VR-ACS6 is an Auxin-Inducible l-Aminocyclopropane-l-carboxylate Synthase Gene in Mungbean (Vigna radiata)

We have isolated four cDNA clones of ACC synthase from etiolatedmungbean seedlings treated with auxin. PVR-ACS2, pVR-ACS3 andpVR-ACS6 contained the same sequences as the previously reportedDNA fragments, pMAC2, pMAC3 (Botella et al. 1992b) and pMBAl(Kim et al. 1992), respectively. pVR-ACSl was identical withpAIM-1 (Botella et al. 1992a). VR-ACS6 was specifically induced in response to the auxin signal.The IAA-induction of VR-ACS6 was very rapid (within 30 min)and insensitive to cycloheximide treatment at concentrationsup to 100 µM. Significant accumulation of VR-ACS6 mRNAwas detected at 1 µM.IAA.The IAA-induced expression ofVR-ACS6 was suppressed by ABA and ethylene, but enhanced byBA. These characteristics of VR-ACS6 expression were well correlatedwith the physiological data of auxin-induced ethylene productionin mungbean hypocotyls. VR-ACS1 was strongly induced by cycloheximide, but was foundto be not auxin-specific. Inhibitors of either ethylene biosynthesis(AOA) or action (NBD) increased the basal level of VR-ACS1 mRNA. (Received May 7, 1996; Accepted November 25, 1996)     

An Auxin-Responsive Promoter Is Differentially Induced by Auxin Gradients during Tropisms

We constructed a chimeric gene consisting of a soybean small auxin up RNA (SAUR) promoter and leader sequence fused to an Escherichia coli [beta]-glucuronidase (GUS) open reading frame and a 3[prime] untranslated nopaline synthase sequence from Agrobacterium tumefaciens. This chimeric gene was used to transform tobacco by Agrobacterium-mediated transformation. In R2 etiolated transgenic tobacco seedlings, GUS expression occurred primarily in elongation regions of hypocotyls and roots. In green plants, GUS was expressed primarily in the epidermis and cortex of stems and petioles, as well as in elongation regions of anther filaments in developing flowers. GUS expression was responsive to exogenous auxin in the range of 10-8 to 10-3 M. During gravitropism and phototropism, the GUS activity became greater on the more rapidly elongating side of tobacco stems. Auxin transport inhibitors and other manipulations that blocked gravitropism also blocked the asymmetric distribution of GUS activity in gravistimulated stems. Light treatment of dark-grown seedlings resulted in a rapid decrease in GUS activity. Light-induced decay in GUS activity was fully reversed by application of auxin. Taken together, our results add support for the formation of an asymmetric distribution of auxin at sites of action during tropism.     

Differential expression of the auxin primary response gene MASSUGU2/IAA19during tropic responses of Arabidopsis hypocotyls

We have examined the expression pattern of an auxin primary response gene, MSG2/IAA19 , during photo- and gravitropic responses of hypocotyls using a transgenic Arabidopsis harboring MSG2/IAA19 promoter::GUS . The upper portion of most etiolated hypocotyls showed uniform β-glucuronidase (GUS) staining with the strongest activity in the pericycle. When hypocotyls were irradiated with unilateral blue light, GUS activity on the concave side of hypocotyls was decreased, resulting in differential GUS staining with a stronger signal on the convex side. The number of differentially stained hypocotyls peaked at 24 h after the onset of the phototropic stimuli, while hypocotyl curvature continued to increase for the entire 36-h experimental period. This result suggests that the MSG2/IAA19 expression precedes the phototropic responses. When seedlings were grown under dim white light, their hypocotyls displayed almost no GUS activity. The light-grown hypocotyls also showed differential GUS staining after phototropic stimuli as result of the increase in GUS activity on the convex side of hypocotyls, especially in the epidermis, the outer cortex and pericycle, although GUS activity was much weaker than that observed in etiolated hypocotyls. Similar but less obvious differential staining was obtained for gravitropic response of hypocotyls. Considering the recent finding that Aux/IAA proteins are immediate targets of the auxin F box receptors, MSG2/IAA19 is likely to act as one of master genes for tropic responses.     

