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1.
There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) inArabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5′ region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants byAgrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5′ coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5′ coding region of PAH or PAI3 was 60–100-fold higher than that without the corresponding 5′ region. However, the effect of 5’ coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by the 5′ region of PAI genes have the function of PTP. 相似文献
2.
There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5' region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5' coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5' coding region of PAI1 or PAI3 was 60—100-fold higher than that without the corresponding 5' region. However, the effect of 5' coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by t 相似文献
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The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed. 相似文献
6.
Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with β-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells
such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization
showed that expression of the serine racemase gene was not induced by l- and d-serine, light irradiation, biotic and abiotic stresses. 相似文献
7.
The tomato geneRSI-1 was previously identified as a molecular marker for auxin-induced lateral root initiation. We have further characterized
the expression mode of theRSI-1 gene in tomato andArabidopsis thaliana. Northern blot analyses revealed that the gene was induced specifically by auxin in tomato roots and hypocotyls. For experiments
with transgenic plants, the 5′ flanking region of theRSI-1 gene was linked to a GUS reporter gene, then transformed into tomato andArabidopsis. In these transgenic tomato plants, GUS activity was detected at the sites of initiation for lateral and adventitious roots.
Expression of the fusion gene was auxin-dependent and tissue-specific. This was consistent with results from the northern
blot analyses. In transgenicArabidopsis, the overall expression pattern of theRSI-GUS gene, including tissue specificity and auxin inducibility, was comparable to that in transgenic tomato seedlings. These results
indicate that an identical regulatory mechanism for lateral root initiation might be conserved in both plants. Thus, the expression
mode of theRSI-CUS gene inArabidopsis mutants defective in lateral root development should be investigated to provide details of this process. 相似文献
8.
Plant mitochondria contain an alternative oxidase (AOX) acting as a terminal electron acceptor of the alternative pathway
in the electron transport chain. Here we describe the production of inducible antisense Aox1a plants of Arabidopsis thaliana (L.) Heynh. and the procedures used to determine the resulting alternative pathway activity. The Arabidopsis Aox1a cDNA sequence was cloned behind a copper-inducible promoter system in the antisense orientation. Arabidopsis thaliana (Columbia) plants were transformed by in-planta vacuum infiltration with Agrobacterium containing the antisense construct. Whole-leaf ethanol production was used as a measure to investigate alternative pathway
activity in the presence of antimycin A. After 24 h, leaves from the copper-induced, antisense line F1.1 produced up to 8.8
times more ethanol (via aerobic fermentation) than the non-induced and wild-type leaves, indicating effective cytochrome pathway
inhibition by antimycin A and a decreased alternative pathway activity in induced F1.1 leaves. Transgene expression studies
also revealed no expression in non-induced leaves and up until 24 h post-induction. Copper-induced transgenic leaves were
less susceptible to alternative pathway inhibition than non-induced transgenic leaves, as seen via tissue-slice respiratory
studies, and mitochondrial respiration, using F1.1 cell cultures, also supported this. These results demonstrate the successful
production of a transgenic line of Arabidopsis in which the alternative pathway activity can be genetically manipulated with an inducible antisense system.
Received: 31 March 2000 / Accepted: 10 May 2000 相似文献
9.
Zhang J. Van Toai T. Huynh L. Preiszner J. 《Molecular breeding : new strategies in plant improvement》2000,6(2):135-144
Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T2 generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants. 相似文献
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11.
Iñaki Alvarez M. Isabel Geli Eulogio Pimentel Dolors Ludevid Margarita Torrent 《Planta》1998,205(3):420-427
We have previously shown that the maize (Zea mays L.) storage prolamine γ-zein, accumulates in endoplasmic reticulum-derived protein bodies in transgenic plants of Arabidopsis thaliana (L.) ecotype R+P. The retention of γ-zein in the endoplasmic reticulum was found to be mediated by structural features contained
in the polypeptide, an N-terminal proline-rich and a C-terminal cysteine-rich domain which were necessary for the correct
retention and assembly of γ-zein within protein bodies (M.I. Geli et al., 1994, Plant Cell 6: 1911–1922). In the present work
we incorporated in the γ-zein gene lysine-rich coding sequences which were positioned after the N-terminal proline-rich domain
and at five amino-acid residues from the C-terminus. The targeting of lysine-rich γ-zeins was analyzed by expression of chimeric
genes regulated by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic Arabidopsis plants. The lysine-rich γ-zeins were detected by immunoblotting and we found that these proteins were modified post-translationally
to reach their mature form. Subcellular fractionation and immunocytochemical studies demonstrated that glycosylated lysine-rich
γ-zeins were secreted to the cell wall of transgenic Arabidopsis leaf cells.
Received: 9 May 1997 / Accepted: 31 October 1997 相似文献
12.
Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase 总被引:3,自引:0,他引:3
Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants,
but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant
J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete
cDNA encoding the enzyme from A. thaliana was constitutively expressed in both A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged
plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels
were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate
levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS
might be a way of controlling defense dynamics in higher plants.
Received: 10 February 2000 / Accepted: 11 March 2000 相似文献
13.
