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The functional role of theNicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools. 相似文献
3.
Transgenic Arabidopsis thaliana plants containingthe Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increasedcytokinin (CK) levels on the development of this rosette plant species. Inthreeindependently transformed lines (ipt-156, 158 and 161),Arabidopsis plants had smaller leaves, an underdevelopedroot system and decreased apical dominance in inflorescence stems. The smallertransgenic leaves were highly serrated along the margins, pale green and hadpointed leaf tips. In cross section, transgenic leaves had smaller cells andirregularly shaped epidermal cells. In the ipt-161 line,leaves and hypocotyls frequently exhibited purple color due to anthocyaninproduction. The most severe phenotype was observed in tissue cultureconditions,while growth in soil reduced or eliminated some phenotypic effects. Compared toC24 wild type plants, ipt-161 plants accumulated zeatinandzeatin riboside with an approximate 10-fold increase in the total pool of CKs.Astudy of the progeny resulting from crosses between theipt-161 transgenic line and the ethylene insensitivemutants ein1, ein2 andeti5 suggested that part of the altered developmentexhibited by the ipt transgenic plants was caused byincreased ethylene levels. 相似文献
4.
We exploited leaves of tobacco (Nicotiana tabacum L., cv. Wisconsin 38) with introduced chimeric construct consisting of SAG12 promoter fused with ipt gene for cytokinin synthesis and therefore prolonged life-span. As a control we used its wild type. In 12-week-old plants,
the first leaves of control plants showed senescence symptoms at the time of sampling. Carotenoid content decreased with increasing
leaf age both in control and in transgenic plants. On the other hand, the first leaves of transgenic plants demonstrated better
antioxidant capacity represented by carotenoids compared to the leaves of control plants of the same age. They stayed still
green at this age. 相似文献
5.
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
6.
Cytokinins play important roles in regulating plant growth and development. A new genetic construct for regulating cytokinin content in plant cells was cloned and tested. The gene coding for isopentenyl transferase (ipt) was placed under the control of a 0.821 kb fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene promoter from Lycopersicon esculentum (LEACO1) and introduced into Nicotiana tabacum (cv. Havana). Some LEACO10.821 kb-ipt transgenic plant lines displayed normal shoot morphology but with a dramatic increase in the number of flower buds compared to nontransgenic plants. Other transgenic lines produced excessive lateral branch development but no change in flower bud number. Isolated leaves of transgenic tobacco plants showed a significantly prolonged retention of chlorophyll under dark incubation (25°C for 20 days). Leaves of nontransformed plants senesced gradually under the same conditions. Experiments with LEACO10.821 kb-gus transgenic tobacco plants suggested auxin and ethylene involvement in induction of LEACO10.821 kb promoter activity. Multiple copies of nucleotide base sequences associated with either ethylene or auxin response elements were identified in the LEACO10.821 kb promoter fragment. The LEACO10.821 kb-ipt fusion gene appears to have potential utility for improving certain ornamental and agricultural crop species by increasing flower bud initiation and altering branching habit. 相似文献
7.
Timothy J. Strabala Sebastian Y. Bednarek Gregory Bertoni Richard M. Amasino 《Molecular & general genetics : MGG》1989,216(2-3):388-394
Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes. 相似文献
8.
A cytokinin biosynthetic gene encoding isopentenyl transferase (ipt) was cloned with its native promoter from Agrobacterium tumefaciens and introduced into tobacco plants. Indolebutyric acid was applied in rooting medium and morphologically normal transgenic
tobacco plants were regenerated. Genetic analysis of self-fertilized progeny showed that a single copy of intact ipt gene had been integrated, and T2 progeny had become homozygous for the transgene. Stable inheritance of the intact ipt gene in T2 progeny was verified by Southern hybridization. Northern blot hybridization revealed that the expression of this ipt gene was confined in leaves and stems but undetectable in roots of the transgenic plants. Endogenous cytokinin levels in
the leaves and stems of the transgenic tobaccos were two to threefold higher than that of control, but in roots, both the
transgenic and control tobaccos had similar cytokinin levels. The elevated cytokinin levels in the transgenic tobacco leaves
resulted in delayed leaf senescence in terms of chlorophyll content without affecting the net photosynthetic rate. The root
growth and morphology of the plant were not affected in the transgenic tobacco. 相似文献
9.
