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1.
The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis
suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal
cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation
method, Arabidopsis transgenic KanR plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls,
upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any
expression in sepals and pistils. 相似文献
2.
We previously cloned and analyzed the 1,893-bp promoter region (−1,915 to −23) of the tomato (Lycopersicon esculentum) Lehsp23.8 gene, whose expression is induced by treatment with high or low temperatures, heavy metal, or abscisic acid (ABA). In our
present work, we examined how this expression is regulated. A comprehensive quantitative promoter deletion and base-substitution
analysis was conducted under various environmental conditions. The proximal region (−565 to −23 bp) of the Lehsp23.8 promoter harbors cis-regulatory elements that conferred high levels of heat-induced expression in transgenic tobacco. Mutation of the five proximal
HSEs (HSE1 to 5) of that promoter led to an absence of heat inducibility. The AT-rich regions between −255 bp and −565 bp
(AT-rich1 to 4) in the promoter might serve as enhancers for such heat-induced expression. Deletion and HSE mutation analysis
indicated that other cis-acting elements also function in response to low temperature, heavy metal, and ABA and that HSE1 to 5 act at least as cis-acting elements in multiple-stress responses of Lehsp23.8. These results reveal that those five proximal HSEs and AT-rich regions function interdependently in the expression of Lehsp23.8 in response to non-heat stresses. Furthermore, the putative elements CRT/DRE, AP-1, and ABRE in that promoter are not required
for multiple-stress induction. 相似文献
3.
Zhao-Jun Wei Miao Yu Shun-Ming Tang Yong-Zhu Yi Gui-Yun Hong Shao-Tong Jiang 《Molecular biology reports》2011,38(2):1121-1127
Prothoracicotropic hormone (PTTH) is one of key players in regulation of insect growth, molting, metamorphosis, diapause,
and is expressed specifically in the two pairs of lateral PTTH-producing neurosecretory cells in the brain. Analysis of cis-regulatory elements of the PTTH promoter might elucidate the regulatory mechanism controlling PTTH expression. In this study,
the PTTH gene promoter of Bombyx mori (Bom-PTTH) was cloned and sequenced. The cis-regulatory elements in Bom-PTTH gene promoter were predicted using Matinspector software, including myocyte-specific enhancer
factor 2, pre-B-cell leukemia homeobox 1, TATA box, etc. Transient transfection assays using a series of fragments linked
to the luciferase reporter gene indicated that the fragment spanning −110 to +33 bp of the Bom-PTTH promoter showed high ability
to support reporter gene expression, but the region of +34 to +192 bp and −512 to −111 bp repressed the promoter activity
in the BmN and Bm5 cell lines. Electrophoretic mobility shift assays demonstrated that the nuclear protein could specifically
bind to the region spanning −124 to −6 bp of the Bom-PTTH promoter. Furthermore, we observed that the nuclear protein could
specifically bind to the −59 to −30 bp region of the Bom-PTTH promoter. A classical TATA box, TATATAA, localized at positions
−47 to −41 bp, which is a potential site for interaction with TATA box binding protein (TBP). Mutation of this TATA box resulted
in no distinct binding band. Taken together, TATA box was involved in regulation of PTTH gene expression in B. mori. 相似文献
4.
A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter (PMagpd) was obtained from Metarhizium acridum and its active region analyzed by 5′-deletion strategy using β-glucuronidase (GUS) as a reporter. Sequence analysis revealed
that typical regulatory elements of PMagpd were included in the 1.7 kb region upstream of the start codon of the Magpd gene. Deletion of the region from −1,691 bp to −1,463 bp, where the gpd box is harbored, did not significantly affect the PMagpd activity. Deletions of the regions upstream of −946 bp and upstream of −684 bp caused a major decrease of GUS activity. Compared
with PgpdA (2.2 kb) in Aspergillus nidulans, PMagpd (1.4 kb) had a shorter sequence and significantly higher activity in M. acridum. This study provides an applicable promoter for over-expression of target genes in M. acridum. 相似文献
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Li Xu Rongjian Ye Yusheng Zheng Zhekui Wang Peng Zhou Yongjun Lin Dongdong Li 《Plant cell reports》2010,29(9):1061-1068
As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of
an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements
including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5′-deletion fragments
were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2
and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty
vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic
lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76-
and 2.8-fold higher than those driven by the −817 bp and −453 bp upstream fragments, and 10.7-fold higher than that driven
by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut
have a significant potential in genetically improving endosperm in main crops. 相似文献
7.
