In Silico and Deletion Analysis of Upstream Promoter Fragment of S-Adenosyl Homocysteine Hydrolase (SAHH1) Gene of Arabidopsis Leads to the Identification of a Fragment Capable of Driving Gene Expression in Developing Seeds and Anthers

S-adenosyl homocysteine hydrolase (SAHH) is a key enzyme in methylation metabolism of eukaryotes. A 1585 by fragment upstream to ATG of SAHH1 gene, was fused with a promoter-less β-Glucuronidase (GUS) gene and mobilized into Arabidopsis by Agrobacterium-mediated floral transformation to generate transgenic Arabidopsis. This fragment was found to drive constitutive expression of GUS in T₂ progeny of transgenic Arabidopsis. In silico analysis of the promoter region of SAHH1 suggested the presence of several cis-regulatory motifs including seed-specific motifs as well as anther-specific motifs in the 376 by (upstream to TSS of SAHH1) promoter fragment. Based on the partial deletion analysis carried out in the promoter region of SAHH1 (At4gl3940) this 376 by promoter fragment was found to be capable of driving GUS expression in developing seeds and in some anthers/micros pores.     

Fruit-specific Over-expression of LeEXP1 Gene in Tomato Alters Fruit Texture

Expansins are cellular proteins with diverse physiological functions. Expression of fruit-specific expansin gene in tomato is associated with fruit softening — a desirable trait from the processing point of view. In the present study, an expansin gene LeEXP1 was introduced via Agrobacterium tumefaciens in sense orientation under the control of a fruit-specific promoter LeACS4 with nptII gene as selection marker in Indian tomato cv Pusa Uphar. PCR detection and Southern blot analysis confirmed the integration of the transgene in the transformed tomato plants. RT-PCR and northern blot analysis using total RNA isolated from leaves and fruits confirmed over-expression of the LeEXP1 gene in transgenic fruits as compared to the wild type plants. Apart from the visual change in increased red colouration of fruits at different stages of ripening, overexpression of the LeEXP1 gene resulted in enhanced fruit softening, as determined by force required to rupture the fruit pericarp, in the transgenic fruits from breaker stage onwards as compared to the non-transformed wild type fruits. The results thus suggest an improvement in texture of the LeEXP1 over-expressing fruits, which might be useful for tomato processing industry.     

Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development

Fruit-specific promoters have been used as genetic engineering tools for studies on molecular mechanism of fruit development and advance in fruit quality and additional value by increasing functional component. Especially fruit-ripening specific promoters have been well utilized and studied in tomato; however, few studies have reported the development of promoters that act at fruit developing stages such as immature green and mature green periods. In this study, we report novel promoters for gene expression during the green to ripening stages of tomato fruit development. Genes specifically expressed at tomato fruit were selected using microarray data. Subsequent to confirmation of the expression of the selected 12 genes, upstream DNA fragments of the genes LA22CD07, Les.3122.2.A1_a_at and LesAffx.6852.1.S1_at which specifically expressed at fruit were isolated from tomato genomic DNA as promoter regions. Isolated promoter regions were fused with the GUS gene and the resultant constructs were introduced into tomato by agrobacterium-mediated transformation for evaluation of promoter activity in tomato fruit. The two promoters of LA22CD07, and LesAffx.6852.1.S1_at showed strong activity in the fruit, weak activity in the flower and undetectable activity in other tissues. Unlike well-known fruit-ripening specific promoters, such as the E8 promoter, these promoters exhibited strong activity in green fruit in addition to red-ripening fruit, indicating that the promoters are suitable for transgene expression during green to ripening stages of tomato fruit development. KEY MESSAGE: Novel fruit-specific promoters have been identified and are suitable for transgene expression during green to ripening stages of tomato fruit development.     

甜蛋白Brazzein基因在番茄果实中的特异表达

西瓜(Citrullus vulgaris S.)来源的AGPL1启动子在番茄(Lycopersicon esculentum L.)果实中具有较强的特异性驱动功能。将该启动子与甜味蛋白基因Brazzein融合构建植物表达载体, 通过根癌农杆菌(Agrobacterium tumefaciens)介导法成功地进行了对番茄的遗传转化, 获得转化植株。组织化学法、PCR特异扩增、Southern杂交分析及RT-PCR检测, 表明Brazzein基因已整合到转基因番茄植株基因组中并且稳定表达。通过AGPL1果实特异启动子的调控, 在不改变果实其他性状的前提下提高了番茄果实甜味品质, 并为甜蛋白的生产提供经验。     

Analysis of banana fruit-specific promoters using transient expression in embryogenic cells of banana cultivar Robusta (AAA Group)

The study reports the transient expression of gusA gene in embryogenic cells using three banana derived fruit-specific promoters. Banana embryogenic cells were transformed with a pCAMBIA-1301 derived plasmid construct harboring gusA gene driven by either chitinase, glucanase or expansin promoters derived from banana. The transient expression of ??-glucuronidase was studied 5?days after co-cultivation with Agrobacterium harboring the expression plasmids. The transformed embryogenic cells were treated with different inducers of ethylene such as ethephon, methyl jasmonate, methyl salicylate, abscisic acid and indole acetic acid. The maximum expression of 64099.78?pmoles 4-MU/h/mg total protein was noted with expansin promoter when the cells were treated with the combination of ethephon (0.25?mM) and MJ (10?mM). The results suggest that these promoters can be used to achieve fruit-specific expression of useful transgenes in banana. The results should prove to be an important guide for short term expression studies for promoter validation and gene screening.     

