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1.
Tae-Geum Kim Ju Kim Dae-Hyuk Kim Moon-Sik Yang 《Biotechnology and Bioprocess Engineering》2001,6(3):173-178
Food yeast.Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins.
AmA1, a seed storage albumin fromAmaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further
improving food and animal feed additives. In order to find an effective means of expressingAmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate
dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter.
The recombinantAmA1 genes were then introduced into the yeastSaccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed thatAmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount
of AmA1 protein in the recombinant yeast was 1.3–4.3% of the total soluble proteins. The highest expression level was obtained
from the hybrid ADH2-GPD promoter. 相似文献
2.
Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions.
chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the
control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction
(PCR) and Southern blot analysis. The inheritance of the transgene in T1 and T2 generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was
observed among the transgenic lines in T0 and a similar range was retained in the T1 and T2 generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant
to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici. 相似文献
3.
Rajasekhar Urla P. Pavan Kumar Prabhakar Sripadi Venkateswara Rao Khareedu Dashavantha Reddy Vudem 《Plant biotechnology reports》2017,11(5):259-270
Flax contains very high levels of alpha-linolenic acid (57%) and a fatty acid desaturase 3-coding sequence (Lufad3) of flax has been amplified from the RNA isolated from developing seeds. The deduced amino acid sequence of LuFAD3 showed the presence of three histidine motifs, six membrane spanning domains and an endoplasmic reticulum (ER) retention signal KSK, indicating its plausible localization into the ER. Flax is not amenable for genetic transformation and not suitable for functional validation of Lufad3 gene. Hence, rice with well-developed genetic transformation has been selected as heterologous host system. Coding sequence of Lufad3 driven by maize Ubi1 promoter has been introduced into indica rice by Agrobacterium tumefaciens-mediated genetic transformation. Southern analysis of putative transformants (T 0) revealed signals at variable lengths at >5 kbp indicating random integration of transgene into the genomes of different transformants. The Mendelian segregation observed for selectable marker gene hyg in both T1 and T2 generations confirmed stable inheritance and single-site integration of transgenes. As compared to untransformed control (UC), homozygous transgenic rice expressing Lufad3 showed higher levels of essential α-linolenic acid in leaves and seeds validating its functionality. 相似文献
4.
Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties
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Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants. 相似文献
5.
Cotton transgenics for resistance against cotton leaf curl disease using antisense movement protein gene (AV2) were developed in an Indian variety (F846) via Agrobacterium-mediated transformation using the protocol developed previously. A binary vector pPZP carrying the antisense AV2 (350 bp) gene along with the nptII gene was used. Transgenic nature of the putative transgenics was confirmed by molecular analysis. Shoots were induced on selection medium and subcultured on rooting medium containing IBA and 75 mg l–1 kanamycin. Transgenic plants were recovered in 12–16 weeks from the time of gene transfer to establishment in pots. Preliminary analysis of the field-established plantlets was conducted by PCR. T1 plants were obtained from T0 seeds, the presence of the AV2 and nptIIgenes in the transgenic plants was verified by PCR and integration of T-DNA with AV2 into the plant genome of putative transgenics was further confirmed by Southern blot analysis. Several T1 lines were maintained in the greenhouse. Progeny analysis of these plants by PCR analysis showed a classical Mendelian pattern of inheritance. 相似文献
6.
Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase 总被引:1,自引:0,他引:1
Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T1 and T2 progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.Abbreviations
2,4-D:
2,4-Diclorophenoxyacetic acid
-
IAA:
Indole acetic acid
-
ICRISAT:
International Crops Research Institute for the Semi-Arid Tropics
-
IZEs
Immature zygotic embryos
Communicated by H. Lörz 相似文献
7.
Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B. Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium. Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure. By Southern blot analysis, integration of the gfp and nptII genes was confirmed. Stable transformation efficiency was 0.1%. From 68 T1 plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene. Expression of gfp was detected in 10 T1 plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis. Expression of the nptII gene was confirmed in 13 T1 plants. The transformation system enables the rapid transfer of agronomically important genes. 相似文献
8.
Ufuk Celikkol Akcay M. Mahmoudian H. Kamci M. Yucel H. A. Oktem 《Plant cell reports》2009,28(3):407-417
A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found
to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application
of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection
regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with
Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced
with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS
assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to
soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T0, T1, T2 and T3 plants. PCR assays of T1, T2 and T3 progenies confirmed the stable transmission of the transgene to the next generations. 相似文献
9.
Tissue‐specific and pathogen‐inducible expression of a fusion protein containing a Fusarium‐specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins
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Wei Cheng He‐Ping Li Jing‐Bo Zhang Hong‐Jie Du Qi‐Yong Wei Tao Huang Peng Yang Xian‐Wei Kong Yu‐Cai Liao 《Plant biotechnology journal》2015,13(5):664-674
Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.‐specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea‐specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea‐specific in transgenic wheat. Single‐floret inoculation of independent transgenic wheat lines of the T3 to T6 generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography–mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB‐susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real‐time PCR analysis revealed that the tissue‐specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue‐specific and pathogen‐inducible expression of this Fusarium‐specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain. 相似文献
10.
11.
