Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants

The expression of foreign genes in pollen may pose potentialproblems in the field release of transgenic plants, since pollenrepresents a route whereby foreign genes and their productsmay escape into the wider environment. The possible risks posedby cross-hybridization with wild relatives have been extensivelyexplored, but problems that may arise due to the expressionof foreign gene products in pollen have not been so widely studied.The activities of the CaMV 35S and nos promoters in pollen inpopulations of stably transformed plants and in transient expressionanalysis are described. These promoters are commonly used inall areas of plant molecular biology research and their expressionpatterns will be of interest to those involved in field releasestudies. The results show that both promoters had no detectablepollen activity in Arabidopsis, but both showed activity intobacco pollen. The CaMV 35S-gus gene fusion showed heritableexpression levels in tobacco pollen of up to a maximum of 64.6pmol 4-MU min^–1 mg ^–1 total protein. nos promoteractivity in transgenic tobacco pollen was highly variable, withGUS activities ranging from undetectable levels up to 2561 pmol4-MU min^–1 mg^–1 total protein within the transgenicpopulation. Histochemical staining of anther sections from 10–12mm buds revealed that the CaMV 35S promoter had some activityin the vascular bundle, stomium and tapetum, while GUS expressionfrom the nos promoter in sporophytic tissues was confined entirelyto the stomium. Key words: CaMV 35S promoter, nos promoter, pollen, transgenic plant release     

Comparing constitutive promoters using CAT activity in transgenic tobacco plants

The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.     

Isolation of two highly active soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system

A novel automated image collection and analysis system was used to compare two new soybean (Glycine max (L.) Merr.) promoters with the cauliflower mosaic virus 35S (CaMV35S) promoter, which was used as an expression standard. For expression comparisons, various permutations of a soybean polyubiquitin (Gmubi) promoter, a soybean heat shock protein 90-like (GmHSP90L) promoter and the CaMV35S promoter were placed upstream of a green fluorescent protein (gfp) gene. DNA constructs were introduced via particle bombardment into excised cotyledons of germinating lima bean (Phaseolus lunatus L.) seeds, which were arranged in Petri dishes for automated image capture and image analysis. The automated system allowed monitoring and quantification of gfp gene expression in the same piece of tissue over time. The Gmubi promoter, with its intronic region intact, showed the highest expression that was over five times stronger than the CaMV35S promoter. When an intronic region was removed from the Gmubi promoter, GFP expression was reduced, but was still over two times greater than with the CaMV35S promoter. The full-length soybean GmHSP90L promoter was four times stronger than the CaMV35S promoter. Truncation of the GmHSP90L promoter resulted in stepwise decreases in promoter strength, which appear to correspond to removal of regulatory elements. Automated image capture and analysis allowed the rapid and efficient evaluation of these new promoters. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Transient expression of a reporter gene in bulbscales and immature embryos of three Lilium species is affected by 5'upstream sequences and culture conditions

In Lilium , a transformation system has not yet been developed. For efficient selection of cells expressing transferred genes following particle bombardment, the effects of 5'upstream regions on the transient expression of the β-glucuronidase gene ( gusA ) were estimated in bulbscales and immature embryos of lily. When four plasmids having the gusA gene under the control of the cauliflower mosaic virus (CaMV) 35S, maize alcohol dehydrogenase gene and rice actin gene ( Actl ) promoters, and the castor bean catalase introm were introduced by particle bombardment, the patterns of transient expression in the bulbscales showed differences among three Lilium species, L. x formolongi, L. dauricum and L. japonicum . In immature embryos of L x formolongi , transient expression was significantly influenced by age of embryos after self-pollination, duration of culture before bombardment, and culture conditions. Moreover, the transient gusA expression driven by six different 5'upstream regions, including the maize ubiquitin gene promoter and a modified CaMV 35S promoter were compared in both bulbscales and immature embryos. Use of the Actl and modified CaMV 35S promoters resulted in the greatest number of cells that transiently expressed gusA in both types of tissue of L. x formolongi . These two promoters are efficient for use in lily transformation.     

Electroporation and PEG delivery of DNA into maize microspores

Summary The ability to deliver and detect reporter gene activity in maize microspores was tested. Tested expression vectors contained the chloramphenicol acetyl transferase (CAT) gene and one of the following promoter-intron combinations: 1) cauliflower mosaic virus (CaMV 35S), 2) CaMV 35S + maize alcohol dehydrogenase 1 intron 6 (Adh1-I6), 3) maize alcohol dehydrogenase 1 + intron 1 (Adh1-I1), or 4) maize ubiquitin 1 + intron 1 (Ubiq 1-I1) promoter + intron. The expression vectors were delivered into maize microspores using electroporation or polyethylene glycol (PEG). Both methods were effective for delivering free DNA into microspores. Although all four promoters were active in maize protoplasts, only two promoters were active in maize microspores. The CaMV 35S and the Adh1 promoters did not promote gene expression in maize microspore. The CaMV 35S + Adh1-I6 and Ubiq1-I1 promoters produced high levels of CAT activity in maize microspores.     