Isolation and Activity Analysis of a Seed-Abundant soyAP1 Gene Promoter from Soybean

The soybean aspartic proteinase gene soyAP1 has previously been shown to be expressed specifically in soybean seeds. To investigate the expression pattern and active cis-elements of the soyAP1 promoter, the 1,650-bp 5??-upstream genomic DNA fragment named PS-552 was isolated by PCR walking. Sequence analysis revealed that this fragment contains a series of motifs related to seed-specific promoters and some pollen-expressed elements. Stable expression in transgenic Arabidopsis thaliana showed that the PS-552 promoter can regulate beta-glucuronidase gene accumulation in mature seeds at much higher levels than other tissues, especially vegetative tissues, and exhibits similar activity to the 35S promoter in mature seeds. These results show that the PS-552 promoter is a highly active promoter controlling downstream gene expression, mainly in mature seeds. The 5??-end deletion studies of PS-552 showed that the cis-elements of CAAACAC, AACA, E-box, and CCAA play a role in increasing the seed-specific activity. The proportion of mature seed activity and flower activity was increased as the deletion fragment lengthened, indicating that seed cis-elements possibly lessen or suppress the effect of pollen-expressed elements, increasing the activity of PS-552 in mature seeds.     

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.     

Isolation and Characterization of OsGSTL1 Promoter from Rice

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the β-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2?155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2?155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1?224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5′-upstream region between −2?155 and −1?224 bp.     

GUS expression driven by constitutive and phloem-specific promoters in citrus hybrid US-802

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated.     

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

Key message

The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit.

Abstract

Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30–630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.     

Seed-specific expression of seven Arabidopsis promoters

Seeds contain storage compounds, from various carbohydrates to proteins and lipids, which are synthesized during seed development. For the purposes of many plant researches or commercial applications, developing promoter systems expressing specifically in seeds or in particular constituents or tissues/compartments of seeds are indispensable. To screen genes dominantly or specifically expressed in seed tissues, we analyzed Arabidopsis ATH1 microarray data open to the public. Thirty-two candidate genes were selected and their expressions in seed tissues were confirmed by RT-PCR. Finally, seven genes were selected for promoter analysis. The promoters of seven genes were cloned into pBI101 vector and transformed into Arabidopsis to assay histochemical β-glucuronidase (GUS) activity. We found that Pro-at3g03230 promoter drove GUS expression in a chalazal endosperm, Pro-at4g27530:GUS expressed in both chalazal endosperm and embryo, Pro-at4g31830 accelerated GUS expression both in radicle and procambium, Pro-at5g10120 and Pro-at5g16460 drove GUS expression uniquely in embryo, Pro-at5g53100:GUS expressed only in endosperm, and Pro-at5g54000 promoted GUS expression in both embryo and inner integument. These promoters can be used for expressing any genes in specific seed tissues for practical application.     

Functional analysis of an <Emphasis Type="Italic">iaaM</Emphasis> gene in parthenocarpic fruit development in transgenic <Emphasis Type="Italic">Physalis pubescens</Emphasis> L. plants

An efficient somatic embryogenesis system for Physalis pubescens L. (husk tomato) was developed prior to transformation. Subsequently, cotyledonary explants of P. pubescens were transformed with a chimeric construct containing an iaaM gene from driven by the fruit-specific promoter 2A12 to develop parthenocarpic fruits. Following selection of explants on Murashige and Skoog (MS) medium containing containing 75 mg l⁻¹ kanamycin (Km), 36 km-resistant callus clusters were recovered, and these were regenerated into whole plants. Expression of the iaaM gene was detected in confirmed transgenic fruits. The 0.9-kb 2A12 promoter was capable of directing expression of the introduced iaaM gene in transgenic P. pubescens fruits, but iaaM expression was absent from both leaves and flowers. Quantitative measurements of indole-3-acetic acid (IAA) content during fruit development indicated that the IAA levels in transgenic lines increased from anthesis through young fruits and peaked at fruit maturity. On average, IAA contents in transgenic fruits were two-fold higher than those in control fruits. Under greenhouse condition, vegetative growth, morphology, and the flowering of transgenic plants were comparable to those of control plants. However, the fruits of transgenic lines ripened earlier and had fewer seeds per fruit than did control plants.