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1.
The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous -glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.Abbreviations AS
Acetosyringone
- CaMV 35S P
Cauliflower mosaic virus 35S promoter
- CaMV 35S poly A
Cauliflower mosaic virus 35S poly A terminator
- CB
Citrus blight
- 2,4-D
2,4-Dichlorophenoxyacetic acid
- FMV
Figwort mosaic virus
- GUS
-Glucuronidase
- GUS gene
uidA
- IBA
Indole-3-butyric acid
- MES
2-(N-Morpholino) ethane sulfonic acid
- MSI
Inoculation medium
- MSP-10M
Plasmolysis solution with 10% maltose
- MSP-8S
Plasmolysis solution with 8% sucrose
- NAA
-Naphthaleneacetic acid
- NOS
Nopaline synthase
- NP
Nopaline synthase promoter
- NT
Nopaline synthase terminator
-
NPTII
Neomycin phosphotransferase II
- p12
Blight-associated protein p12 相似文献
2.
Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer 总被引:5,自引:0,他引:5
By using highly regenerative calluses, we developed a stable transformation system in garlic (Allium sativum L.). The temperature and number of days of co-cultivation with Agrobacterium tumefaciens was shown to be an important factor in transient expression of the uid A gene. After a culture period of 5 months in selection medium containing hygromycin, 20 shoots were induced from ca. 1000
calluses, among which 15 plants expressed β-glucuronidase activity upon staining with X-Gluc. Shoots developed into transgenic garlic after 1 month. Integration of the uid A gene was confirmed by Southern blot analysis for genomic DNA of transgenic garlic plants.
Received: 25 October 1999 / Revision received: 16 February 2000 / Accepted: 22 February 2000 相似文献
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5.
Shirasawa-Seo N Sano Y Nakamura S Murakami T Gotoh Y Naito Y Hsia CN Seo S Mitsuhara I Kosugi S Ohashi Y 《Plant cell reports》2005,24(3):155-163
The activity of a predicted promoter, PMC8, from Milk vetch dwarf virus was evaluated by comparing it with the cauliflower mosaic virus 35S RNA promoter (P35S) and PNCR, a promoter from Soybean chlorotic mottle virus. When the GUS fusion gene was introduced into tobacco, PMC8 showed a similar expression profile to P35S but with a more intense expression in proliferating tissues. The usefulness of PMC8 was confirmed by driving NPTII for selection of kanamycin-resistant tobacco plants with improved transformation efficiency. PMC8 was also effective in transgenic rice plants. Thus, PMC8 is useful as an alternative to P35S in both dicotyledonous and monocotyledonous plants, especially for gene expression in proliferating tissues. 相似文献
6.
Indrajit Dutta Prasenjit Saha Sampa Das 《In vitro cellular & developmental biology. Plant》2008,44(5):401-411
Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige
and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver
nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency
of stable transformation was found to be approximately 19% in the T
0 generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern
blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of
T
1 seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency
over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea. 相似文献
7.
The DIANTHIN gene encoding a ribosome-inactivating protein (RIP) from Dianthus caryophyllus L. was tested for negative selection in tobacco and rice. Tobacco leaf discs and scutellum-derived callus of rice were transformed
with Agrobacterium tumefaciens strain LBA4404 (pSB1, pJAS1). pJAS1 harbors the DIANTHIN gene under the control of the CaMV 35S promoter. Tobacco transformation efficiency, in comparison to pCAMBIA1301, was reduced
by 87 % in pJAS1-transformed leaf discs. The DIANTHIN gene proved to be completely toxic to tobacco as all the recovered hygromycin-resistant transgenic plants harbored truncated
T-DNAs with deletions of the DIANTHIN gene. Transformation of the DIANTHIN gene under a Mungbean yellow mosaic virus (MYMV)-inducible promoter did not cause any toxicity in tobacco as reflected by the recovery of transgenic tobacco plants
with the complete DIANTHIN gene. Transformation efficiency of pJAS1 did not decline in rice. Interestingly, all transgenic rice plants harbored the
complete DIANTHIN gene and expressed the gene. The T1 transgenic lines showed reduction of sheath blight symptom in the range of 29 to 42 %. The difference in the sensitivity
to DIANTHIN between tobacco and rice provides a new direction to study the mechanisms underlying RIP toxicity in plants. 相似文献
8.
C. Walter L. J. Grace A. Wagner D. W. R. White A. R. Walden S. S. Donaldson H. Hinton R. C. Gardner D. R. Smith 《Plant cell reports》1998,17(6-7):460-468
A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered
was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature
somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both
genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones.
Received: 9 May 1997 / Revision received: 18 September 1997 / Accepted: 18 October 1997 相似文献
9.
