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1.
Hong-Yu Lu Jing-Mei Liu Hai-Chao Zhang Tao Yin Shan-Lin Gao 《Plant Molecular Biology Reporter》2008,26(1):1-11
Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS)
plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from
G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog (1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates
were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance
liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight
and was about 2.6 times higher than control hairy roots.
The nucleotide sequences GuSQS1 and GUSQS2 reported in this paper appear in the EMBL nucleotide sequence database with the
accession number AM182329 and AM182330, respectively. 相似文献
2.
3.
Hai-Chao Zhang Jing-Mei Liu Hai-Min Chen Chun-Chun Gao Hong-Yu Lu Hua Zhou Yi Li Shan-Lin Gao 《Molecular biotechnology》2011,47(1):50-56
We evaluated the effect of Tween 80 as elicitor on licochalcone A from hairy root cultures of Glycyrrhiza uralensis Fisch. After a 15-days treatment with 2% Tween 80, hairy roots still grew well and produced higher levels of licochalcone
A and total flavonoids than the control (without treatment). Licochalcone A content and total flavonoid content were 3.103
and 127.095 mg per flask (9- and 11-fold higher), respectively, compared with controls. Secretion of licochalcone A and total
flavonoids into the culture medium was remarkably high, up to 98 and 94% of the total production, respectively. The enhanced
flavonoid production was associated with elevated mRNA levels and enzyme activities of phenylalanine ammonia-lyase (PAL),
4-coumarate:coenzyme A ligase (4CL), and cinnamate-4-hydroxylase (C4H). These results clearly demonstrated that Tween 80 treatment
permeabilized the roots to enhance secretion, but also acted as an efficient elicitor of licochalcone A and total flavonoid
production in hairy roots of G. uralensis Fisch. 相似文献
4.
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
5.
6.
Ok-Sun?Lee Young-Min?Kang Hee-Young?Jung Ji-Yun?Min Seung-Mi?Kang Chandrakant?S.?Karigar D.?Theertha?Prasad Jung-Dong?Bahk Myung-Suk?Choi
Summary In wild-type Scopolia parvilfora (Solanaceae) tissues, only the roots express the enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53), which is the first specific precursor of the tropane alkaloids. Moreover, the tropanane
alkaloid levels were the highest in the root (0.9 mg g−1 on a dry weight basis), followed by the stem and then the leaves. We metabolically engineered S. parviflora by introducing the tobacco pmt gene into its genome by a binary vector system that employs disarmed Agrobacterium rhizogenes. The kanamycin-resistant hairy root lines were shown to bear the pmt gene and to overexpress its mRNA and protein product by at least two-fold, as determined by polymerase chain reaction (PCR)
and Northern and Western blottings, respectively. The transgenic lines also showed higher PMT activity and were morphologically
aberrant in terms of slower growth and the production of lateral roots. The overexpression of pmt markedly elevated the scopolamine and hyoscyamine levels in the transgenic lines that showed the highest pmt mRNA and PMT protein levels. Thus, overexpression of the upstream regulator of the tropane alkaloid pathway enhanced the
biosynthesis of the final product. These observations may be useful in establishing root culture systems that generate large
yields of tropane alkaloids.
These authors contributed equally to this paper (co-first authors). 相似文献
7.
Veena Veena Christopher G. Taylor 《In vitro cellular & developmental biology. Plant》2007,43(5):383-403
Agrobacterium rhizogenes is the etiological agent for hairy-root disease (also known as root-mat disease). This bacterium induces the neoplastic growth
of plant cells that differentiate to form “hairy roots.” Morphologically, A. rhizogenes-induced hairy roots are very similar in structure to wild-type roots with a few notable exceptions: Root hairs are longer,
more numerous, and root systems are more branched and exhibit an agravitropic phenotype. Hairy roots are induced by the incorporation
of a bacterial-derived segment of DNA transferred (T-DNA) into the chromosome of the plant cell. The expression of genes encoded
within the T-DNA promotes the development and production of roots at the site of infection on most dicotyledonous plants.
A key characteristic of hairy roots is their ability to grow quickly in the absence of exogenous plant growth regulators.
As a result, hairy roots are widely used as a transgenic tool for the production of metabolites and for the study of gene
function in plants. Researchers have utilized this tool to study root development and root–biotic interactions, to overexpress
proteins and secondary metabolites, to detoxify environmental pollutants, and to increase drought tolerance. In this review,
we provide an up-to-date overview of the current knowledge of how A. rhizogenes induces root formation, on the new uses for A. rhizogenes in tissue culture and composite plant production (wild-type shoots with transgenic roots), and the recent development of
a disarmed version of A. rhizogenes for stable transgenic plant production. 相似文献
8.
Konstantin V. Kiselev Anna V. Turlenko Galina K. Tchernoded Yuri N. Zhuravlev 《Plant cell reports》2009,28(8):1273-1278
It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P.
ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1–4 nucleotide substitutions were found per sequence in the
540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted
gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted
from P.
ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 ± 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased
(1.37 ± 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to
C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P.
ginseng
actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred
genes in transgenic plant cell cultures. 相似文献
9.
