Transgene Expression in the Vegetative Tissues of Apple Driven by the Vascular-specific rolC and CoYMV Promoters

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of -glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.     

A seed coat-specific promoter for canola

The canola industry generates more than $11 billion of yearly income to the Canadian economy. One problem of meal quality is the dark polyphenolic pigments that accumulate in the seed coat. Seed coat-specific promoters are a pre-requisite to regulate the genes involved in seed coat development and metabolism. The β-glucuronidase (GUS) reporter gene was used to test an Arabidopsis promoter in developing and mature seeds of canola (Brassica napus). The promoter tested is the regulatory region of the laccase gene (AtLAC15) from Arabidopsis thaliana. The AtLAC15 promoter::GUS construct was inserted into canola double haploid line DH12075 using Agrobacterium-mediated transformation. Southern blot analysis using a 536 bp GUS probe showed variation among the transformed plants in the T-DNA copy numbers and the position of the insertion in their genomes. Histochemical assay of the GUS enzyme in different tissues (roots, leaves, stem, pollen grains, flowers, siliques, embryos and seed coats) showed ascending GUS activity only in the seed coat from 10 days after pollination (DAP) to the fully mature stage (35 DAP). GUS stain was observed in the mucilage cell layer, in the outer integument layer of the seed coat but not in the inner integument. The AtLAC15 promoter exhibited a specificity and expression level that is useful as a seed coat-specific promoter for canola.     

Expression of a polyubiquitin promoter isolated from Gladiolus

A polyubiquitin promoter (GUBQ1) including its 5′UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5′ and 3′ intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5′UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5′UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.     

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。     

Comparative expression of β-glucuronidase with five different promoters in transgenic carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) root and leaf tissues

Tissue-specific patterns and levels of protein expression were characterized in transgenic carrot plants transformed with the β-glucuronidase (GUS) gene driven by one of five promoters: Cauliflower mosaic virus 35S (35S) and double 35S (D35S), Arabidopsis ubiquitin (UBQ3), mannopine synthase (mas2) from Agrobacterium tumefaciens or the rooting loci promoter (rolD) from A. rhizogenes. Five independently transformed carrot lines of each promoter construct were assessed for GUS activity. In leaves, activity was highest in plants with the D35S, 35S and UBQ3 promoters, while staining was weak in plants with the mas2 promoter, and only slight visual staining was present in the leaf veins of plants containing rolD promoter . Strong staining was seen in the lateral roots, including root tips, hairs and the vascular tissues of plants expressing the 35S, D35S and UBQ3. Lateral roots of plants containing the rolD construct also showed staining in these tissues while the mas2 promoter exhibited heightened staining in the root tips. Relatively strong GUS staining was seen throughout the tap root with all the promoters tested.. When GUS expression was quantified, the UBQ3 promoter provided the highest activity in roots of mature plants, while plants with the D35S and 35S promoter constructs had higher activity in the leaves. Although plants containing the mas2 promoter had higher levels of activity compared to the rolD plants, these two promoters were significantly weaker than D35S, 35S and UBQ3. The potential for utilization of specific promoters to target expression of desired transgenes in carrot tissues is demonstrated.     

大麦与油菜种皮特异启动子表达模式的比较

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件;转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高;油菜Bntt种皮特异启动子表达较弱;成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性;Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。     

Tissue specificity of the sugarcane bacilliform virus promoter in oat,barley and wheat

The tissue-specificity of the sugarcane bacilliform virus (SCBV) promoter was investigated in oat, barley, and wheat to determine whether its expression pattern in one species was predictive of promoter specificity in the other closely related Gramineae species. Progeny of transgenic plants produced using constructs containing the SCBV promoter driving gusA were sampled at different stages of plant development and stained for GUS activity using a histochemical assay. Overall, the GUS staining patterns were most similar between oat and barley. In all three species, similar GUS staining patterns were observed in mature endosperms, leaves, and floral bracts of developing infloresences. No GUS staining was detected in oat embryos whereas the entire barley embryo was stained, and GUS staining was confined to the scutellum of wheat embryos. Oat and barley stems exhibited GUS staining whereas no GUS staining was observed in stems of the transgenic wheat plants. The SCBV promoter conferred strong GUS staining intensity in most tissues of oat and barley but was generally weaker in wheat. These differences in SCBV promoter specificity indicate that promoter evaluation should be conducted in the target species of interest rather than by extrapolation from expression patterns in other species.     

拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。     

Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants

Tomato (Lycopersicon esculentum Mill. cv. Better Boy) plants were transformed with a fused gene containing a 2.2-kb promoter fragment of the tomato prosystemin gene and the coding region of the β-glucuronidase (GUS) reporter gene. The transgenic plants exhibited a low constitutive level of prosystemin-β-glucuronidase gene expression, assayed by histochemical staining and GUS enzyme activity, that was associated in the vascular bundles of leaf main veins, petiolules, petioles and stems. The GUS activity in the vascular bundles in each tissue was increased by wounding and by treatment of the plants with methyl jasmonate, similar to the induction of prosystemin in wild-type plants. The increase in GUS activity in the vascular bundles of leaves in response to wounding correlated with the wound-inducible increase in prosystemin mRNA. Tissue printing, using rabbit anti-serum prepared against prosystemin, confirmed that inducible prosystemin protein was localized in vascular bundles of petiolules, petioles and stems of wild-type tomato plants. The evidence indicates that the 2.2-kb promoter region of the tomato prosystemin gene contains elements conferring its correct temporal and spatial expression in the vascular bundles of transgenic tomato plants. Received: 7 January 1997 / Accepted: 2 April 1997     

Promoter Activity of Phenylalanine Ammonia-Lyase Gene of Pharbitis nil in Arabidopsis

Promoter activity of phenylalanine ammonia-lyase (PAL) gene of Pharbitis nil was examined by introducing a PAL:GUS construct into Arabidopsis. GUS staining was observed in vascular bundles of hypocotyl and cotyledons, endodermal cells of the primary root, hydathodes, stigma and pollens of mature flower, abscission zones of petals and sepals and inner layer of seed coat. Light induced GUS expression in cotyledons and the upper part of hypocotyl in which anthocyanin was accumulated. Wounding also induced GUS expression. Endogenous PAL activity increased earlier than the GUS activity directed by the PAL promoter.     

Abscisic acid-regulated Glb1 transient expression in cultured maize P3377 cells

In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression. Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997     

Evaluation of microprojectile-mediated DNA delivery and reporter genes for genetic transformation of the Mediterranean cypress (Cupressus sempervirens L.)

Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria. Received: 18 November 1996/ Revision received: 19 February 1997/ Accepted: 20 November 1997     

Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment

A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG) expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was generally present at a higher copy number than the selected (HYG) gene. Received: 10 October 1997 / Revision received: 18 March 1998 / Accepted: 29 November 1998     

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the ,-glucuronidase gene in tomato

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity. Received: 20 October 1998 / Revision received: 1 December 1998 / Accepted: 14 December 1998     

The tobacco extensin gene Ext 1.4 is expressed in cells submitted to mechanical restraints and in cells proliferating under hormone control

The promoter region of a Nicotiana tabacum extensin gene (Ext 1.4) was studied in tobacco transgenic plants carrying Ext 1.4/GUS (-glucuronidase) chimeric genes. The pattern of expression could be defined and cis-regulatory elements were localized in small regions of the promoter. In healthy plants, expression was essentially found in cells under mechanical stress, that is at the emergence of lateral roots, at the junction between stem and petiole and at the fusion of carpels. In roots of germinating plantlets, expression was found in the piliferous zone. In flowers, expression was found on the one hand in the placenta, in the locular tissue of ovaries and in the zone of carpel fusion, and on the other hand in the connective tissue of anthers, in mature and in germinating pollen. A developmental regulation during seed germination, where the gene fusion is transiently expressed in the endosperm and in the root tip before its expression becomes similar to that found in mature plants has also been shown. The expression of the Ext 1.4/GUS chimeric gene was also induced during cell proliferation under hormone control, for example in response to Agrobacterium tumefaciens infection and in calli. However, when organogenesis occurred under hormone control, expression was never found in root or shoot primordia. Cis-regulatory elements important for expression of the Ext 1.4 GUS gene fusion in germinating seeds, in mature plants or in proliferating cells have been localized in the proximal promoter region whereas enhancer elements have been located further upstream.     

Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco

The β-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5′ deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA₃, ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.     

Combination of viral promoter sequences to generate highly active promoters for heterologous therapeutic protein over-expression in plants

To develop strong promoters for protein over-expression in both dicots and monocots, we constructed a new family of chimeric promoters using sequences of the Commelina Yellow Mosaic Virus (CoYMV), of the Cassava Vein Mosaic Virus (CsVMV) and activating sequences from the CaMV 35S promoter. The chimeric promoters were cloned upstream from the gusA reporter gene. The constructs were used in transient expression experiments, via DNA-coated gold particle delivery to tobacco leaves and maize endosperms. The results showed that candidates among the chimeric promoters could drive expression of the reporter gene to very high levels in the dicot plant tobacco, and all chimeric promoters showed higher expression in maize endosperm than the maize γ-zein promoter used as reference for the monocot expression. Expression cassettes were then used in stable tobacco transformation. Determination of GUS activity throughout growth of the primary transformants showed that two promoters (MPr1163 and MPr1165) could drive expression three to five-fold higher than the highly efficient enhanced 35S promoter. The use of MPr1163 was additionally validated for successful heterologous protein production of human lactoferrin in tobacco via agroinfiltration.