The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl-transferase (FTase) gene was examined using transgenic expression of the β-glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the -glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, -glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.     

Light-regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene

Expression of the pea plastocyanin gene ( PetE ) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5' untranslated leader region does not direct light-regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light-regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5' untranslated leader region, the coding region and the 3' region of the PetE gene to light-regulated expression. Both the coding region and the 5' untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5' leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5' end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.     

A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells

A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5-deletion mutants of the SWPA2 promoter fused to the -glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the –1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the –1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the –1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.     

Analysis of the Tissue-SpecificS RNase gene promoter associated with self-incompatibility in tomato (Lycopersicon peruvianum L.)

To understand the expression pattern of theS RNase gene in the floral tissues associated with self-incompatibility (SI), promoter region of S₁₁ RNase gene was serially deleted and fused GUS. Five chimeric constructs containing a deleted promoter region of the S₁₁ RNase gene were constructed, and introduced intoNicotiana tabacum using Agrobacterium-mediated transformation. Northern blot analysis revealed that the GUS gene was expressed in the style, anther, and developing pollen of all stages in each transgenic tobacco plant The developing pollen expressed the same amount of GUS mRNA in all stages in transgenic tobacco plants. In addition, histochemical analysis showed GUS gene expression in vascular bundle, endothecium, stomium, and tapetum cells during pollen development in transgenic plants. From these results, it is speculated that SI ofLycopersicon peruvianum may occur through the interaction ofS RNase expressed in both style and pollen tissues.     

Increased stable inheritance of herbicide resistance in transgenic lettuce carrying a petE promoter-bar gene compared with a CaMV 35S-bar gene

Inheritance of resistance to herbicide (300 mg/l glufosinate ammonium) up to the third (T3) seed generation was compared in two populations of transgenic lettuce (Lactuca sativa L. cv ’Evola’) harbouring a T-DNA containing the bar gene, linked to either the Cauliflower Mosaic Virus (CaMV) 35S promoter, or a –784-bp plastocyanin promoter from pea (petE). Only 2.5% (4/163) of CaMV 35S-bar plants, selected by their kanamycin resistance(T0 generation), transmitted herbicide resistance at high frequency to their T3 seed generation compared with 97% (29/30) for kanamycin resistant petE-bar plants. In the case of 35S-bar transformants, only 16% (341/2,150) of the first seed generation (T1) plants, 22% (426/1,935) T2 plants and 11% (1,235/10,949) T3 plants were herbicide-resistant. In contrast, 63% (190/300) T1 plants, 83% (2,370/2,845) T2 plants and 99% (122/123) T3 petE-bar transformed plants were resistant to glufosinate ammonium. The T-DNAs carrying the petE-bar and CaMV 35S-bar genes also contained a CaMV 35S-neomycin phosphotransferase (nptII) gene. ELISA showed that NPTII protein was absent in 29% (45/156) of the herbicide-resistant T2 plants from 8/19 herbicide-resistant petE-bar lines. This indicated specific inactivation of the CaMV 35S promoter on the same T-DNA locus as an active petE promoter. The choice of promoter and T-DNA construct are crucial for long-term expression of transgenes in lettuce. Received: 13 November 1998 / Accepted: 20 February 1999     

