天然反义转录物及其调控基因的表达机制

天然反义转录(NATs)是一组编码蛋白质或非编码蛋白质的RNAs, 与其他(有义)转录物具有互补序列, 可以调节有义链的表达。这种调节可以发生在转录水平或转录后水平, 调节方式有转录干扰、RNA封闭、双链依赖机制或染色质重建(修饰)等。正义链和反义链分别加工成小RNAs调节基因表达, 也是NATs调节基因表达的重要方式, 如piRNAs的“乒乓机制”。实验或计算机研究已经证明了NATs在生物中广泛存在, 是一种重要的基因表达调节方式。文章论述了NATs的重要作用和机理, 重点论述了NATs的调节机制和相关的小RNAs。     

Flower-specific expression of the Agrobacterium tumefaciens isopentenyltransferase gene results in radial expansion of floral organs in Petunia hybrida

Biotechnology has the potential to modify commercially important traits of crops, such as fruit size and stress tolerance. To date, the floricultural industry has not profited significantly from these possibilities to manipulate, for example, flower size. Cytokinins are known to be involved in many aspects of plant development, including cell division. Increasing the amount of cytokinins has the potential to increase the size of an organ, such as the flower or the fruit. The Agrobacterium tumefaciens cytokinin biosynthesis gene isopentenyltransferase ( ipt ) has been shown to increase cytokinin levels when introduced into plants. Moreover, it has a dramatic effect on the vegetative development of plants. The expression of the ipt gene under the control of the flower-specific Arabidopsis APETALA3 promoter in petunia ( Petunia hybrida ) increases the flower size dramatically, but with no effect on vegetative development. The resulting transgenic plants produced flowers with larger corolla diameter and greater total floral fresh weight. This strategy has the potential for use in the production of ornamental crops with large flowers and crop species with larger fruit.     

Centromeric localization of an S-RNase gene in Petunia hybrida Vilm.

S-RNase has been identified to be an S-allele-specific stylar determinant contributing to the self-incompatibility response in Solanaceae. In order to examine the physical location of the S-RNase gene, multi-color fluorescence in situ hybridization (FISH) using the S ^B1 -RNase cDNA probe and ribosomal RNA gene (rDNA) probe was performed on an S ^B1 S ^B2 heterozygote of Petunia hybrida. The S ^B1 -RNase gene was detected as a doublet signal close to the centromere of chromosome III. Next, we performed FISH using a large genome probe prepared from a λSB1–311 clone (20 kb) which contains the S ^B1 -RNase gene and its 3′ flanking region. This probe hybridized to the centromeric regions of all P. hybrida chromosomes. Sequence analysis of the λSB1–311 clone revealed the presence of a repetitive sequence consisting of a novel 666 bp unit sequence. A subclone (pBS-SB1B5) containing this unit sequence also hybridized to all of the centromeric regions, confirming that this unit is the centromeric specific repetitive sequence. These data suggested that the S ^B1 -RNase gene is located very close to (within a distance of 12 kb from) the centromeric-specific repetitive sequence. Likewise, the pBS-SB1B5 probe hybridized to the centromeric regions of all chromosomes in P. littoralis, another Petunia species. However, the probe did not hybridize to the centromere of the chromosomes from other species in Solanaceae. These results suggested that this centromeric repetitive sequence might be a genus-specific one. Received: 3 December 1998 / Accepted: 8 December 1998<@head-com-p1a.lf>Communicated by F. Mechelke     

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon‐limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light‐dependent manner and may be required for processing the L11 r‐operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ～10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks.     

The role of SPY and its TPR domain in the regulation of gibberellin action throughout the life cycle of Petunia hybrida plants

SPY acts as a negative regulator of gibberellin (GA) action in Arabidopsis, but its mode of action and regulation are still unknown. SPY over-expression in transgenic petunia plants affected various GA-regulated processes, including seed germination, shoot elongation, flower initiation, flower development and the expression of a GA-induced gene, GIP. A similar phenotype was obtained when wild-type petunia plants were treated with the GA-biosynthesis inhibitor, paclobutrazol. The N-terminus of SPY contains tetratricopeptide repeats (TPR). TPR motifs participate in protein-protein interactions, suggesting that SPY is part of a multiprotein complex. To test this hypothesis, we over-expressed the SPY's TPR region without the catalytic domain in transgenic petunia and generated a dominant-negative SPY mutant. The transgenic seeds were able to germinate on paclobutrazol, suggesting an enhanced GA signal. We cloned the petunia SPY homologue, PhSPY, and showed that its mRNA level is not affected by GA or ABA. The results of this study support the role of SPY as a negative regulator of GA action, suggest that the TPR domain is required for the interaction with other proteins to form an active complex and indicate that different plants use similar mechanisms to transduce the GA signal.     

