The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants

A series of 5′ deletions of the pea plastocyanin gene (petE) promoter fused to the β-glucuronidase (GUS) reporter gene has been examined for expression in transgenic tobacco plants. Strong positive and negative cis-elements which modulate quantitative expression of the transgene in the light and the dark have been detected within the petE promoter. Disruption of a negative regulatory element at ?784 bp produced the strongest photosynthesis-gene promoter so far described. Histochemical analysis demonstrated that all petE-GUS constructs directed expression in chloroplast-containing cells, and that a region from ?176 bp to +4 bp from the translation start site was sufficient for such cell-specific expression. The petE-promoter fusions were expressed at high levels in etiolated transgenic tobacco seedlings but there was no marked induction of GUS activity in the light. The endogenous tobacco plastocyanin genes and the complete pea plastocyanin gene in transgenic tobacco plants were also expressed in the dark, but showed a three- to sevenfold increase in the light. This indicates a requirement for sequences 3′ to the promoter for the full light response of the petE gene.     

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.     

Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection.     

Isolation and heterologous transformation analysis of a pollen-specific promoter from wheat (Triticum aestivum L.)

The promoter of a pollen-specific gene TaPSG719 was isolated from wheat (Triticum aestivum L.) by inverse-PCR (IPCR). Sequence analysis revealed that the promoter contains two cis-acting elements (AGAAA and GTGA) known to confer anther/pollen-specific gene expression which suggests that the promoter of TaPSG719 gene is a pollen-specific one. To ascertain the regulatory function of TaPSG719 promoter, two deleted fragments (?1,776 to ?1 bp and ?1,019 to ?1 bp) were fused to the β-glucuronidase (GUS) gene and transformed into tobacco plants. Similar GUS expression patterns were observed in all transformed plants and its activity was detected exclusively in pollen. No GUS activity in any other floral or vegetative tissue was observed. The results confirm that TaPSG719 promoter is pollen-specific and active during the middle stages of pollen development till anther matured, and it can drive pollen-specific gene expression across the species.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

Partial Characterization of a Gene for a 31 kD Protein of Pseudomonas syringae pv. syringae Apparently Involved in Pathogenicity

P. syringae pv. syringae strain R32 causes the bacterial brown spot disease on bush beans. A 31 kD protein was detected which is involved in the pathogenic response. Monospecific antibodies directed against this 31 kD-protein were used to screen a protein expression gene bank made from the wild type strain R32. A 0.8 kb DNA insert of a clone which gave a positive reaction with the monospecific antibodies was used in hybrizations to clone a larger chromosomal fragment. A km-cassette (kmresistance) was integrated into this chromosomal DNA-fragment preventing the expression of the 31 kD-protein. This construct was integrated into the chromosomal of the wild type strain R32 viahomologous recombination resulting in the 31 kD-protein deficient mutant LMI. Biotests with the host plant (bean) and with tobacco leaves showed no symptoms or hypersensitive reaction (HR) when the mutant LMI was inoculated. However, atypical chlorotic and necrotic lesions compared to the wild type strain R32 were found on tobacco leaves when the mutant LMI was incubated for more than 2 weeks. Complementation of the LMI mutant with a plasmid harbouring the corresponding wild type R32 DNA fragment resulted in an isolate (LMIC) which showed a partial restoration of the HR on tobacco but no brown spot disease symptoms on bush beans. The 31 kD-protein could be detected serologically in LMIC.     

Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco

Summary Maturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumintype genes of several legume species. Constructs containing-833 and-1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions-97 and-549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions-549 and-1203 bp appear to function as enhancer-like elements, to increase expression.     

花生小GTP结合蛋白基因AhRabG3f启动子的盐胁迫响应元件分析

为研究花生小GTP结合蛋白基因AhRabG3f对盐胁迫响应的分子机制,文中克隆了花生AhRabG3f基因起始密码子上游1 914 bp的启动子片段(3f-P)。将该启动子5''末端截短获得5个片段(3f-P1-3f-P5),长度分别为1 729、1 379、666、510、179 bp。构建了将这6个启动子片段与gus基因融合的植物表达载体,利用农杆菌介导法转化烟草。对转基因烟草进行GUS表达分析和酶活性检测,结果表明,在转入各启动子片段的烟草中,都能检测到gus基因的表达,其中全长启动子3f-P的驱动活性最弱,而截短片段3f-P3的驱动活性最强。对转基因烟草进行盐胁迫处理后,3f-P、3f-P1、3f-P2和3f-P3所驱动GUS酶活性是未经盐诱导的3.3、1.2、1.9、1.2倍,表明AhRabG3f启动子是盐诱导型的,在3f-P至3f-P3之间可能存在对盐响应的正调控元件。通过对盐胁迫处理后各启动子片段驱动的GUS活性分析,推测在AhRabG3f启动子上游1 930–1 745 bp、682–526 bp之间存在可能对盐响应的正调控元件MYB、GT1和富含TC的重复序列,1 395–682 bp之间存在可能对盐响应的负调控元件MYC。研究结果可为利用诱导型启动子调控花生的耐盐性提供指导。