竹节花黄斑驳病毒启动子的缺失分析及功能

竹节花黄斑驳病毒(CoYMV)是侵染单子叶植物竹节花的一 种双链环状DNA病毒,它的启动子可介导外源基因在烟草韧皮部特异表达。为了研究其组织 特异性表达的最佳启动子区域,对CoYMV启动子进行了5′端五种不同长度的缺失分析,用不同长度的启动子片段与GUS基因及NOS3′端转录中止序列构建了全长启动子及5 个缺失启动子序列的六个嵌合GUS基因植物表达载体。利用农杆菌将上述嵌合基因转化烟草 外植体后,每种表达载体都获得了一批转基因烟草植株。转化再生烟草植株的PCR分析、GUS 酶活测定及GUS组织染色的结果表明六种类型的嵌合基因已整合到烟草染色体中,并有五种 表达出GUS活性。缺失到870bp的启动子比全长启动子(1040bp)的活性约高78%,870bp比585bp启动子介导的GUS活性略高但差别不明显,缺失到447和232时GUS活性有明显下 降,但仍具有韧皮部特异表达的特性。当缺失到TATA box附近的44bp时启动子丧失组织特 异性,GUS活性也降低到测不出来的水平。以上结果表明CoYMV启动子从转录起始位点上游 870bp～230bp及232bp下游区分别与启动子的活性和韧皮部组织特异性密切相关,870bp上游可能存在一个负调控序列,所以该启动子的活性和组织特异性的最佳调控区应在87 0bp或585bp的下游区。CoYMV启动子与35S启动子驱动GUS基因在烟草中表达的活性相比, 前者为后者的70%左右,考虑到前者仅在韧皮部细胞表达而后者为组成型表达,所以CoYMV启 动子在韧皮部的活性可能与35S启动子相当或更高。CoYMV启动子在其它转基因植物中驱动外 源基因表达的特点正在研究中。     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.     

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。     

Differential in vitro DNA binding activity to a promoter element of the gn1β-1,3-glucanase gene in hypersensitively reacting tobacco plants

In a hypersensitive reaction to pathogen infection, expression of the β-1,3-glucanase gn1 gene is induced in cells surrounding the necrotic lesions. The 5′-flanking sequence of gn1 was examined to investigate the molecular basis controlling activation of gene expression during this plant defense response. Studies on transgenic tobacco plants containing gn1 promoter deletions fused to the β-glucuronidase reporter gene revealed the presence of negative and positive regulatory sequences mediating both the level and the spatial distribution of gn1 expression. Promoter sequences to ?138 bp were sufficient to confer increased gene expression around the necrotic lesions produced in response to Pseudomonas syringae pv. syringae inoculation. It is demonstrated by electrophoretic mobility shift assays that nuclear proteins in both healthy and hypersensitively reacting tobacco leaves interact with DNA sequences within the regulatory elements identified. Among the binding sequences characterized, the promoter region extending from ?250 to ?217 bp contained the DNA motif -GGCGGC- found to be conserved in most if not all promoters of genes encoding pathogenesis-related basic proteins. The activity bound by this promoter sequence was stronger in hypersensitively responding tissues than in healthy untreated tobacco leaves.     

玉米19kD醇溶贮藏蛋白基因启动子种子特异性表达控制区段的分析

为研究玉米（Ｚｅａｍａｙｓ　Ｌ．）１９ｋＤ醇溶贮藏蛋白（ｚｅｉｎ）基因启动子种子特异性表达的控制区段,将全长６９４ｂｐ的启动子进行５’端缺失,共得到６个缺失突变体,长度分别为４８８ｂｐ、３７８ｂｐ、３０２ｂｐ、１５２ｂｐ、１２４ｂｐ和８５ｂｐ。将６个片段分别与报告基因ｇｕｓ连接构建成表达载体ｐＤＧＢ系列,经土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ）介导转化,引入烟草。ＧＵＳ活性检测证明,４８８ｂｐ启动子片段能促使ｇｕｓ基因在种子中特异表达。３７８ｂｐ、３０２ｂｐ、１５２ｂｐ和１２４ｂｐ片段启动子引导的ｇｕｓ基因在烟草根、叶柄、种子中均可表达。     

Analysis of the Maize 19 kD Zein Gene Promoter Fragment Driving Seed specific Expression

A deletion works of a maize 19 kD zein gene promoter in the 5'end was performed and six promoter fragments of different length were obtained. A series of expression vectors was constructed and then transferred into tobacco ( Nicotiarta tabacum L. ) plants. GUS activity assays indicated that the expression of 488 bp promoter was tissue-specific, for which GUS was active only in transgenic tobacco seeds. The other four fragments containing 378 bp,302 bp,152 bp and 124 bp also have the activity of promoter. They could drive gus gene expressed not only in seeds but also in roots and petioles.