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。     

Variation in GUS activity in vegetatively propagated Hevea brasiliensis transgenic plants

Hevea brasiliensis transgenic plants are regenerated from transgenic callus lines by somatic embryogenesis. Somatic embryogenesis is not yet available for commercial propagation of Hevea clones, which requires conventional grafting of buds on rootstock seedlings (budding). The stability of transgene expression in budded plants is therefore necessary for further development of genetic engineering in rubber trees. Transgene expression was assessed by fluorimetric beta-glucuronidase (GUS) activity in fully developed leaves of in vitro plants from transgenic lines and their sub-lines obtained by budding. A large variation in GUS activity was found in self-rooted in vitro plants of five transgenic lines, and the absence of activity in one line suggested transgene silencing. Beyond confirming transmissibility of the reporter gene by budding and long-term expression, a quantification of GUS activity revealed that greater variability existed in budded plants compared to self-rooted mother in vitro plants for three transgenic lines. Although somatic embryogenesis provided more stable GUS activity, budding remained an efficient way of propagating transgenic plants but transgene expression in budded plants should be verified for functional analysis and further development.     

Genetic analysis and epigenetic silencing of At4CL1 and At4CL2 expression in transgenic Arabidopsis

4-coumarate::CoA ligase (4CL) gene family members are involved in channeling carbon flow into branch pathways of phenylpropanoid metabolism. Transgenic Arabidopsis plants containing the At4CL1 or At4CL2 promoter fused to the beta-glucuronidase (GUS) reporter gene show developmentally regulated GUS expression in the xylem tissues of the root and shoot. To identify regulatory genes involved in the developmental regulation of At4CL and other phenylpropanoid-specific genes, we generated ethyl methyl sulfate mutagenized populations of At4CL1::GUS and At4CL2::GUS transgenic lines and screened approximately 16,000 progeny for reduced or altered GUS expression. Several lines with reproducible patterns of reduced GUS expression were identified. However, the GUS-expression phenotype segregated in a non-Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5-azacytidine (aza) treatment, suggesting inhibitory DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation of At4CL::GUS lines with further At4CL promoter constructs enhanced the GUS-silencing phenotype. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two different modes of silencing were engaged in the At4CL1::GUS and At4CL2::GUS silenced lines. While silencing in the seedlings of the At4CL1::GUS lines was root specific in seedlings, it affected all organs in the At4CL2::GUS lines. Also, At4CL1::GUS transgene silencing was confined to the transgene but At4CL2::GUS silencing extended to the endogenous At4CL2 gene. Organ-specific silencing of the At4CL1::GUS transgene cannot be explained by current models in the literature.     

The Effects of Chromosomal Rearrangements on the zeste-white Interaction in Drosophila melanogaster

Three gene systems have been shown to exhibit proximity-dependent phenotypes in Drosophila melanogaster: bithorax (BX-C), decapentaplegic (DPP-C) and white (w). In structurally homozygous genotypes, specific allelic combinations at these loci exhibit one phenotype, while in certain rearrangement heterozygotes the same allelic combinations exhibit dramatically different phenotypes. These observations have led to the suggestion that, through the process of somatic chromosome pairing, such loci are brought into sufficient proximity to permit effective passage of molecular information between homologues; rearrangement heterozygosity would then displace the homologues relative to one another such that this trans-communication is obviated. The genetic properties of the proximity-dependent allelic complementation (termed transvection effects) at the BX-C and DPP-C, are quite similar. Chromosomal rearrangements which disrupt transvection possess a breakpoint in a particular segment of the chromosome arm bearing the transvection-sensitive gene (arm 2L for the DDP-C and 3R for the BX-C); this segment of each arm has been termed the critical region by Lewis (1954). As determined by cytogenetic analysis of transvection-disrupting rearrangements, the critical regions for the BX-C and DDP-C transvection effects extend proximally from these loci for several hundred polytene chromosome bands (Lewis 1954; Gelbart 1982). The interaction between the zeste and white loci appears to depend upon the proximity of the two w+ alleles. By use of insertional duplications, displacement of w+ homologues has been shown to interfere with the zeste-white interaction. In contrast to transvection at bithorax and decapentaplegic, however, only breakpoints in the immediate vicinity of the white locus can disrupt the zeste-white interaction (Gans 1953; Green 1967; Gelbart 1971; this report). In this report, we investigate the basis for the difference in the size of the BX-C and DPP-C critical regions from that of white. We test and eliminate the possibility that the difference is due to the presence near the white locus of a site which mediates somatic chromosome pairing. Rather, our evidence strongly suggests that the zeste-white interaction is, at the phenotypic level, much less sensitive to displacement of the homologous genes than is transvection at either the BX-C or DPP-C. We also show that many of the breakpoints in the vicinity of the white locus do not behave as if they are disrupting a critical region for somatic chromosome pairing. Given these results, we suggest that the zeste-white interaction and transvection are two different proximity-dependent phenomena.     

Effect of mutations in the genes su(Hw) and mod(mdg4) on transvection between alleles of the Yellow locus in Drosophila melanogaster]

A typical example of transvection is a complementation between alleles in the yellow locus: y2 (mdg4 insertion inactivating certain y-enhancers) and y1 (deletion of the y-promoter but not of the enhancer). Transvection was explained by trans-activation of promoter in y2-allele by enhancer of y1-allele. Here we found that the mutation mod(mdg4)1u1 in the modifier of mdg4 locus (a regulatory gene controlling, together with suppressor of Hairy wing) expression of (mdg4) completely suppress the complementation. Removal of an acidic domain from su(Hw) protein product in su(Hw)j mutation partially suppress the complementation. We also have found that mod(mdg4)1u1 mutation trans-inactivates the yellow allele with a wild type phenotype (y+2MC) in heterozygote with the y2 allele, i.e. the negative transvection takes place. In this case, deletion removing an acidic domain even in one copy of su(Hw) suppresses the effect of mod(mdg4)1u1 mutation.