Transgenic tomato plants were produced with the isopentenyl transferase gene (ipt) ligated to a promoter that is active exclusively in sink tissue. Initially, transgenic plants had smaller, round-scale leaves,
swollen stems, and exhibited early development of lateral shoots compared to wild type. Expression of the ipt gene resulted in the formation of unbranched roots on cuttings and delayed senescence in excised leaves. Callus and root
formation occurred on excised leaves and leaf discs during dark incubation. The retention percentage of chlorophyll, as well
as cytokinin in excised leaves or discs was significantly greater than wild type. Transgenic tomato fruit had elevated levels
of cytokinins in the first days after fruit set and these levels were maintained longer during fruit development. 相似文献
10.
Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers. 相似文献
11.
Samanta Zelasco Valentina Ressegotti Massimo Confalonieri Daniela Carbonera Paolo Calligari Martina Bonadei Stefano Bisoffi Keiko Yamada Alma Balestrazzi 《Plant Cell, Tissue and Organ Culture》2007,91(1):61-72
Genetic transformation of an elite white poplar genotype (Populus alba L., cv. ‘Villafranca’) was performed with MAT vectors carrying the ipt and rol genes from Agrobacterium spp. as morphological markers. The effects associated with the use of different gene promoters and distinct in vitro regeneration
protocols were evaluated. Poplar plantlets showing abnormal ipt and rol phenotypes were produced only in the presence of exogenous growth regulators. The occurrence of abnormal ipt and rol phenotypes allowed the visual selection of transformants. The ipt-type MAT vector pEXM2 was used to monitor the activity of the yeast site-specific recombination R/RS system in the transformed
white poplar cells. Results from these experiments demonstrated that recombinase-mediated excision events occurred during
the early stages of in vitro culture, thus causing the direct production of ipt marker-free transgenic plants with normal phenotype at an estimated frequency of 36.4%. Beside this unexpected finding, transgenic
ipt-shooty plants were obtained at a frequency of 63.6% and normal shoots were subsequently recovered after a prolonged period
of in vitro culture. Although the transformation efficiency observed in this study, using both ipt and nptII genes as selection markers, was similar to that previously reported with standard vectors carrying only the nptII gene, the easy identification of ipt transformants, the early recombinase-mediated excision events and finally the relatively short time period required to produce
ipt marker-free transgenic plants support for the choice of MAT vectors as a reliable strategy for the future production of marker-free
GM poplars. 相似文献
12.
Jane Murfett Paul R. Ebert Volker Haring Adrienne E. Clarke 《Plant molecular biology》1995,28(5):957-963
Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S
2 clone, with 1.6 kb of 5 sequence, nor the S
6 clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S
2 clone was not detected, however the S
6 clone was expressed (at low levels) in three out of four transgenic plants. An S
6-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves. 相似文献
13.
Sasha Daskalova Alex McCormac Nigel Scott Harry Van Onckelen Malcolm Elliott 《Plant Growth Regulation》2007,51(3):217-229
A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6 kb wheat high molecular weight
(HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco
the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20 days after anthesis (DAA), corresponding
to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble
carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on
protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in
the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to
increase storage assimilate accumulation without a detrimental effect on development. 相似文献
14.
The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade
adenine
- Ados
adenosine
- BA
6-benzylaminopurine
- C
control
- Con A
concanavallin A
- CP
cellulose phosphate
- IPT
isopentenyl transferase
- NAA
1-naphthylacetic acid
- NP
normal phase
- NPPU
N-(3-nitrophenyl)-N-phenylurea
- RIA
radioimmunoassay
- RP
reversed phase
We wish to thank Dr. J. Zwar for supplying phenylurea derivitives. 相似文献
15.