The TACPyAT repeats in the chalcone synthase promoter of Petunia hybrida act as a dominant negative cis-acting module in the control of organ-specific expression

Analysis of the expression of the GUS reporter gene driven by various regions of the Petunia hybrida chalcone synthase (chsA) promoter revealed that the developmental and organ-specific expression of the chsA gene is conferred by a TATA proximal module located between -67 and -53, previously designated as the TACPyAT repeats. Histochemical analysis of GUS reporter gene expression revealed that the organ-specific 67 bp promoter fragment directs the same cell-type specificity as a 530 bp promoter, whereas additional enhancer sequences are present within the more TATA distal region. Moreover, the region between -800 and -530 is also involved in extending the cell-type specificity to the trichomes of flower organs and of young seedlings. The mechanism by which the TACPyAT repeats modulate expression during plant development was studied by analysing the expression of the GUS gene driven by chimeric promoters consisting of the CaMV 35S enhancer (domain B, -750 to -90) fused to various chsA 5' upstream sequences. Detailed enzymatic and histochemical analysis revealed that in the presence of the TACPyAT module the CaMV 35S region only enhances GUS activity in those organs in which the chsA promoter is normally active. Furthermore, this analysis shows that enhancement in the presence of the CaMV 35S domain B is accomplished by increasing the number of cell types expressing the GUS gene within the organ, rather than enhancement of the chsA cell-type-specific expression within these organs. Deletion of the TACPyAT sequences in the chimeric promoter construct completely restores the well-documented CaMV 35S domain B cell-type specificity, showing that the TACPyAT module acts as a dominant negative cis-acting element which controls both organ and developmental regulation of the chsA promoter activity.     

Transformation of chicory and expression of the bacterial uidA and nptll genes in the transgenic regenerants

Transgenic chicory plants were obtained from different explantsco-cultured with Agrobacterium tumefaciens. Among tap-root,leaf and cotyledonary tissues, etiolated cotyledons showed thegreatest competence for transformation. The Agrobacterium strainsused contained either pGSGLUC1 or pTDE4 as a vector which carryboth the neomycin phosphotransferase II gene (nptll) for kanamycinresistance and ß-glucuronidase gene (uidA) under thecontrol of different promoters. Transformation was confirmedby NPTII enzymatic assay, histochemical analysis of GUS activityand DNA hybridization. Transgenic plants expressed both markergenes in root and shoot tissues. In leaves, GUS activity wasexpressed in all tissue types, whatever the nature of the promoter.Nevertheless, variable heterogeneous patterns of expressionwere observed in the different root tissues. Differential expression of the GUS fusions controlled by thedual TR or the CaMV 35S promoters are discussed. Key words: Chicory, genetic transformation, GUS activity, kanamycin resistance     

Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation

We have constructed a set of plant transformation vectors, promoter cassettes, and chimeric antibiotic-resistance genes for the transformation and expression of foreign genes in plants sensitive to Agrobacterium infection. The different vectors allow for either concurrent or consecutive selection for kanamycin and hygromycin resistance and have a number of unique restriction sites for the insertion of additional DNA. The promoter cassettes utilize the CaMV 19S and CaMV 35S promoters and are constructed to allow for the easy insertion of foreign genes. The cloned gene can then easily be inserted into the transformation vectors. We have utilized the promoter cassettes to express the hygromycin-resistance gene either from the CaMV 35S or the CaMV 19S promoters, with both chimeric resistance genes allowing for the selection of hygromycin-resistant tobacco plants.     

Transformation and evaluation of different transgenic lines for Glyphosate tolerance and cane borer resistance genes in sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.)

The aim of this study was to ensure the systematic protein expression of two genes (GTG and Cry1Ac) under the influence of two different constitutive promoters i.e. Ubiquitin-1 and CaMV 35S promoters in different sugarcane lines. PCR amplification of GTG and Cry1Ac was achieved from putative transgenic plants through gene specific primers. Qualitative comparisons of GTG and Cry1Ac genes expression under two different promoters were obtained through protein dot blot and dipstick assay. The appearance of comparatively dark color dots in dot blot and dark color bands on dipstick with Ubiquitin as compared to light color bands with CaMV35S promoter, qualitatively confirmed high protein expression of two genes under Ubiquitin promoter. In quantitative gene expression comparisons maximum optical density (OD) at 450 nm of UV-light was obtained for GTG (3.7 OD) and Cry1Ac (3 OD) under Ubiquitin promoter, while for GTG (1.6 OD) and Cry1Ac (2.5 OD) with CaMV 35S promoter. The results indicated higher expression of two genes under Ubiquitin-1 promoter in sugarcane was found as compared to CaMV 35S promoter. This study provides a guide for stable and high expression of transgenes with reference to Ubiquitin-1 promoter which can be utilize in sugarcane as well as in other monocots.     

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots.

The rice (Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5'-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB's 5'-flanking DNA fragments (nucleotides -1066 to +298) had about 20 and 3-4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (-56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (-56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5'-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from -329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (-329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。