Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes

2008 ◽  
Vol 88 (4) ◽  
pp. 593-598 ◽  
Author(s):  
Huaijun Si ◽  
Jun Liu ◽  
Jian Huang ◽  
Conghua Xie
Keyword(s):  
Escherichia Coli ◽  
Cdna Clone ◽  
Antisense Rna ◽  
Cauliflower Mosaic Virus ◽  
Class I ◽  
35S Promoter ◽  
Transformed Plants ◽  
Transgenic Potatoes ◽  
Class I Patatin

Expression of a class I patatin cDNA clone, SK24-1, in Escherichia coli revealed that the cDNA clone possessed lipid acyl hydrolase (LAH) activity. Transformed potato plants were obtained via Agrobacterium-mediated transformation using the chimeric constructs containing the sense and antisense cDNA under the control cauliflower mosaic virus 35S (CaMV 35S) promoter. In some sense transformed plants, both sense patatin RNA and LAH activity were increased and further resulted in a significant increase of percentage of plantlets that formed microtubers and numbers of microtubers per plantlet in vitro. All antisense plants displayed a reduction in LAH activity. Both sense and antisense RNA could be detected in antisense plants, but transcripts of antisense RNA resulted in a reduction of endogenous sense RNA. Moreover, expression of antisense cDNA in some antisense transformed plants led to a significant decrease in the number of microtubers formed. These results suggest that SK24-1 was involved in regulating microtuber formation. Key words: Patatin, potato, Escherichia coli, sense RNA, antisense RNA

Transformation of potato using an antisense class I patatin gene and its effect on microtuber formation

2005 ◽  
Vol 2 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Si Huai-Jun ◽  
Liu Jun ◽  
Xie Cong-Hua
Keyword(s):  
Pcr Amplification ◽  
Transgenic Potato ◽  
Class I ◽  
Total Soluble Protein ◽  
Northern Hybridization ◽  
35S Promoter ◽  
Antisense Gene ◽  
Potato Genome ◽  
Transformed Plants ◽  
Class I Patatin

AbstractAn antisense class I patatin gene under control of the CaMV 35S promoter was introduced into potato (Solanum tuberosum) cultivar E-potato 3 using the Agrobacterium tumefaciens system. PCR amplification and PCR–Southern blot analysis indicated that the antisense class I patatin gene had been integrated into the potato genome. Northern hybridization analysis showed that the antisense gene transcribed normally in the transgenic potato plants and resulted in a reduction of endogenous class I patatin mRNA. Total soluble protein content and lipid acyl hydrolase activity of microtubers, derived from transformed plants, decreased by a maximum of 36.4% and 31.4%, respectively, compared with control plants. The expression of this antisense gene also resulted in reductions of the plantlets forming tubers, tubers per plantlet and the effective tubers (≥50 mg) of the transformed plants.

scholarly journals Uropathogenic Escherichia coli Flagella Aid in Efficient Urinary Tract Colonization

2005 ◽  
Vol 73 (11) ◽  
pp. 7657-7668 ◽  
Author(s):  
Kelly J. Wright ◽  
Patrick C. Seed ◽  
Scott J. Hultgren
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Class I ◽  
Class Iii ◽  
Epithelial Surface ◽  
Fitness Advantage ◽  
Regulatory Cascade ◽  
Tract Infections

ABSTRACT In the murine model of urinary tract infections (UTI), cystitis by uropathogenic Escherichia coli (UPEC) occurs through an intimate relationship with the bladder superficial umbrella cell entailing cycles of adherence, invasion, intracellular bacterial community (IBC) formation, and dispersal (fluxing) from the intracellular environment. IBC dispersal is a key step that results in the spread of bacteria over the epithelial surface to initiate additional rounds of IBC formation. We investigated the role of flagella in mediating adherence and motility during UTI, hypothesizing that the dispersion of the IBC would be incomplete in the absence of motility, thus interrupting the IBC pathway and attenuating the infection. Using gfp reporter fusions, the expression of the flagellar class I flhDC and class III fliC genes was monitored to track key points of regulation throughout the pathogenic cascade. In vitro, growth under conditions promoting motility resulted in the robust expression of both fusions. In contrast, only the class I fusion produced significant expression throughout early stages of IBC development including the dispersion stage. Thus, unlike in vitro modeling of motility, the regulatory cascade appeared incomplete in vivo. Throughout IBC formation, nonmotile ΔfliC mutants achieved the same number of IBCs as the wild-type (wt) strain, demonstrating that flagella are neither essential nor required for first- or second-generation IBC formation. However, in competition experiments between wt and ΔfliC strains, the wt was shown to have a fitness advantage in persisting throughout the urinary tract for 2 weeks, demonstrating a subtle but measurable role for flagella in virulence.

