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.     

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 。     

RNA沉默机制及其抗病毒应用

RNA沉默是发生在植物 (转录后基因沉默或共抑制 )、动物 (RNA干扰 )和真菌 (消除作用 )等真核生物细胞中的一种对外源遗传因子 (转座子、转基因或病毒 )的特异性和高效率的降解机制。随着对植物病毒分子遗传学认识的加深和对寄主防御系统研究的深入 ,发现了许多控制植物病毒病的方法 ,不过迄今为止最为成功的是通过RNA沉默机制获取抗病毒工程植株。在陈述了RNA沉默机制的研究最新进展基础上 ,提出了如何充分利用该机制进行植物抗病毒转基因研究。     

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus

Constructs carrying the entire or part of the tobacco nitrate reductase cDNA (NIA) cloned between the promoter and terminator sequences of the 35S RNA of the cauliflower mosaic virus were introduced into tobacco, in an attempt to improve nitrate assimilation. Several transgenic plants that had elevated NIA mRNA and nitrate reductase (NR) activity were obtained. In addition, a few plants that exhibited a chlorotic phenotype characteristic of NR-deficient mutants were also obtained. One of these plants contained no NIA mRNA, no NR activity and accumulated nitrate. This phenotype was therefore assumed to result from co-suppression of 35S-NIA transgenes and host NIA genes. NR-deficient plants were also found among the progeny of transformants overexpressing NIA mRNA. Genetic analyses indicated that these NR-deficient plants were homozygous for the 35S-NIA transgene, although not all homozygous plants were deficient for NR. The ratio of normal to NR-deficient plants in the progeny of homozygous plants remained constant at each generation, irrespective of the state of expression of the NIA genes (active or inactive) in the previous generation. This ratio also remained unchanged when field trials were performed in two areas of France: Versailles and Bergerac. The analysis of homozygous plants revealed that co-suppression was reversible at some stage of sexual reproduction. Indeed, host genes and transgenes reactivated at each generation, and co-suppression always appeared after a lag period of normal growth, suggesting that the phenomenon is developmentaly regulated. We observed that the triggering of cosuppression was delayed when plants were initially grown under limited light and/or watered with limited nitrate supply (light and nitrate both being required for the expression of the host NIA genes). However, this delay did not affect the final ratio between normal and NR-deficient plants after transfer to nitrate-fertilized fields. Independent transformants exhibited either different co-suppression ratios or no co-suppression at all, irrespective of the transgene copy number, suggesting that genomic sequences surrounding the transgene might play a role in determining co-suppression.     

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。     

Transgene Induced Co-Suppression during Vegetative Growth in Cryptococcus neoformans

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (～50%), but at a much lower frequency during vegetative growth (～0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ～90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.