A novel endosperm transfer cell-containing region-specific gene and its promoter in rice

The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT-PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and β-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack.     

Expression pattern and activity of six glutelin gene promoters in transgenic rice

The shortage of strong endosperm-specific expression promoters for driving the expression of recombinant protein genes in cereal endosperm is a major limitation in obtaining the required level and pattern of expression. Six promoters of seed storage glutelin genes (GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC) were isolated from rice (Oryza sativa L.) genomic DNA by PCR. Their spatial and temporal expression patterns and expression potential in stable transgenic rice plants were examined with beta-glucuronidase (GUS) used as a reporter gene. All the promoters showed the expected spatial expression within the endosperm. The GluA-1, GluA-2, and GluA-3 promoters directed GUS expression mainly in the outer portion (peripheral region) of the endosperm. The GluB-5 and GluC promoters directed GUS expression in the whole endosperm, with the latter expressed almost evenly throughout the whole endosperm, a feature different from that of other rice glutelin gene promoters. The GluB-3 promoter directed GUS expression solely in aleurone and subaleurone layers. Promoter activities examined during seed maturation showed that the GluC promoter had much higher activity than the other promoters. These promoters are ideal candidates for achieving gene expression for multiple purposes in monocot endosperm but avoid promoter homology-based gene silencing. The GluC promoter did not contain the endosperm specificity-determining motifs GCN4, AACA, and the prolamin-box, which suggests the existence of additional regulatory mechanism in determining endosperm specificity.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

The scutellar vascular bundle-specific promoter of the wheat HD-Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice

An HD‐Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD‐Zip IV family. The 3′ untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full‐length gene containing a 3‐kb‐long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter‐GUS fusion constructs in transgenic wheat, barley and rice plants. Whole‐mount and histochemical GUS staining patterns revealed grain‐specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endosperm during germination and the endosperm‐dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage.     

Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains

The feasibility of producing plant cell wall polysaccharide-hydrolysing feed enzymes in the endosperm of barley grain was investigated. The coding region of a modified xylanase gene (xynA) from the rumen fungus, Neocallimastix patriciarum, linked with an endosperm-specific promoter from cereal storage protein genes was introduced into barley by Agrobacterium-mediated transformation. Twenty-four independently transformed barley lines with the xylanase gene were produced and analysed. The fungal xylanase was produced in the developing endosperm under the control of either the rice glutelin B-1 (GluB-1) or barley B1 hordein (Hor2-4) promoter. The rice GluB-1 promoter provided an apparently higher expression level of recombinant proteins in barley grain than the barley Hor2-4 promoter in both transient and stable expression experiments. In particular, the mean value for the fungal xylanase activity driven by the GluB-1 promoter in the mature grains of transgenic barley was more than twice that with the Hor2-4 promoter. Expression of the xylanase transgene under these endosperm-specific promoters was not observed in the leaf, stem and root tissues. Accumulation of the fungal xylanase in the developing grains of transgenic barley followed the pattern of storage protein deposition. The xylanase was stably maintained in the grain during grain maturation and desiccation and post-harvest storage. These results indicate that the cereal grain expression system may provide an economic means for large scale production of feed enzymes in the future.     

两个水稻胚乳启动子的克隆及表达分析

启动子的克隆对基因表达及基因工程研究有重要意义。根据数据库中EST丰度,从水稻中克隆了两个预测在水稻胚乳中高效表达的启动子Os772和Os359,并将启动子片段与GUS报告基因融合,构建了重组表达载体。通过农杆菌介导方法将其导入水稻愈伤组织细胞。转基因水稻经GUS组织化学分析显示,Os772和Os359能启动GUS基因在水稻胚乳中表达但不能在根、茎、叶和花中表达。该结果表明Os772和Os359为两个水稻胚乳特异性启动子。     