16.
Belinda Martineau Catherine M. Houck Raymond E. Sheehy William R. Hiatt 《The Plant journal : for cell and molecular biology》1994,5(1):11-19
This paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the ipt gene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit-specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to 100-fold higher than in control fruit. In the leaves of a fruit-bearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR-1 and chitinase mRNAs, which encode defense-related proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described. 相似文献
17.
Selma Gulbitti-Onarici Mohsin Abbas Zaidi Ibrahim Taga Sebahattin Ozcan Illimar Altosaar 《Molecular biotechnology》2009,42(3):341-349
Expression of cry1Ac gene from Bacillus
thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1
promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic
plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of
Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance
against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they
were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded
leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without
pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay. 相似文献
18.
The bacterial isopentenyl transferase (ipt) gene involved in cytokinin biosynthesis was fused with a seed-specific lectin promoter from soybean and introduced into
tobacco. Under the control of the lectin promoter, the expression of the ipt gene increased cytokinin levels and promoted cell division in the embryo in transgenic tobacco seeds. Compared with controls,
the number of plerome cell layers and the cell number of cotyledons and pleromes were significantly increased from 16 DAF
(days after flowering); the embryo diameter of transgenic tobacco was enlarged at 16, 19, and 21 DAF (16.1%, 12.7%, and 13.9%
increase, respectively). Furthermore, the soluble protein content of the transgenic mature seeds was increased by 9.8–22.2%
and the dry weight of transgenic tobacco seeds was increased by 8.8–21.8% compared with that of controls. The transgenic tobacco
seedlings also grew quickly and a greater increase in fresh weight compared with controls was observed at 20 and 35 days after
germination (average 14% and 8% increase above controls, respectively). 相似文献
19.
Wolfgang Heinemeyer Ilka Buchmann Dave W. Tonge John D. Windass Juliane Alt-Moerbe Elmar W. Weiler Thomas Botz Joachim Schröder 《Molecular & general genetics : MGG》1987,210(1):156-164
Summary
Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.Abbreviations DMAPP
dimethylallylpyrophosphate
- iP
isopentenyladenine
- iPA
isopentenyladenosine
- iPMP
isopentenyladenosine 5-monophosphate
- IPT
isopentenyltransferase
-
trans-Z
trans-zeatin 相似文献
20.
Mariya Khodakovskaya Radomira Vaňková Jiři Malbeck Aizhen Li Yi Li Richard McAvoy 《Plant cell reports》2009,28(9):1351-1362
The cytokinin biosynthesis gene, isopentenyl transferase (ipt), under the control of an 821 bp fragment of the LEACO1 gene promoter (from Lycopersicon esculentum) was introduced into Dendranthema × grandiflorium ‘Iridon’ (chrysanthemum). LEACO10.821kb-ipt transgenic lines grown in the vegetative state, exhibited a range of phenotypic changes including increased branching
and reduced internode lengths. LEACO10.821kb-ipt transgenic lines grown in the generative state, exhibited increased flower bud count that ranged from 3.8- to 6.7-times
the number produced by wild-type plants. Dramatic increases in flower number were associated with a delay of flower bud development
and a decrease in flower bud diameter. RT-PCR analysis indicated differences in ipt gene expression between individual transgenic lines that exhibited a range of phenotypes. Within an individual transgenic
line, RT-PCR analysis revealed changes in ipt gene expression at different stages of generative shoot development. Expression of ipt in transgenic lines correlated well with high concentrations of the sum total to bioactive cytokinins plus the glucosides
and phosphate derivatives of these species, under both vegetative and generative growth conditions. In general, transgenic
lines accumulated higher concentrations of both storage-form cytokinins (O-glucosides) and deactivated-form cytokinins (N-glucosides) in generative shoots of than in vegetative shoots. Based on the range of phenotypes observed in various transgenic
chrysanthemum lines, we conclude that the LEACO1
0.821kb
-ipt gene appears to have great potential for use in ornamental crop improvement.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献