Co-silencing of homologous transgenes in tobacco

Two transgenes inserted into different genomic positions can co-inactivate each other when they share homologous sequences while each of the two homologous transgenes is stably expressed in the absence of a second homologous copy. To evaluate the efficiency of such homology-dependent gene silencing (HDGS) effects, we have produced 19 tobacco transformants that contained a stably expressed NPTII transgene inserted into a single genomic locus, and have analysed the stability of each transgene in the presence of a second stably expressed homologous transgene. All transformants shared the coding region of the NPTII gene but individual transformants differed in transgene copy number, expression levels and in the continuity of the transgene homology due to the insertion of introns into the NPTII region as well as the use of different promoters and terminators for the design of the transgene constructs. We generated 189 progeny populations representing all possible dual combinations among the 19 lines and analysed the kanamycin resistance of 400 seedlings of each cross. Our data show (1) that gene silencing occurs at a relative low frequency when transgenic loci sharing an homology at the coding sequence level are combined, and (2) that neither the variation of this homology by insertion of introns in the coding sequence, or by changing the promoter and terminator of the construct, nor the variation in the expression level of the transgene, are decisive parameters modifying the efficiency of co-silencing between two NPTII transgenes.     

转基因植物内源基因与外源基因共抑制问题研究进展

在植物基因工程研究中,有时外源转化基因和植物中同源的内源基因的表达均被抑制了,这种现象被称为共抑制。引起该现象的原因包括许多因素,有外源基因和与之同源的内源基因RNA的转录量,DNA的甲基化,T-DNA的结构形式,RNA依赖的RNA聚合酶,非正常RNA以及非等位配对等。其具体机制仍不清楚。最近的研究结果表明转基因植物对病毒的抗性在某些方面与共抑制有相似之处。两者都表现为外源转化基因转录水平很高而稳定RNA的水平很低。本文综述了这方面的最新研究进展。     

植物转基因沉默研究进展

随着转基因技术在植物中的广泛应用,转基因沉默受到越来越多的重视。转基因沉默可发生在转录和转录后两种水平,其基本特征就是依赖于同源的重复序列。转基因的重复拷贝间,转基因与同源的内源基因间及RNA病毒与同源转基因间都会发生基因沉默。可能有不同的机制导致转基因沉默,本文综述了转基因沉默的机理研究及转基因沉默在植物抗病基因工程和植物功能基因组学方面的应用 。     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

Regulation of I-transposon activity in Drosophila: evidence for cosuppression of nonhomologous transgenes and possible role of ancestral I-related pericentromeric elements

We have previously shown that the activity of functional I retrotransposons (I factors) introduced into Drosophila devoid of such elements can be repressed by transgenes containing an internal fragment of the I factor itself and that this repressing effect presents the characteristic features of homology-dependent gene silencing or cosuppression. Here we show that the same transgenes can induce silencing of a nonhomologous reporter gene containing as the sole I-factor sequence its 100-bp promoter fragment. Silencing of the nonhomologous reporter gene shows strong similarities to I-factor cosuppression: It does not require any translation product from the regulating transgenes, sense and antisense constructs are equally potent, and the silencing effect is only maternally transmitted and fully reversible. A search for genomic I-like sequences containing domains with similarities to those of both the regulating and the reporter transgenes led to the identification of four such elements, which therefore could act as intermediates-or relays-in the cosuppression machinery. These results strongly suggest that ancestral transposition-defective I-related elements, which are naturally present in the Drosophila genome, may participate per se in the natural conditions of I-factor silencing.     

Transgene Coplacement and High Efficiency Site-Specific Recombination with the Cre/Loxp System in Drosophila

Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mos1 promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F(1) progeny. Excision in germ cells of the F(1) yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F(2) generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.     

Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants

Silencing ofNia host genes and transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobaccoNia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whetherNii host genes and transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a transgene consisting of the tobaccoNii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of theNii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing ofNii host genes and transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of transgene loci and/or on certain allelic or ectopic combinations of transgene loci. In one transformant carrying a single transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this transgene locus is brought into the presence of a non-allelic transgene locus by crossing, suggesting that a homozygous state is not absolutely required.