Manju Sharma Aditi Kothari-Chajer Swati Jagga-Chugh S. L. Kothari 《Plant Cell, Tissue and Organ Culture》2011,105(1):93-104
Agrobacterium-mediated transformation protocol has been developed for Eleusine coracana (var. PR-202) by varying several factors which influence T-DNA delivery. Green nodular regenerative calli with meristematic
nodules of seed origin were used as the target tissue for Agrobacterium
tumefaciens-mediated gene transfer. The highest frequency of transformation (44.4%) was observed when callus was infected, co-cultivated
and incubated at 22°C. Incorporation of higher level of CuSO4 in the regeneration medium had significantly positive effect on the recovery of transformed plants. PCR analysis of T
0 and T
1 generation plants with nptII-specific primers revealed the amplification of nptII gene. Southern blot analysis of six regenerated plants confirmed selectable marker gene integration in three plants. This
is a first report on Agrobacterium-mediated genetic transformation of finger millet and will pave the way for further studies in this and other millet crops. 相似文献
12.
Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker 总被引:13,自引:0,他引:13
W. P. Chen X. Gu G. H. Liang S. Muthukrishnan P. D. Chen D. J. Liu B. S. Gill 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(8):1296-1306
Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression
of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was
to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter
and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded
with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration
medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T0) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected
35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal
and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T0 and T1 transgenic plants. Mendelian segregation of herbicide resistance was observed in some T1 progenies. Interestingly, a majority of the T1 progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression
under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated
in T1 transgenic wheat plants.
Received: 12 May 1998 / Accepted: 15 May 1998 相似文献
13.
D. N. Miroshnichenko G. N. Poroshin S. V. Dolgov 《Applied Biochemistry and Microbiology》2011,47(8):767-775
A protocol for biolistic transformation of bread wheat based on using mature seed tissues as explants has been developed.
Embryogenic callus obtained from mature seed tissues was transformed with a psGFP-BAR plasmid containing gfp reporter gene and bar selectable marker gene. The influence of hormone composition of the medium on the efficiency of transformation of mature
wheat seed tissues has been demonstrated. The use of auxin 2,4-D resulted in the formation of transgenic plants with a frequency
of 0.75%, while the use of Dicamba auxin for the regeneration of plants did not result in transformant development. The transgenic
status of the plants obtained in the experiments has been confirmed by PCR and RT-PCR. Stable inheritance of transgenic features
in the following generations of wheat (T1, T2) has been demonstrated and transgenic plants exhibiting high resistance to herbicides have been obtained. The protocol developed
allows for a simplified transformation of wheat in order to obtain transgenic plants with novel features. 相似文献
14.
15.
The carboxypeptidase gene from rice and corresponding cDNA clones were isolated. The SalI 11.2 kb fragment of DNA cloned from a size-fractionated genome library contained eight introns and an open reading frame that encoded 500 amino acids (M
r 55445). The structure deduced for the carboxypeptidase from rice was very similar to those of type III serine carboxypeptidases from barley and wheat. The extent of homology of the amino acid sequence to that of these carboxypeptidases from barley and wheat was 92.3% and 87.2%, respectively. The accumulation of mRNA for the rice carboxypeptidase was conspicuous in germinating endosperms that contained aleurone layers, but levels were lower in leaves and roots. The abundance of the mRNA in endosperms was enhanced by gibberellic acid (GA) and accumulation of the mRNA was inhibited by abscisic acid (ABA). The rice gene for carboxypeptidase contained some pyrimidine boxes (T
CCTTTTT
C), in the 5 flanking region, which are a characteristic of a GA-responsive gene. 相似文献
16.
Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling 总被引:1,自引:0,他引:1
To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying β-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2∼5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T0 transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T1 plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T2 plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that β-1, 3-glucanase gene was normally expressed in the T2 plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.Tong-Jin Zhao and Shuang-Yi Zhao contributed equally to this paper. 相似文献
17.
<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>) 总被引:1,自引:1,他引:0
Byoung-Kyu?Lee Seung-Hee?Yu Yul-Ho?Kim Byung-Ohg?Ahn Han-Sun?Hur Sang-Chul?Lee Zhanyuan?Zhang Jang-Yong?Lee
Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l−1 phosphinothricin (PPT) and 150 mg l−1 kanamycin, respectively, for shoot induction and 1 mg l−1 PPT and 125 mg l−1 kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T0 and T1 plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T0 and T1 transgenic plants. 相似文献
18.
19.
Luo H Hu Q Nelson K Longo C Kausch AP Chandlee JM Wipff JK Fricker CR 《Plant cell reports》2004,22(9):645-652
Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60–65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.Abbreviations
2,4-D:
2,4-Dichlorophenoxyacetic acid
-
bar:
Bialaphos resistance gene
-
GUS:
-Glucuronidase
-
PPT:
Phosphinothricin
-
ubi:
Ubiquitin
Communicated by J.M. Widholm 相似文献
20.
Jianzhong Lin Bo Zhou Yuanzhu Yang Jin Mei Xiaoying Zhao Xinhong Guo Xingqun Huang Dongying Tang Xuanming Liu 《Plant cell reports》2009,28(7):1065-1074
Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method
was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T
0) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T
0 plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation
was further confirmed by the genetic and molecular analyses of T
1 transformants. 相似文献