A novel petal up-regulated PhXTH7 promoter analysis in Petunia hybrida by using bioluminescence reporter gene

Flower opening is an important phenomenon in plant that indicates the readiness of the flower for pollination leading to petal expansion and pigmentation. This phenomenon has great impact on crop yield, which makes researches of its mechanism attractive for both plant physiology study and agriculture. Gene promoters directing the expression in petal during the petal cell wall modification and expansion when flower opens could be a convenient tool to analyze or monitor gene expression targeting this event. However, there are no reports of isolated gene promoters that can direct gene expression in petal or petal limb during the rapid cell wall dynamics when the flower opens. Xyloglucan endotransglucosylase/hydrolase 7 (XTH7), a cell wall modifying enzyme, was reported having up-regulated gene expression in the petal of Arabidopsis thaliana and Petunia hybrida. In this study, we fused a 1,904 bp length P. hybrida XTH7 promoter with a gene encoding a bright bioluminescent protein (Green enhanced Nano-lantern) to report gene expression and observed petal up-regulated bioluminescence activity by means of a consumer-grade camera. More importantly, this novel promoter demonstrated up-regulated activity in the petal limb of P. hybrida matured flower during flower opening. P. hybrida XTH7 promoter would be a useful tool for flowering study, especially for petal expansion research during flower opening.     

NEC1, a novel gene, highly expressed in nectary tissue of Petunia hybrida

To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RT-PCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating that the protein is incorporated into the cell membrane or the cytoplast membrane. Immunolocalization revealed that NEC1 protein is present in the nectaries. Northern blot analyses showed that NEC1 is highly expressed in nectary tissue and weakly in the stamen. GUS expression driven by the NEC1 promoter revealed GUS activity in the outer nectary parenchyma cells, the upper part of the filament and the anther stomium. The same expression pattern was observed in Brassica napus. GUS expression was observed as blue spots on the surface of very young nectaries that do not secrete nectar and do accumulate starch. GUS expression was highest in open flowers in which active secretion of nectar and starch hydrolysis had taken place. Ectopic expression of NEC1 resulted in transgenic plants that displayed a phenotype with leaves having 3-4 times more phloem bundles in mid-veins than the wild-type Petunia. The possible role of NEC1 gene in sugar metabolism and nectar secretion is discussed.     

Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants

Cytokinins (CKs) are phytohormones that play an important role in plant growth and development. Although the first naturally produced CK, zeatin, was isolated almost four decades ago, no endogenous gene has been shown to produce active CKs in planta. In an activation tagging experiment we have identified a petunia line that showed CK-specific effects including enhanced shooting, reduced apical dominance and delayed senescence and flowering. This phenotype correlated with the enhanced expression of a gene we labelled Sho (Shooting). Sho, which encodes a protein with homology to isopentenyl transferases (IPTs), also causes CK-specific effects when expressed in other plant species. In contrast to the ipt gene from Agrobacterium, which primarily increases zeatin levels, Sho expression in petunia and tobacco especially enhances the levels of certain N6-(delta2-isopentenyl) adenosine (2iP) derivatives. Our data suggest that Sho encodes a plant enzyme whose activity is sufficient to produce active CKs in plants.     

Regulation of gene action in Petunia hybrida: Unstable alleles of a gene for flower colour

Summary In a progeny of a selfed individual of the dark red-flowered cultivar Roter Vogel some white-flowered plants appeared as the result of a mutation of the genetic factor Anl involved in anthocyanin synthesis. The white flowers of these plants had red spots owing to back-mutations in the dermal cells of the young corolla.Owing to a striking unstability of the new allele of Anl, a number of mutants originated which differ mutually in the frequency of reversion, which expressed itself in the very substantial differences in the spot density of the limb of the corolla. Between a mean number of less than one spot per cm² of the limb and a mean number of over 10.000 spots/cm², a series of transitions was found.The reversions did not remain restricted to the young epidermis but also occurred in sporogenous tissues. This resulted in the appearance of selfcoloured red descendants of plants with red-spotted white flowers. There is a positive correlation between the spot density of the parent plants and the percentage of plants with completely red corollas.The red spots on the corolla usually have the same colour as the wild type (Roter Vogel), but occasionally mutants occur with paler spots, the colour varying from a very pale pink to a red nearly as deep as in the wild type. The selfcoloured descendants of such mutants also show this colour variation from pale pink to red.On the grounds of these observations a theory was formulated which postulates that the Anl locus consists of a structural gene responsible for an enzyme active during anthocyanin synthesis and a regulatory element built up from intermediate repetitive DNA. This regulatory element in turn is built up of two components, one of which, the mutator, decides the activation of the structural gene while the other, the expressor, modifies the rate of activation. The mutations must be considered representative of larger or smaller deletions within one or both of these components. Reversions are the result of the restoration of the deletions by means of an amplification of the repetitive DNA in dividing cells of the developing flower buds.     