scholarly journals Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
Author(s):  
Christine M. Beatty ◽  
Douglas F. Browning ◽  
Stephen J. W. Busby ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class I ◽  
Receptor Protein ◽  
Class Iii ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

scholarly journals Paxillus involutus Forms an Ectomycorrhizal Symbiosis and Enhances Survival of PtCOMT-modified Betula pendula in vitro

2008 ◽  
Vol 57 (1-6) ◽  
pp. 235-242 ◽  
Author(s):  
H. Tiimonen ◽  
T. Aronen ◽  
T. Laakso ◽  
P. Saranpää ◽  
V. Chiang ◽  
...  
Keyword(s):  
Populus Tremuloides ◽  
Betula Pendula ◽  
Root Formation ◽  
Lateral Roots ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Paxillus Involutus ◽  
Transgenic Lines ◽  
G Ratio

Abstract The ability of the PtCOMT (caffeate/5-hydroxyferulate O-methyltransferase from Populus tremuloides L.) - modified Betula pendula Roth. lines to form symbiosis with an ectomycorrhizal (ECM) fungus Paxillus involutus Batsch Fr. was studied in vitro. Lignin precursor gene PtCOMT was introduced into two B. pendula clones under the control of the cauliflower mosaic virus 35S promoter or the promoter of the sunflower polyubiquitin gene UbB1. Of the four transgenic lines, one 35SPtCOMT line (23) had a decreased syringyl/guaiacyl (S/G) ratio of root lignin, and two UbB1-PtCOMT lines (110 and 130) retarded root growth compared to the control clone. Both control clones and all transgenic lines were able to form ECMs with P. involutus, but the transgenic lines differed from the controls in the characteristics of the ECMs. The number of lateral roots covered with fungal hyphae and/or development of a Hartig net (HN) were reduced in line 23 with a decreased S/G ratio, and in lines 110 and 130 with slower root formation and changed root morphology, respectively. However, line 23 benefited more from the inoculation in lateral root formation than the control, and in lines 110 and 130 the percentage of viable plants increased most due to inoculation. The results show that B. pendula plants genetically transformed with the lignin gene PtCOMT could form mycorrhizal symbiosis regardless of changes in either the root S/G ratio or development. The benefits of the symbiosis were variable even in the closed in vitro system, and dependent on the clone or transgenic line and the ECM fungal symbiont.

scholarly journals Functional Expression of hMYH, a Human Homolog of the Escherichia coli MutY Protein

1999 ◽  
Vol 181 (19) ◽  
pp. 6210-6213 ◽  
Author(s):  
Malgorzata M. Slupska ◽  
Wendy M. Luther ◽  
Ju-Huei Chiang ◽  
Hanjing Yang ◽  
Jeffrey H. Miller
Keyword(s):  
Escherichia Coli ◽  
Cdna Clone ◽  
Functional Expression ◽  
Human Homolog ◽  
Mutator Phenotype ◽  
Genomic Clones

ABSTRACT We have previously described the hMYH cDNA and genomic clones (M. M. Slupska et al., J. Bacteriol. 178:3885–3892, 1996). Here, we report that the enzyme expressed from an hMYH cDNA clone in Escherichia coli complements the mutator phenotype in a mutY mutant and can remove A from an A · 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A · G mismatch in vitro.

Expression of a Chicken Ovalbumin Gene in Three Lucerne Cultivars

1991 ◽  
Vol 18 (5) ◽  
pp. 495 ◽  
Author(s):  
HE Schroeder ◽  
MRI Khan ◽  
WR Knibb ◽  
D Spencer ◽  
TJV Higgins
Keyword(s):  
Transgenic Plants ◽  
Alternative Interpretation ◽  
Cauliflower Mosaic Virus ◽  
Vector System ◽  
35S Promoter ◽  
Restriction Enzyme Digestion ◽  
Flanking Sequence ◽  
Gene Encoding ◽  
Transformed Plants ◽  
Chicken Ovalbumin