Xue-Feng Wu Chun-Lian Wang En-Bei Xie Ying Gao Ying-Lun Fan Pi-Qing Liu Kai-Jun Zhao 《Planta》2009,229(6):1231-1242
We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009–1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection.
These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing
the BjCHI1 promoter or its derivatives to β-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating
that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from −695 to −620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at −353 to −348
of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter.
Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA. 相似文献
8.
In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These
plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected
under other abiotic stresses. Deletion analysis indicated that the region from −178 to −771 was essential for the promoter’s
response to high temperature. 相似文献
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10.
Muzna Zahur Asma Maqbool Muhammad Irfan Muhammad Younas Khan Barozai Bushra Rashid Shiekh Riazuddin Tayyab Husnain 《Molecular Biology》2009,43(4):578-585
The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboreum. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter
region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid
and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were
affected by the deletion between −208 and −949 bp and showed 2–4-fold induction. However, in response to dark, gibberellic
acid and heavy metals the induction was only 2-fold. These findings further our understanding of the regulation of GUSP expression. This is an important study as no report of this universal stress protein promoter is available in literature. 相似文献
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Soluble glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays important roles in the synthesis of triacylglycerol
and in the glycerol-3-phosphate shutter. Though GPD1 is expressed in most adult tissues, little is known about the regulation of its expression. In this study, we analyzed the
characters, organization and core region of the promoter of pig GPD1 gene by in silico analysis and activity detection of deletion mutants. We also identified and testified the negative regulation effect of C/EBP
β on pig GPD1 gene by Chromatin immunoprecipitation (ChIP) assay and over-expression experiments in cultured pig kidney cells. Compared
to that of human, pig GPD1 gene promoter has three conserved regions and one deletion region. In silico analysis indicated that pig GPD1 promoter was TATA-less with at least 3 CpG islands of over 200 bp in length and over 60% in GC content. The activity detection
of deletion mutants suggested that the essential elements required for the optimal promoter activity scatter in the promoter
region, while the core promoter region was from -422 bp to -1 bp. Chromatin immunoprecipitation (ChIP) assay results indicated
that C/EBP β had plenty of binding sites in pig GPD1 promoter with the common cis-element (5’- TKNNGCAAK -3’). The over-expression examination of C/EBP β showed that the expression of GPD1 was negatively regulated by C/EBP β in pig kidney cells. Overall, our study revealed that the pig GPD1 promoter is a TATA-less promoter, and in promoter region, the binding sites of C/EBP β share common motif of (5’-TKNNGCAAK
-3’). We also showed that pig GPD1 gene is regulated negatively by C/EBP β in cultured kidney cells. 相似文献
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16.
Xin-Wen Hu Si-Xin Liu Jian-Chun Guo Ji-Tao Li Rui-Jun Duan Shao-Ping Fu 《Functional & integrative genomics》2009,9(3):351-361
Mabinlin II is one of the major sweet proteins stored in the seeds of Capparis masaikai Lévl. Its promoter region (779 bp) located 5′ upstream of the mabinlin II gene has been isolated and named as MBL-779 (GenBank
accession number, EU014073). This promoter contains two typical TATA box regions and a series of motifs related to seed-specific
promoters, such as ACGT motifs, RY motif, napin motif, and G box. The MBL-779 promoter drove GUS gene to transiently express in the embryos of bean, maize, and rice seeds or to constantly express in the embryos and anthers
of the transgenic Arabidopsis. The MBL-779 promoter regulated gene expression from approximately the 12th day and peaked on approximately the 16th day
after flowering in Arabidopsis. The −300-bp promoter region is a minimal sequence required to functionally regulate gene expression. The CAATs at −325 to
−322 bp and −419 to −416 bp and the region at −485 to −770 bp play a role in the quantitative regulation of gene expression.
