoi on Yield Attributes of Potatoes     

M. Fladung 《Plant Breeding》1993,111(3):242-245

The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase which conjugates free indoleacetic acid (IAA) with lysine. lAA-lys is biologically less active than free IAA. The iaaL coding region was expressed under the control of the cauliflower mosaic virus 35S promoter and transgenic potato plants were produced (Spena et al. 1991). 35S iaaL potato plants are characterized by increased internodal length and epinastic bending of older leaves. In three greenhouse experiments with plants grown in pots of different size and in two growth chamber experiments tuber number increased in iaaL transgenic plants compared to untransformed and vector-transformed controls of the same genotype. The increase in tuber numbers observed under controlled conditions was reflected in tuber yield which increased in the pot grown transgenics.  相似文献   

Genetic Engineering Approaches to Pig Production*     

B. Brenig  G. Brem 《Reproduction in domestic animals》1991,26(1):14-21

Modern biotechnology promises a number of new applications in animal breeding and production. Although conventional pig breeding has achieved a high level of efficiency and productivity numerous problems have been encountered with animal health and the loss of meat quality. Selection based on phenotypic performance data of individual animals does not take into account the importance of specific genes and their relevance within a complex regulatory system. In most cases it is therefore difficult to trace back the genetic origins of clinically important disorders. The application of genetic engineering techniques in pig production will facilitate diagnosis, improvement of productivity, and animal health by allowing direct genetic manipulation. Attention must be focussed on the physical and genetic analysis of the procine genome. The isolation and characterisation of genes, DNA-markers, polymorphic DNA-fragments, and their chromosomal assignment will be important prerequisites and tools for the elucidation of genetic disorders. Especially the detection of heterozygous carriers of recessive disorders and their elimination from the breeding stock will increase selection accuracy and decrease the generation intervals. But also the rapid and simple detection of infectious diseases, which is sometimes difficult if not impossible at present, will improve animal health and welfare. Although the production of transgenic animals either by DNA-microinjection into zygotes or the use of embryonal stem cells manipulated in vitro is less straightforward than DNA-based diagnosis it will play an important role in the direct manipulation of the porcine genome and genes. Breeding programmes including the use of transgenic livestock have already been developed. There is no doubt that genetic engineering has reached a degree of practical feasibility, allowing it to play an important role in pig breeding in particular and animal production in general.  相似文献   

Biochemical properties of the pathogenesis-related proteins from tobacco     

J. F. Antoniw  R. F. White 《European journal of plant pathology / European Foundation for Plant Pathology》1983,89(6):255-264

This review describes the discovery and identification of the pathogenesis-related proteins (PRs) from tobacco. In crude leaf extracts the PRs are distinguished from the proteins in uninfected plants by their solubility at pH 3, resistance to a range of proteases, and mobility in polyacrylamide gels upon electrophoresis (PAGE) in non-denaturing conditions. PAGE has been used as a qualitative and semi-quantitative assay for PRs, and their migration in gels made from different acrylamide concentrations has been used to identify charge and size isomers and electrophoretically identical PRs in different tobacco cultivars. The subunit composition and molecular weight (mol. wt) of the four PRs identified first in Xanthi-nc were determined by SDS-PAGE; staining the gels has shown that these same four proteins in Samsun NN did not contain carbohydrate, lipid or nucleic acid, nor were they isozymic forms of twenty five enzymes known to increase in activity following infection with TMV. Evidence suggests that most of the PRs in Xanthi-nc and Samsun NN are extracellular.The purification of several PRs from Xanthi-nc, Samsun NN and other tobaccos is described, as well as their mol. wt, subunit and amino acid composition. PRs 1a, b and c consist of a single polypeptide and have similar mol. wt and amino acid compositions. Antisera prepared against purified Xanthi-nc b₁ protein have been used to determine serological relationships between PRs and form the basis of a very sensitive quantitative assay using ELISA. The regulation of synthesis of some PRs has been shown to involve translational control.  相似文献   

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors     

S. Kreiah  M. L. Edwards  W. S. Hawes  A. T. Jones  D. J. F. Brown  W. J. McGavin  J. I. Cooper 《European journal of plant pathology / European Foundation for Plant Pathology》1996,102(3):297-303

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG initiation codon sequences added at the 5-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5 S/G site and a TAG stop codon sequence added at the 3-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.  相似文献   

Comparison of the effects of salicylic acid and ethephon with virus-induced hypersensitivity and acquired resistance in tobacco   总被引：4，自引：0，他引：4  

L. C. Van Loon  J. F. Antoniw 《European journal of plant pathology / European Foundation for Plant Pathology》1982,88(6):237-256

