Molecular cloning and characterization of the promoter for the multiple stress-inducible gene <Emphasis Type="Italic">BjCHI1</Emphasis> from <Emphasis Type="Italic">Brassica juncea</Emphasis>

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.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

The promoter of the pepper pathogen-induced membrane protein gene <Emphasis Type="Italic">CaPIMP1</Emphasis> mediates environmental stress responses in plants

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number DQ356279.     

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

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 Kan^R 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.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Combinatorial Interactions of Two <Emphasis Type="Italic">cis-</Emphasis>Acting Elements,AT-Rich Regions and HSEs,in the Expression of Tomato <Emphasis Type="Italic">Lehsp23.8</Emphasis> upon Heat and Non-Heat Stresses

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.     

Molecular cloning and characterization of a jasmonate biosynthetic pathway gene for allene oxide cyclase from <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Herein, we cloned a full-length cDNA encoding allene oxide cyclase (AOC, EC 5.3.99.6) that is a key enzyme in jasmonates (JAs) biosynthetic pathway from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels, named as JcAOC (GenBank accession no. FJ874630). The cDNA was 924 bp in length with a complete open reading frame of 750 bp, which encoded a polypeptide of 250 amino acids including a putative signal peptide of 65 amino acid residues and a mature protein of 185 amino acids with a predicted molecular mass of 20.7 kDa and a isoelectric point of 6.24. Phylogenetic analysis indicated that JcAOC belonged to the AOC superfamily. Semi-quantitative RT-PCR analysis revealed that JcAOC mRNA was expressed in roots, stems, leaves, young seeds, endosperms, and flowers, but that the expression level was highest in leaves and lowest in seeds, and mRNA expression of JcAOC could be induced by salt stress (300 mM NaCl) and low temperature (4°C). Furthermore, the full-length coding region of JcAOC excluding signal peptide sequence was inserted into pET-30a and was successfully expressed in Escherichia coli. Overexpression of JcAOC in E. coli conferred its resistance to salt stress and low temperature.     

The Tall Fescue Turf Grass Class I Chitinase Gene <Emphasis Type="Italic">FaChit1</Emphasis> Is Activated by Fungal Elicitors,Dehydration, Ethylene,and Mechanical Wounding

The cDNA, genomic DNA, and promoter sequence of FaChit1, a class I chitinase gene from Festuca arundinacea, were isolated and characterized in the present work. The deduced amino acid sequence of FaChit1 contains the chitin binding, catalytic, and proline and glycine-rich domains characteristic for most class I chitinases, but no C-terminal extension region. FaChit1 is induced effectively by fungal elicitors, dehydration, and ethylene, but only slightly by mechanical wounding. To identify potential stress-related cis-acting elements, 5′ sequences 935, 651, and 233 bp upstream of the FaChit1 start codon were fused to the GUS reporter gene and analyzed in transgenic tobacco. The results indicated that the 935 bp fragment closely mirrored endogenous gene expression and that the 651 bp fragment was sufficient to direct reporter the gene expression in response to fungal elicitors, ethylene, dehydration, or mechanical wounding due to both known and presently uncharacterized cis-acting elements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transcriptional regulation of the gene for prothoracicotropic hormone in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

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.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Embryo and anther regulation of the mabinlin II sweet protein gene in Capparis masaikai Lévl

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.     

Isolation and functional analysis of the Catharanthus roseus deacetylvindoline-4-O-acetyltransferase gene promoter

Madagascar periwinkle (Catharanthus roseus) produces many therapeutically valuable terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine derived from the monomers vindoline and catharanthine. Deacetylvindoline-4-O-acetyltransferase (DAT) is a key enzyme for the terminal step of vindoline biosynthesis. In this research, the DAT gene promoter was cloned, sequenced, and analyzed. An approximately 1,773 bp genomic DNA fragment of DAT promoter was obtained. Sequence analysis revealed that DAT promoter contained several potential regulatory elements which were involved in the regulation of gene expression. To investigate its function, the promoter fragments with 5′ deletions and gain-of-function deletions were fused to GUS reporter gene, and their expressions were analyzed by transient expression in C. roseus cell suspensions. The regulatory activity of DAT promoter was identified with fluorescence quantitative assays. Three TGACG motifs and one inverted motif (CGTCA) between −808 and −1,086 bp in the DAT promoter were found to be involved in methyljasmonate signal transduction pathway.