Gauri Saxena Suchitra Banerjee Laiq-ur-Rahman Praveen Chandra Verma G. R. Mallavarapu Sushil Kumar 《Plant Cell, Tissue and Organ Culture》2007,90(2):215-223
Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A4 and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles
and internodal segments, respectively. The A4 strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy
root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16%
and 12% root lines of A4 and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A4 and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as
against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype
having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root
system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer
to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild
type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters
signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value
represent an improvement over that of the wild type parent from the commercial point of view. 相似文献
10.
Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l−1. HS and OS at 30 mg l−1, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70%
to ∼20 g l−1 from 13 g l−1 and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides
may have plant growth-regulatory activity in plant tissue cultures. 相似文献
11.
The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However,
the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies
by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic
screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These
results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous
tau genes are conserved.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain
and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600;
2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the
Chinese Academy of Sciences (KJCX1-09-03). 相似文献
12.
Gentiana dahurica Fisch is one of four important commercial Radix Gentianae stipulated by the Chinese Pharmacopoeia. We have established a rapid and effective regeneration system using zygotic embryo-derived
callus of G.
dahurica Fisch. Using this regeneration system, Agrobacterium
tumefaciens-mediated transformation of G. dahurica Fisch has been developed. Zygotic embryo-derived callus was infected with an A.
tumefaciens strain (GV3130) harboring a pBI121 vector that contained an nptII selective marker gene. In a total of 60 zygotic embryo-derived calli assayed, frequency of calli with nptII gene PCR-positive transgenic plantlets is 5%. Transient glucuronidase (GUS) expression was observed from these transgenic
plantlets. Frequency of GUS-positive transgenic plantlets (4/78) was approximately consistent with that of PCR assay (3/60).
Our data indicate that we have successfully established a stable G. dahurica Fisch transformation system by A.
tumefaciens. In general, this transformation system we have established might be useful for modifying physiological, medicinal and horticultural
traits. 相似文献
13.
Y. Q. Zhou H. Y. Duan C. E. Zhou J. J. Li F. P. Gu F. Wang Z. Y. Zhang Z. M. Gao 《Russian Journal of Plant Physiology》2009,56(2):224-231
The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from
hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige
and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots
and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes
and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that
rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis
revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants
of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time.
This text was submitted by the authors in English.
Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 247–255. 相似文献
14.
Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties.
To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source
of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction
and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled
to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01–0.02 μmol/g of DW) and
4-methoxyglucobrassicin (0.06–0.18 μmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06–0.21 μmol/g of
DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate
biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive
agent (indole-3-carbinol). 相似文献
15.
The balloon flower (Platycodon grandiflorum) is a popular traditional medicinal plant used in Korea to treat conditions such as bronchitis, asthma, tuberculosis, diabetes,
and inflammatory diseases. Recently, immunopharmacological research identified triterpenoid and saponin as important active
compounds in P. grandiflorum. To study and extract these compounds and other metabolites from P. grandiflorum, a technique was developed for producing hairy root cultures, which are a reliable source of plant compounds. To achieve this,
the activity of Agrobacterium rhizogenes was exploited, which can transfer DNA segments into plant genomes after infecting them. In this study, the A. rhizogenes strain R1000 was determined that had the highest infection frequency (87.5%) and induced the most hairy roots per plant,
and the concentration of antibiotics (75 mg/l kanamycin) was elucidated for selection after transformation. Wild-type and
transgenic hairy roots contained various phenolic compounds, although both of them had similar concentrations of phenolic
compounds. In the future, the protocols described here should be useful for studying and extracting valuable metabolites such
as phenolic compounds from P. grandiflorum hairy root cultures. 相似文献
16.
Y. Bao P. Dharmawardhana R. Arias M. B. Allen C. Ma Steven H. Strauss 《Plant cell reports》2009,28(6):947-962
We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs
to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common
in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems
as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were
placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary
growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response
regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events
recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended
to stimulate meristem development to promote clonal propagation and genetic transformation.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
17.
Pramila Shah N. K. Singh Neeraj Khare Meenal Rathore S. Anandhan M. Arif Rupesh Kumar Singh S. C. Das Z. Ahmed Narendra Kumar 《Plant Cell, Tissue and Organ Culture》2008,95(3):363-371
Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore,
experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation.
6-benzylaminopurine at 0.05 mg l−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets
in indole-3-aceticacid 0.5 mg l−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after
co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by
polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent. 相似文献
18.
Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×106 per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts
reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented
with 2 mgl−1 (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl−1 (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl−1 casein hydrolyzate at a plating density of 1.0×105 per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency
was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators.
Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast
culture system would be valuable for further somatic hybridization in forage legumes. 相似文献
19.
Stefanie Kimbacher Ingrid Gerstl Branko Velimirov Sylvia Hagemann 《Molecular genetics and genomics : MGG》2009,282(2):165-172
P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding
region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
20.
To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation
with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation,
when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated
roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight
of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa.
Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation
of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms. 相似文献