Extracellular Calmodulin stimulates RbcS-GUS expression of etiolated transgenic tobacco plants in full darkness

We constructed a plant transformation intermediate vector pBZ3, which contains the RbcS-GUS chimeric gene and this was transferred into tobacco NC89 leaf discs. The RbcS promoter maintained photospecificity since we observed strong GUS-staining from light-grown transgenic seedlings while no visible GUS activity was found from etiolated seedlings. When purified CaM was injected into the hypocotyls of these etiolated seedlings, the promoter became functional even under complete darkness. Northern blot analysis showed that both green seedlings and CaM-treated etiolated seedlings contained significantly higher amount of GUS mRNA when compared to that of untreated etiolated seedlings. The level of active calmodulin in etiolated seedlings was found much lower than that in green seedlings as determined by Western blot analysis. Our results indicated that CaM is involved in photoregulated gene expression.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl-transferase (FTase) gene was examined using transgenic expression of the β-glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

The first intron of rice EPSP synthase enhances expression of foreign gene

Translatable exon sequences in pre-mRNA often are separated by non-coding introns in eu-karyotic genomes. The removal of non-coding introns from pre-mRNA and the splicing together of translatable exons sequence is an essential requirement of gene expression. DNA size of introns in a gene is 5—10 times larger than that of exon, which can store more information and is helpful for a gene during evolution[1]. In many experiments on gene expression, it is indispensable for a gene to be expresse…     

The first intron of rice EPSP synthase enhances expression of foreign gene

The first intron (EPI) of rice 5-enolpyruvylshikimate 3-phosphate synthase gene was isolated by PCR from one clone with genomic EPSP synthase gene. Sequence analysis showed that the first intron is 704 bp in length with 36.2% G+C content. To investigate its effect on expression of foreign gene, we inserted the first intron between CaMV35S promoter and β-glucuronidase (GUS) gene. The transient expression results showed that GUS could be expressed effectively with EPI. The GUS activity in transgenic tobacco shows that the EPI can greatly enhance the expression level of β-glucuronidase (P < 0.01) compared with transgenic tobacco without the first intron, and 3-to 6-fold increase in GUS activity in some transgenic tobaccos. Northern blot indicated the first intron was spliced from GUS pre-mRNA, and the steady-state mRNA levels of GUS with EPI in transgenic tobaccos were higher than that in transgenic tobacco without EPI, which suggested that the first intron of EPSP was a non-translated intron.     

高羊茅FaChit1基因启动子的功能分析

用含有不同长度FaChitl基因启动子区域与GUS基因融合构建植物表达载体pFaChitlP—I、pFaChitlP-Ⅱ以及pFaChitlP-Ⅲ并分别对烟草进行转化,经真菌激发子、干旱、机械损伤以及乙烯等多种胁迫处理后测定GUS活性。启动子缺失分析实验结果显示,真菌激发子对FaChitl基因启动子所介导的GUS诱导表达效果最强,而机械损伤只能微弱地诱导GL靥基因表达;FaChitl基因启动子-651bp以内的序列均能介导GUS基因的诱导表达,同时-935bp与-233bp之间的区域是该启动子响应真菌激发子、乙烯以及机械损伤胁迫所必需的。表明FaChitl启动子是一个多胁迫诱导型启动子。     

The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants

A series of 5′ deletions of the pea plastocyanin gene (petE) promoter fused to the β-glucuronidase (GUS) reporter gene has been examined for expression in transgenic tobacco plants. Strong positive and negative cis-elements which modulate quantitative expression of the transgene in the light and the dark have been detected within the petE promoter. Disruption of a negative regulatory element at ?784 bp produced the strongest photosynthesis-gene promoter so far described. Histochemical analysis demonstrated that all petE-GUS constructs directed expression in chloroplast-containing cells, and that a region from ?176 bp to +4 bp from the translation start site was sufficient for such cell-specific expression. The petE-promoter fusions were expressed at high levels in etiolated transgenic tobacco seedlings but there was no marked induction of GUS activity in the light. The endogenous tobacco plastocyanin genes and the complete pea plastocyanin gene in transgenic tobacco plants were also expressed in the dark, but showed a three- to sevenfold increase in the light. This indicates a requirement for sequences 3′ to the promoter for the full light response of the petE gene.     

Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco

The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter. To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco. The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves. The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.