In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These
plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected
under other abiotic stresses. Deletion analysis indicated that the region from −178 to −771 was essential for the promoter’s
response to high temperature. 相似文献
14.
Vorwerk S Biernacki S Hillebrand H Janzik I Müller A Weiler EW Piotrowski M 《Planta》2001,212(4):508-516
Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three
nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active
proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences.
All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective
substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low
affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate
breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion
to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum
around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed
(IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for
NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform
in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under
the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development
rather than during stratification.
Received: 13 May 2000 / Accepted: 14 August 2000 相似文献
15.
E. Van der Eycken N. Terryn J. L. Goeman G. Carlens W. Nerinckx M. Claeyssens J. Van der Eycken M. Van Montagu M. Brito-Arias G. Engler 《Plant cell reports》2000,19(10):966-970
Synthesis of five different Sudan-β-d-glucuronides (I, II, III, IV, and RedB) was performed by condensation of a set of red Sudan diazo dyes with methyl (1-deoxy-2,3,4-tri-O-acetyl-1-trichloroacetimidoyl-α-d-glucopyran)uronate. After the acid and alcohol groups had been deprotected, the resulting compounds were used for histochemical
localization of β-glucuronidase (GUS) activity in transgenic plants (Petunia hybrida, Arabidopsis thaliana, and Nicotiana tabacum) that contained the GUS reporter system. Because the cleavage of the β-glucuronide results in the liberation of an insoluble
Sudan dye, Sudan substrates gave no diffusion artifacts as described for the commonly used 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc). A comparison of assays with different Sudan glucuronides and X-gluc demonstrated that the SudanIV variant
is a valuable glucuronide substrate for the precise histochemical localization of GUS activity in transgenic plants.
Received: 9 December 1999 / Revision received: 25 January 2000 / Accepted: 26 January 2000 相似文献
16.
In silico analysis showed that the differentially expressed type 3 oil palm metallothionein-like genes MT3-A and MT3-B share at least 11 common putative promoter regulatory elements. The identified motifs include W-boxes, TATCCA element, binding
element for cytokinin response regulators and pollen-specific elements. A high degree of conservation was observed in their
genomic organisation where the coding regions are divided at two identical positions in both genes by two AT-rich introns.
Promoter activity of the MT3-B gene was analysed using a transient assay by bombarding oil palm tissue slices with a β-glucuronidase (GUS) gene construct
and a stable reporter assay by analysing GUS expression in transformed Arabidopsis thaliana plants. Transient expression analysis revealed MT3-B promoter activity in oil palm root tissues but not in fruit mesocarp at 12 weeks after anthesis and spear leaves. The T3
homozygous transgenic Arabidopsis plants, harbouring the MT3-B promoter/GUS construct, showed reporter activity in cotyledons and mature leaves with lower expression levels in root tissues.
The expression levels in the roots of the T3 homozygous transgenic plants increased five- and 2.5-folds when treated with
80 μM of Zn2+ and Fe2+, respectively. Altogether, these results indicate that the MT3-A and MT3-B promoter activities may be regulated by a variety of abiotic factors and MT3-B promoter may potentially be manipulated for use in plant genetic engineering for induced synthesis of gene product. 相似文献
17.
A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not
increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and
about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased
anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results
suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component
involved in regulating P nutrition.
Received: 21 March 2000 / Accepted: 15 August 2000 相似文献
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19.
The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their
light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile,
phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear
to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light,
as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome
B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported
that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants.
Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in
phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark
of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in
the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red
ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings.
Received: 12 July 1998 / Accepted: 13 August 1998 相似文献
20.
Studies of abscisic acid (ABA) and auxin have revealed that these pathways impinge on each other. The Daucus carota (L.) Dc3 promoter: uidA (-glucuronidase: GUS) chimaeric reporter (ProDc3:GUS) is induced by ABA, osmoticum, and the auxin indole-3-acetic acid (IAA) in vegetative tissues of transgenic Arabidopsis thaliana (L.) Heynh. Here, we describe the root tissue-specific expression of ProDc3:GUS in the ABA-insensitive-2 (abi2-1), auxin-insensitive-1 (aux1), auxin-resistant-4 (axr4), and rooty (rty1) mutants of Arabidopsis in response to ABA, IAA and synthetic auxins naphthalene acetic acid (NAA), and 2, 4-(dichlorophenoxy) acetic acid. Quantitative analysis of ProDc3:GUS expression showed that the abi2-1 mutant had reduced GUS activity in response to ABA, IAA, or 2, 4-d, but not to NAA. Similarly, chromogenic staining of ProDc3:GUS activity showed that the aux1 and axr4 mutants gave predictable hypomorphic ProDc3:GUS expression phenotypes in roots treated with IAA or 2, 4-d, but not the diffusible auxin NAA. Likewise the rty mutant, which accumulates auxin, showed elevated ProDc3:GUS expression in the absence or presence of hormones relative to wild type. Interestingly, the aux1 and axr4 mutants showed a hypomorphic effect on ABA-inducible ProDc3:GUS expression, demonstrating that ABA and IAA signaling pathways interact in roots. Possible mechanisms of crosstalk between ABA and auxin signaling are discussed. 相似文献