The RY motif, CATGAC, at −117 to −112 bp and the ACGT within the G box (CACGTG) at −126 to −123 bp positively regulate gene
expression.
X.-W. Hu and S.-X. Liu have the same contribution as first author. 相似文献
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18.
The expression of nitrite reductase (NiR; EC 1.7.7.1), the second enzyme in the nitrate assimilatory pathway, is regulated
by nitrate as well as by end-products of nitrate assimilation, namely, glutamine (Gln) and asparagine (Asn). Nitrate induces
expression of the NiR gene. Previously, using deletion analysis of the spinach (Spinacia oleracea L.) NiR gene promoter in transgenic tobacco (Nicotiana tabacum L.) and in-vivo dimethyl sulfate footprinting, we had identified the region between −230 bp and −180 bp as being critical
for nitrate inducibility of this gene. In the present study, we show that the region from +1 to +67, which forms part of its
untranslated leader, is important for minimal induction in the presence of nitrate. Electrophoretic mobility shift assays
reveal concentration-dependent and competitive binding of a factor in tobacco nuclear extracts to this region. In the presence
of Gln or Asn, the expression of spinach NiR is repressed. This repression is observed with the full-length NiR promoter (−3100 bp)
as well as with the shortest promoter (−230 bp) that gives nitrate induction, which includes the +67 bp leader sequence. The
repressed expression of the gene is not the result of reduced nitrate accumulation in the presence of the nitrogen metabolites.
Received: 2 December 1997 / Accepted: 20 January 1998 相似文献
19.
A 1.6 kb upstream regulatory sequence (GenBank accession no. AF472487) of plasma membrane aquaporinBnPIP1 gene fromBrassica napus was obtained by genomic walking based on ligation-mediated PCR method. Sequence analysis indicated that this fragment contained
seed germination specific and vascular specific sequences. The 1.6 kb upstream sequence and various 5′ end deleted sequences
were fused withuidA gene and constructed into plant expression vectors which were used for tobacco transformation. GUS histochemical assay showed
that the 1.6 kb fragment had high levels of promoter activity and the GUS staining was mainly distributed in vascular systems
and tissues with rapid expanding and proliferating cells. Promoter deletion analysis showed that the deletion of -1610 — -1030
bp resulted in a dramatic reduction in GUS activity. It was assumed that there might be cis-acting element(s) existing in
this region. Whereas, the region located at -1030 — -902 bp strongly inhibited the expression ofgus and probably contained negative regulatory element(s). The fragment of -902 — -19 bp could also directgus expression at high level. 相似文献
20.
Chun-xiao Yu Tong Jin Wei-wen Chen Peng-ju Zhang Wen-wen Liu Heng-yun Guan Ju Zhang Qing-wei Liu An-li Jiang 《Molecular biology reports》2009,36(8):2353-2360
NKX3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. However, little is known
about the mechanism for regulation of NKX3.1 in prostate cancer. With RT-PCR and western blot, we found that NKX3.1 expression was enhanced by over-expression of Sp1 at both the mRNA and protein levels in prostate cancer LNCaP cells. To
identify the Sp1-elements in the promoter region of NKX3.1, a 521 bp-promoter of human NKX3.1 gene containing three possible Sp1-elements was cloned into the upstream of the luciferase reporter gene in pGL3-basic plasmid. With deletion mutation analysis, plasmid construction, EMSA and oligonucleotide decoy technique, two Sp1-elements
which located between +29 to +43 and −60 to −46 of NKX3.1 gene were identified and proven to be functional elements. It will be important to further study on the functions and the
regulatory mechanisms of Sp1 element in NKX3.1 gene expression. 相似文献