On the origin of anthocyanin methyltransferase isozymes of Petunia hybrida and their role in regulation of anthocyanin methylation

Summary Four genes controlling anthocyanin methylation in flowers of Petunia hybrida have been described. Three of them, Mt2, Mf1 and Mf2, caused a dosage effect on anthocyanin methyltransferase activity and degree of methylation of anthocyanins. Antiserum raised against partially purified Mf2-enzyme precipitated three of the four anthocyanin methyltransferases. In two subspecies of one of the ancestral species of P. hybrida: Petunia integrifolia, different anthocyanin methyltransferases were found as determined by immunoprecipitation. The methyltransferase isozymes showed no differences in subcellular or tissue location, and had no physiologically important difference in time course of activity during bud development. The methylation-system in Petunia is discussed with regard to anthocyanin methylation in other plant species.     

A gene controlling rate of anthocyanin synthesis and mutation frequency of the gene An1 in Petunia hybrida

Summary The difference in colour intensity between flowers of sporogenic revertants of the white flowering lines W17 and W28 is caused by an incompletely dominant gene Inl. This gene is not linked to the anthocyanin gene Anl. In the dominant state Inl causes a 50% decrease in colour intensity of selfcoloured red flowers.Chromatographic analysis of anthocyanins of plants homozygous recessive or dominant for Inl showed that the same anthocyanins are produced in both genotypes (cyanidin-3-glucoside and cyanidin-3-diglucoside). Anthocyanin synthesis starts at the same stage of development of the flower in both genotypes. When the bud reaches a length of approximately 45 mm, however, anthocyanin synthesis in the Inl Inl line slows down.No influence of the gene Inl on the concentration of dihydroquercetin-7-glucoside in buds and flowers could be observed, which indicates that the influence of Inl on flower colour development is restricted to the last part of the biosynthesis of anthocyanins, i.e. the conversion of dihydroflavonols into anthocyanins.In addition to Inl having a decreasing effect on flower colour intensity, evidence is produced that the gene Inl also influences the reversion frequency of unstable alleles of the gene Anl.     

Inhibition of tobacco NADH-hydroxypyruvate reductase by expression of a heterologous antisense RNA derived from a cucumber cDNA: Implications for the mechanism of action of antisense RNAs

Tobacco plants were genetically transformed to generate antisense RNA from a gene construct comprised of a full-length cucumber NADH-dependent hydroxypyruvate reductase (HPR) cDNA placed in reverse orientation between the cauliflower mosaic virus 35S promoter and a nopaline synthase termination/polyadenylation signal sequence. In vivo accumulation of antisense HPR RNA within eight independent transgenic tobacco plants resulted in reductions of up to 50% in both native HPR activity and protein accumulation relative to untransformed tobacco plants (mean transgenote HPR activity=67% wild type, mean transgenote HPR protein=63% wild type). However, in contrast to previous reports describing antisense RNA effects in plants, production of the heterologous HPR antisense RNA did not systematically reduce levels of native tobacco HPR mRNA (mean transgenote HPR mRNA level=135% wild type). Simple regression comparison of the steady-state levels of tobacco HPR mRNA to those of HPR antisense RNA showed a weak positive correlation (r value of 0.548, n=9 ; n is wild type control plus eight independent transformants; significant at 85% confidence level), supporting the conclusion that native mRNA levels were not reduced within antisense plants. Although all transgenic antisense plants examined displayed an apparent reduction in both tobacco HPR protein and enzyme activity, there is no clear correlation between HPR activity and the amount of either sense (r=0.267, n=9) or antisense RNA (r=0.175, n=9). This compares to a weak positive correlation between HPR mRNA levels and the amount of HPR activity observed in wild-type SRI tobacco plants (r=0.603, n=5). The results suggest that in vivo production of this heterologous HPR antisense RNA is inhibitory at the level of HPR-specific translation and produces its effect in a manner not dependent upon, nor resulting in, a reduction in steady-state native HPR mRNA levels. In this context, the observed antisense effect appears to differ mechanistically from most antisense systems described to date.     

Embryo sac development is affected in Petunia inflata plants transformed with an antisense gene encoding the extracellular domain of receptor kinase PRK1

 In a previous study of the function of a pollen-expressed receptor kinase of Petunia inflata, PRK1, it was found that transgenic plants carrying an antisense-PRK1 gene were unable to transmit the transgene through either the male or, unexpectedly, the female. In this report, the nature of this female phenotype was studied using one of the transgenic plants, ASRK-13. Electron and light microscopic examination of the embryo sac and seed development of ASRK-13 and a wild-type plant revealed that embryo sac development of approximately half of the ovules of ASRK-13 was abnormal. The development of the affected embryo sacs was arrested at the late stages of megagametogenesis. The majority of the affected embryo sacs completed three rounds of mitosis normally, but failed to progress through the maturation stages when cell expansion, nuclear migration, and differentiation take place. The remaining small number of abnormal embryo sacs were arrested at either the four- or eight-nucleate stages. The ovules containing the defective embryo sacs apparently failed to be fertilized, resulting in degeneration of half of the seeds produced by ASRK-13. RNA gel blot analysis suggests that the PRK1 gene is expressed in the ovary, albeit at a much lower level than in the anther. The possibility that the antisense PRK1 gene is responsible for the abnormal embryo sac development is discussed. Received: 25 April 1997 / Revision accepted: 25 June 1997