水稻 ADP-葡萄糖焦磷酸化酶小亚基 I 基因的启动子分析

水稻（Oryce sativa L．）基因组中的ADP-葡萄糖焦磷酸化酶小亚基（ADP-Glucose pyrophosphorylase small subunit,OsAgpS）由两个基因编码,即OsAgpSl和OsAgpS2。其中OsAgpSl基因产生两个转录本OsAgpSla和OsAgpSlb,区别在第一个外显子的位置不同。通过RT—PCR方法分析了两个转录本在水稻组织和胚乳不同发育时期的表达特性;同时通过报告基因GUS检测了两个转录本上游转录调节区DNA片段的转录启动特性。结果表明,两个启动子与其下游转录本的表达模式完全一致,即OsAgpSla转录本和OsAgpSla上游启动子控制的GUS基因主要在胚乳中高水平表达,在叶片中有很低水平的表达;而OsAgpSlb转录本和OsAgpSlb上游启动子控制的GUS基因主要在叶片和胚乳发育早期低水平表达。这说明OsAgpSl基因产生的两个转录本是由不同的启动子控制转录的,OsAgpSla上游启动子可以作为胚乳表达用启动子。     

水稻病程相关基因OsPR1b启动子的表达特性研究

目的:分析水稻病程相关基因OsPR1b的表达特性,以进一步了解其表达和调控机制。方法:利用PCR技术从水稻日本晴基因组中扩增OsPR1b基因的启动子片段,命名为OsPR1bp,并构建相应的OsPR1bp::GUS融合表达载体,采用农杆菌介导的转基因技术获得转基因植株,进行GUS组织化学分析;利用Real-time PCR对OsPR1b基因在植物激素、非生物因子和水稻白叶枯菌(Xoo)毒性菌株P10(PXO124)处理下的表达水平进行分析。结果:GUS组织染色结果表明OsPR1b在水稻叶片中的表达量较高,而在茎、根、愈伤和花器中的表达量较低;植物激素水杨酸(SA)、茉莉酸甲酯(MeJA)、激动素(KT)、脱落酸(ABA)及NaCl、PEG均可不同程度地提高OsPR1b在叶片中的表达水平,Me-JA、KT和NaCl的处理能提高其在根部的表达水平,但这些激素在诱导OsPR1b在叶片和根部的表达程度上存在明显差异;单独接种Xoo毒性菌株P10 24 h对OsPR1b表达的影响不大,而MeJA与其共同处理后则可显著增强其在叶片中的表达。结论:作为一种防卫基因,OsPR1b在健康植株中的表达水平较低,容易受盐/干旱胁迫及Xoo病原菌的诱导,多种植物激素如JA、KT和ABA很可能作为信号分子参与激活和介导了这种系统性的反应。     

参与水稻光合作用的PsbR3基因启动子的功能分析

本实验旨在研究水稻光合作用蛋白中各基因的表达模式. 采用RT-PCR和定量real-time PCR数据分析水稻不同组织的mRNA表达水平.结果显示,PsaK和PsbR3基因仅在茎、叶等绿色组织表达,而胚、胚乳部分均不表达.通过其启动子克隆、植物表达载体构建,以及农杆菌介导转化后,GUS组织染化分析和GUS荧光定量分析表明,两启动子均为组织特异性优势表达,PsbR3启动报告酶GUS在叶片中的表达活性为Actin启动子的3.29倍,而PsaK启动报告酶GUS在叶片中的表达活性低于Actin启动子的.这些初步结果提示,PsbR3启动子决定水稻绿色组织茎叶的优势表达,PsbR3基因可能参与水稻光合作用.     

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.     

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.     

Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene.

We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene.     

Promoters of Rice Seed Storage Protein Genes Direct Endosperm-Specific Gene Expression in Transgenic Rice

The promoters of genes encoding rice seed storage proteins (glutelin,prolamin, globulin and albumin) were analyzed for their abilityto direct rß-gIucuronidase (GUS) gene expression intransgenic rice plants. All promoters tested could direct endosperm-specificexpression of the GUS reporter gene irrespective of variableactivities and patterns in the endosperm. (Received February 27, 1998; Accepted May 16, 1998)