Routine procedures have been developed for the transformation of lucerne (Medicago sativa cv. Rangelander) with foreign genes using the Agrobacterium tumefaciens binary vector system and for the regeneration of transgenic plants from tissue culture, via somatic embryogenesis. Lucerne transformation was carried out with a gene encoding neomycin phosphotransferase (npt), which conferred resistance to the antibiotic kanamycin, together with a cDNA clone encoding chicken ovalbumin which was modified for expression in plant cells. The ovalbumin cDNA protein coding sequence was combined with the cauliflower mosaic virus 35S promoter and the nopaline synthase 3' flanking sequence to make a chimeric ovalbumin gene. A DNA construct containing both these genes was transferred to lucerne, and ovalbumin was detected in leaves of regenerated plants using protein immunoblots. Pulse-chase labelling experiments and analysis of leaves from the top to bottom of the transformed plants indicated that ovalbumin, once formed, was stable in the leaves of transgenic lucerne. A wide variation in ovalbumin level was frequently observed in plants regenerated from multiple embryos on a single transformed callus. This variation correlated with changes in the restriction enzyme digestion pattern of the ovalbumin DNA from the transgenic plants. These results indicate that each transformed callus may have arisen from more than one transformation event. An alternative interpretation is that the callus may have arisen from a single transformed cell but during cell proliferation the DNA in some cells may have undergone rearrangement prior to embryogenesis. Transformation and regeneration procedures were also developed for two Australian commercial cultivars of lucerne. Although the frequency of recovery of transformed plants was lower than with cv. Rangelander, these protocols open the way for a relatively rapid

Research note: The 35S promoter is not constitutively expressed in the transgenic tropical actinorhizal tree Casuarina glauca

2002 ◽  
Vol 29 (5) ◽  
pp. 649 ◽  
Author(s):  
Aziz Smouni ◽  
Laurent Laplaze ◽  
Didier Bogusz ◽  
Fathia Guermache ◽  
Florence Auguy ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
Gus Expression ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Casuarina Glauca ◽  
Camv 35S ◽  
Highly Active ◽  
One Year

The tropical nitrogen-fixing tree, Casuarina glauca Sieb. ex Spreng. was genetically transformed using Agrobacterium tumefaciens C58C1(pGV2260; pBIN19GUSINT). We report on the expression pattern conferred by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic C. glauca plants grown in vitro, and for one year in a greenhouse. Histochemical assays in shoots from in vitro plants revealed β-glucuronidase (GUS) staining in apical and axillary buds, and in nearly all tissues near the base of the stem. In roots, the CaMV 35S drove strong GUS expression in the apex and vascular tissue. In 1-year old plants grown in a greenhouse, the CaMV 35S promoter was highly active, except in peripheral suberized tissues. Transgenic C. glauca plants were nodulated by the actinomycete Frankia. Histochemical assays on vibratome sections of transgenic nodules demonstrated intense GUS activity in the vascular bundle, the phellogen, and in strands of uninfected cells filled with polyphenols. GUS expression was undetectable in Frankia-infected cells.

scholarly journals Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

2002 ◽  
Vol 184 (8) ◽  
pp. 2273-2280 ◽  
Author(s):  
Nigel J. Savery ◽  
Georgina S. Lloyd ◽  
Stephen J. W. Busby ◽  
Mark S. Thomas ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class Ii ◽  
Class I ◽  
Receptor Protein ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain

ABSTRACT Alanine scanning of the Escherichia coli RNA polymerase α subunit C-terminal domain (αCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of αCTD affected class I CRP-dependent transcription from the CC(−61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for αCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in αCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

Recombinant expression and characterization of lemon (Citrus limon) peroxidase

2020 ◽  
Vol 27 ◽  
Author(s):  
Veda P. Pandey ◽  
Apoorvi Tyagi ◽  
Shagoofa Ali ◽  
Kusum Yadav ◽  
Anurag Yadav ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plant Defense ◽  
Cdna Clone ◽  
Protein Function ◽  
In Silico ◽  
Full Length ◽  
Function Annotation ◽  
Full Length Cdna ◽  
Protein Function Annotation

Background: Class III plant peroxidases play important role in a number of physiological processes in plant such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen species metabolism and plant defense against pathogens. Peroxidases are also of significance in several industrial applications. In view of this, the production and identification of novel peroxidases having resistance towards temperature, pH, salts is desirable. Objective: The objective of the present work was to clone and characterize a novel plant peroxidase suitable for industrial application. Methods: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches, characterized and heterologously expressed in Escherichia coli using standard protocols. The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well as protein levels using standard protocols. Results: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously expressed in Escherichia coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively. The LPRX was found to be activated by metal ions (Na+ , Ca2+, Mg2+ and Mn2+) at lower concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates guaiacol, and pyrogallol and results show the favourability of pyrogallol over guaiacol, which is in agreement with the in-vitro findings. The protein function annotation analyses suggested the involvement of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms. Conclusion: Based on the biochemical characterization, the purified peroxidase was found to be resistant towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico protein function annotation and transcript analyses highlighted the possible involvement of the lemon peroxidase in plant defense response.

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