The induction of a hypersensitive reaction in Samsun NN tobacco by tobacco mosaic virus (TMV) at 20°C leads to the development of both localized and systemic acquired resistance, and is associated with the appearance of pathogenesis-related proteins (PR's) and large increases in peroxidase activity and ethylene production. Salicylic acid (SA) induced a similar resistance in treated plant parts and occasionally also in untreated upper leaves of plants of which three lower leaves had been injected. SA also induced the same four PR's, but these were confined to the treated leaves. Thus, the connection between the presence of PR's and the reduction of TMV multiplication and spread may not be direct.In contrast to TMV, SA did not stimulate ethylene production and hardly increased peroxidase activity. Induction of acquired resistance and PR's by SA developed equally well at 20°C and at 32°C. However, pricking leaves with needles moistened with the ethylene-releasing compound ethephon mimicked TMV infection in inducing acquired resistance and PR's in both treated and untreated leaves at 20°C, but not at 32°C. Ethephon increased peroxidase activity at both temperatures, but only at 20°C dit it induce changes in both the anodic and the cathodic isoenzymes that were similar to those induced as a result of TMV infection. SA induced PR's and reduced TMV multiplication in Samsun tobacco, and inhibited virus spread in Samsun NN at 32°C.These observation indicate that neither the induction of PR's, nor the development of acquired resistance is temperature-sensitive. On the other hand, the effects of ethephon are temperature-sensitive in the same way as the hypersensitive response to TMV. It can thus be hypothesized that ethylene, produced naturally during the hypersensitive reaction of tobacco to TMV, leads to the temperature-sensitive synthesis or release of a presumably benzoic acid-type compound that functions as the natural inducer of PR's and acquired resistance. Although vanillic acid has been shown to accumulate in hypersensitively reacting tobacco leaves, it produced none of the effects of SA, and thus cannot be the natural inducer.Samenvatting Inductie van een overgevoeligheidsreactie in Samsun NN-tabak door tabaksmozaïekvirus (TMV) bij 20°C leidt tot de ontwikkeling van een verworven resistentie die zowel lokaal als systemisch werkzaam is, en gaat samen met het verschijnen van pathogenesis-related proteins (PR's) en sterke toename in de activitieit van peroxidase en de produktie van ethyleen. Salicylzuur (SA) induceerde een vergelijkbare resistentie in behandelde plantedelen en af en toe ook in niet behandelde bovenbladeren van planten waarvan drie onderbladeren waren ingespoten. SA induceerde ook dezelfde vier PR's, maar deze waren beperkt tot de behandelde bladeren. Er bestaat dus geen directe samenhang tussen de aanwezigheid van PR's en de remming van de vermeerdering en uitbreiding van TMV in de plant.In tegenstelling tot TMV stimuleerde SA de ethyleenproduktie niet en verhoogde het de peroxidaseactiviteit nauwelijks. Inductie van verworven resistentie en PR's door SA trad even goed op bij 32°C als bij 20°C. Net als infectie met TMV leidde aanprikken van bladeren met naalden die gedoopt waren in een oplossing van ethefon — waaruit in het blad ethyleen vrijkomt — echter tot inductie van verworven resistentie en PR's in zowel behandelde als onbehandelde bladeren bij 20°C, maar niet bij 32°C. Ethefon verhoogde de peroxidaseactiviteit bij beide temperaturen, maar alleen bij 20°C induceerde het veranderingen in zowel de anodische als de kathodische isoënzymen die vergelijkbaar waren met die welke geïnduceerd werden als gevolg van infectie met TMV. SA induceerde PR's en verminderde de vermenigvuldiging van TMV in Samsun tabak, en remde de uitbreiding van het virus in Samsun NN bij 32°C.Deze waarnemingen tonen dat noch de inductie van PR's, noch de ontwikkeling van verworven resistentie een temperatuurgevoelig proces is. Daarentegen zijn de effecten van ethefon op dezelfde wijze temperatuurgevoelig als de overgevoeligheidsreactie op TMV. Men kan daarom veronderstellen dat ethyleen, dat op natuurlijke wijze geproduceerd wordt tijdens de overgevoeligheidsreactie van tabak op TMV, aanleiding geeft tot een temperatuurgevoelig proces, namelijk de synthese of het vrijkomen van een verbinding, vermoedelijk een benzoëzuurderivaat, dat fungeert als de natuurlijke inductor van PR's en verworven resistentie. Hoewel is aangetoond dat vanillinezuur zich ophoopt in overgevoelig reagerende tabaksbladeren, veroorzaakte deze verbinding geen enkel van de effecten van SA. Vanillinezuur kan dus niet de natuurlijke inductor zijn.  相似文献