Summary An elite aspen hybrid (Populus × canescens × P. grandidentata) was transformed with Agrobacterium tumefaciens strain EHA105 that harbored a binary vector (pBI121) carrying the nptII gene under the nos promoter and tandem rolB-uidA (GUS) genes with the CaMV 35S or heat shock promoter. Among 32 independent kanamycin-resistant plants, 25 plants were confirmed
by polymerase chain reaction and Southern blot analyses to contain all three genes, whereas five plants contained only nptII or/and uidA genes and two plants had both the rolB and nptII or uidA genes. Integration of the rolB gene significantly increased rooting ability of hardwood cuttings. Heat shock-rolB-transformed plants rooted at significantly higher percentage than the CaMV 35S-rolB-transformed plants. Heat shock treatment further enhanced rooting of heat shock-rolB-transformed plants. Exposure to exogenous auxin did not significantly increase the rooting percentage of transgenic hardwood
cuttings, but increased the number of roots induced. This research shows great potential to improve rooting of hardwood cuttings
of difficult-to-root woody plants which are commercially important to the horticultural and forestry industry. The transgenic
plants with gain-of-function in hardwood-cutting rooting can facilitate research in the understanding of adventitious rooting
from hardwood cuttings of recalcitrant woody plants. 相似文献
10.
11.
The merC gene from Acidithiobacillus ferrooxidans functions as a mercury uptake pump. MerC protein localizes in the cytoplasmic membrane of plant cells. When Arabidopsis thaliana and tobacco plants were transformed with the merC gene under the control of the Cauliflower mosaic virus 35S promoter, the resulting overexpression of merC rendered the host plants hypersensitive to Hg2+ and they accumulated approximately twice as much Hg2+ ion as the wild type plants. Thus, bacterial mercuric ion transporters such as MerC may be useful molecular tools for producing transgenic plants that hyperaccumulate Hg2+ ion. 相似文献
12.
We modulated the level of a hormone gene expression in poplars using either 35S promoter (p35S) of cauliflower mosaic virus
(CaMV) or aux promoter (pAUX) of A. rhizogenes. The transgenic poplars (Populus alba × P.
tremula var. glandulosa), in which the bacterial trans-zeatin secretion (tzs) gene was attached either to the 35S promoter or to the aux promoter, were compared for their performance in tissue culture as well as in nursery. Northern blot analysis of total RNA
probed with tzs coding region showed that the total tzs mRNA expression by p35S was approximately 200–300-fold higher than that driven by pAUX. In contrast, the cellular zeatin
content of p35S-tzs transgenic poplars was merely 13-fold of those found in pAUX-tzs plants. Due to different levels of cellular zeatin levels, the two types of transgenic poplars showed different morphogenetic
as well as growth responses. The p35S-tzs transgenic plants showed morphological characteristics typical of those treated with cytokinin in culture. These include
multiple axillary shoot formation, thick stems, narrow leaves and absence of roots. In contrast, the pAUX-tzs plants had slightly higher cellular cytokinin levels than did control plants and showed a lower degree of cytokinin-related
phenotypes, including a few axillary shoots in root-inducing media. Since p35S-tzs did not develop roots, only pAUX-tzs transgenic poplars could be transplanted to the nursery where they resumed a close-to-normal growth. Nevertheless, pAUX-tzs plants transferred to the nursery developed cytokinin-related phenotypes, including greater number of shoots, smaller leaves
and slightly retarded growth in height, but with a high total biomass. 相似文献
13.
Nagadhara D Ramesh S Pasalu IC Rao YK Sarma NP Reddy VD Rao KV 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2004,109(7):1399-1405
Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T1 to T5 generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper . 相似文献
14.
B. R. Kuluev A. V. Knyazev A. A. Iljassowa A. V. Chemeris 《Russian Journal of Plant Physiology》2011,58(3):507-515
The auxin-inducible gene ARGOS from Arabidopsis thaliana is expressed in growing tissues and controls the plant organ size by regulating cell proliferation and meristematic competence.
The promoter of the dahlia (Dahlia pinnata Cav.) mosaic virus (DMV) resembles the well-known cauliflower mosaic virus 35S promoter but shows a higher activity in transgenic
tobacco plants (Nicotiana tabacum L.). We obtained transgenic tobacco plants expressing the Arabidopsis ARGOS gene under the control of the DMV promoter. Several of the T0 generation plants exhibited an accelerated transition to flowering,
a slight increase in flower size, and a significant increase in the leaf size. The T1 transgenic plants were characterized
by faster growth, the increased leaf size, and somewhat enlarged flowers as compared with control plants. These phenotypic
traits, as well as stability and inheritance of the transgene were demonstrated also in T2 transgenic plants. 相似文献
15.
Sergei F. Krasnyanski Jagdeep Sandhu Leslie L. Domier Dennis E. Buetow Schuyler S. Korban 《In vitro cellular & developmental biology. Plant》2001,37(4):427-433
Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa
mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their
effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable
regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation
of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed
the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T0 and T1 plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic
plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative
tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression
of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than
when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T1 plants segregated in a 3∶1 Mendelian ratio. 相似文献
16.
H. A. Richards V. A. Rudas H. Sun J. K. McDaniel Z. Tomaszewski B. V. Conger 《Plant cell reports》2001,20(1):48-54
A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived
embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from
the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l–1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red
to yellow. Basta tolerance was observed in T1 plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene.
Received: 11 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000 相似文献
17.
