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。     

Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing

Tobacco plants transformed with the RNA polymerase (RdRp) gene of potato virus X (PVX) that are extremely resistant to infection by potato virus X have previously been described. The PVX-resistant plants accumulated the RdRp protein at a lower level than fully susceptible plants transformed with the same RdRp construct. In this paper the difference between the PVX-resistant and susceptible transformed plants is investigated and it is demonstrated that there are three associated phenotypes of the RdRp transgene that vary in parallel between transformed lines. These phenotypes are: accumulation of the transgenic RdRp RNA at a low level; strain-specific resistance to PVX; and the ability of the transgene to trans -inactivate homologous transgenes. This gene-silencing potential of the transgenes conferring PVX resistance was illustrated by analysis of progeny from a cross between a transformant that was extremely resistant to PVX and a second PVX-susceptible transformant. In other transformants, in which the resistance was less extreme, the same three phenotypes were associated but in a transgene dosage-dependent manner. The same association of strain-specific resistance and low-level accumulation of the transgenic RdRp RNA was observed with plants that were transformed with mutant or wild-type versions of the RdRp gene. Strain-specific resistance was also produced in plants transformed with untranslatable versions of the RdRp transgene. Based on these data it is proposed that homology-dependent gene silencing and transgenic resistance to PVX may be due to the same RNA-based mechanism. An undefined genomic feature is proposed to account for the variation in the resistance and trans -inactivation phenotypes of different transformants. It is further proposed that this genome feature influences a cytoplasmic mechanism that degrades RNA with sequence homology to the silencing transgene.     

Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes

Insertion of foreign DNA into plant genomes frequently results in the recovery of transgenic plants with silenced transgenes. To investigate to what extent regeneration under selective conditions limits the recovery of transgenic plants showing gene silencing in woody species, Mexican lime [ Citrus aurantifolia (Christm.) Swing.] plants were transformed with the p25 coat protein gene of Citrus tristeza virus (CTV) with or without selection for nptII and uidA. Strikingly, more than 30% of the transgenic limes regenerated under non-selective conditions had silenced transgenes, and in all cases silencing affected all the three transgenes incorporated. These results indicate that the frequency of transgene silencing may be greatly underestimated when the rate of silencing is estimated from the number of regenerants obtained under selective conditions. To our knowledge, this is the first report in which the frequency of gene silencing after transformation has been quantified. When the integration pattern of T-DNA was analyzed in silenced and non-silenced lines, it was observed that inverted repeats as well as direct repeats and even single integrations were able to trigger gene silencing. Gene silencing has often been associated with the insertion of DNA sequences as inverted repeats. Interestingly, here, direct repeats and single-copy insertions were found in both silenced and non-silenced lines, suggesting that the presence of inverted-repeat T-DNAs and the subsequent formation of dsRNAs triggering gene silencing cannot account for all silencing events.     

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.