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

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。     

人血栓调节蛋白基因的表达及转基因小鼠的建立

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。     

药物防治蝇害的研究进展

蝇害已经成为危害规模化畜禽养殖的重要问题。文章回顾了防治蝇害的三代杀虫剂的发展过程和存在的问题 ;展望了作为新一代环境友好防治蝇害药物的昆虫生长调节剂的开发前景。     

转基因植物对蜜蜂的安全性（下）

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     

我国南方部分地区实蝇记述及2种中国新记录

1980～1988年,曾用地中海实蝇、瓜实蝇和桔小实蝇(Trimedlure,Cuelure 和 Methy eugonol)性引诱剂,和野外采果相结合的办法,进行了实蝇的调查,所采集的标本,已鉴定出16种,其名称是;桔小实蝇、芒果实蝇、番石榴实蝇、辣椒实蝇、颜带实蝇、鳄梨实蝇、瓜实蝇、南瓜实蝇、颜黑纹实蝇、具条实蝇、拟具条实蝇、腹眼实蝇、二态实蝇、蜜柑大实蝇、桔大实蝇和黑肩光顶实蝇.内鳄梨实蝇和腹眼实蝇2种为中国新记录.在这次调查中,未发现地中海实蝇(Ceratitiscapitata Wied.)。     

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

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.     

Genetic Engineering Approaches to Pig Production*

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.     

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

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.     

Engineering of a Single Chain Variable Fragment Antibody Specific for the Citrus tristeza virus and its Expression in Escherichia coli and Nicotiana tabacum

Citrus tristeza virus (CTV) is one of the most destructive citrus virus diseases in the world. The construction of an engineered antibody, EMBL accession number AJ278109, able to specifically recognize its antigen, i.e. the coat protein of CTV, directly on infected plant material without any purification or manipulation of the entire woody plant. The potential uses of this engineered antibody are discussed.