Etr1-1 Gene Expression Alters Regeneration Patterns in Transgenic Lettuce Stimulating Root Formation 总被引:1,自引:0,他引:1
We have evaluated the transformation efficiency of two lettuce (Lactuca sativa L.) cultivars, LE126 and Seagreen, using Agrobacterium tumefaciens-mediated gene transfer. Six-day-old cotyledons were co-cultivated with Agrobacterium cultures carrying binary vectors with two different genetic constructs. The first construct contained the β-glucuronidase
gene (GUS) under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S), while the second construct contained
the ethylene mutant receptor etr1-1, which confers ethylene insensitivity, under the control of a leaf senescence-specific promoter (sag12). Tissues co-cultivated with the GUS construct showed strong regeneration potential with over 90% of explants developing
callus masses and 85% of the calli developing shoots. Histochemical GUS assays showed that 85.7% of the plants recovered were
transgenic. Very different results were observed when cotyledon explants were co-cultivated with Agrobacteria carrying the etr1-1 gene. There was a dramatic effect on the regeneration properties of the cultured explants with root formation taking place
directly from the cotyledon tissue in 34% of the explants and no callus or shoots observed initially. Eventually callus formed
in 10% of cotyledons and some organogenic shoots were obtained (2.86%). These results indicate that the ethylene insensitivity
conferred by the etr1-1 gene alters the normal pattern of regeneration in lettuce cotyledons, inhibiting the formation of shoots and stimulating
root formation during regeneration. 相似文献
18.
Multiple virus resistance in transgenic plants conferred by the human dsRNA-dependent protein kinase
Pyung Ok Lim Ung Lee Jong Sang Ryu Jang Kyung Choi Ara Hovanessian Cheol Soo Kim Baik Ho Cho Hong Gil Nam 《Molecular breeding : new strategies in plant improvement》2002,10(1-2):11-18
We have developed a new strategy for engineering resistance to multipleviruses in plants. The strategy exploits the human double stranded (ds)RNA-dependent protein kinase (PKR). PKR is one of theinterferon-induced enzymes. It confers viral resistance in mammals byinhibitingviral replication through the inactivation of the translational initiationfactor, eIF-2, upon activation by dsRNA. The humanPKR gene was fused to the promoter of theArabidopsis blue copper binding protein gene(BCB) that is induced rapidly in response to wounding. Thechimeric gene cassette was introduced into tobacco plants. Expression of thePKR gene in transgenic tobacco plants was demonstrated byRNA gel blot analysis and autophosphorylation assay of anM
r 68,000 protein. The transgenic plantsexpressing the PKR gene showed significantly reduced viralsymptoms or no viral symptoms at all, when challenged by different plant RNAviruses, such as Cucumber mosaic virus, Tobaccoetch virus, or Potato virus Y. Thus, expressionof a single component in the human interferon pathway, thePKR gene, can effectively confer resistance to multipleviruses in transgenic plants. 相似文献
19.
Jean H. Gould Yuanxiang Zhou Veeraragavan Padmanabhan Maria E. Magallanes-Cedeno Ronald J. Newton 《Molecular breeding : new strategies in plant improvement》2002,10(3):131-141
Loblolly pine (Pinus taeda L.) is the mostimportant tree species in US commerce and has much to gain through geneticengineering. This species can be transformed using particle bombardment andAgrobacterium; however, the regeneration of plants fromtransgenic tissues has been difficult and the recovery of transgenic plants hasbeen rare. A shoot-based and genotype-independent transformation methodemploying Agrobacterium tumefaciens was used to facilitaterecovery of plants and permit the transformation of elite germplasm. Shootsfrom4–6 week old seedlings and adventitious shoots from culture wereinoculated with A. tumefaciens EHA101 (pGUS3), or EHA105(pSSLa.3), subjected to selection and regenerated. Shoots that survivedexhibited expression of the uidA gene (GUS) in a patterncharacteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcSpromoter (pSSLa.3) transferred. Recovered plants were screened using PCRamplification. Southern DNA analyses and amplification of the T-DNA borderjunction confirmed genomic integration of both transferreduidA and nptII genes. In this proofofconcept study, the overall recovery of P. taeda shoots wasfair (10–20%), while recovery of intact rooted plants was poor (>1%)due to difficulty in rooting. Recovery of intact rooted plants from inoculatedshoots of P. eldarica and P. radiatawas more efficient (10–30%). The addition of a shoot multiplication stepand effective rooting protocols will improve the efficiency of this genotypeindependent transformation method in P. taeda, and inotherPinus spp. 相似文献
20.
Amber Afroz Zubeda Chaudhry Umer Rashid Ghulam Muhammad Ali Farhat Nazir Javaid Iqbal Muhammad Rashid Khan 《Plant Cell, Tissue and Organ Culture》2011,104(2):227-237
To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tumefaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma,
Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture
of the explants with acetosyringone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after
co-cultivation without selective agent. One week of pre-selection following selection along with 400 μM acetosyringone resulted
in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the
Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5–42.12%
PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T1 plants (resulting from self-pollination of transgenic plants) tested against Pseudomonas solanacearum inoculation in glasshouse, showed Mendelian segregation. 相似文献