Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G_M1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.     

Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue

We report the investigation of an endosperm-specific promoter from the rsus3 gene from rice (Oryza sativa). The promoter was characterized by deletion analysis and transient expression in guar (Cyamopsis tetragonoloba) seed-tissue. Transient expression was monitored by histochemical GUS assay, and quantitative dual reporter assays comprising firefly luciferase as a test reporter, and Renilla luciferase and GUS as reference reporters. These revealed high expression levels of the reporter genes directed by the rsus3 promoter in guar endosperm. Specificity for this tissue in seeds was apparent from a virtual absence of reporter activity in guar cotyledons. Removal of a putative intron region only slightly raised the expression level, whereas duplication of the minimal promoter region, in a tandem-repeat rsus3 promoter construct, retained endosperm specificity in guar, and displayed three times the reporter activity observed with the single copy construct.     

Intron-mediated enhancement of the banana bunchy top virus DNA-6 promoter in banana (Musa spp.) embryogenic cells and plants

 Intron-containing fragments derived from the 5′ untranslated regions of the maize ubi1, maize adh1, rice act1 and sugarcane rbcS genes were tested for their enhancing effects on the banana bunchy top virus DNA-6 promoter (BT6.1) in banana (Musa spp. cv. Bluggoe) embryogenic cells. The rice act1 and maize ubi1 introns provided the highest levels of intron-mediated enhancement of GUS expression, increasing native BT6.1 promoter activity by about 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about tenfold, whereas the adh1 intron had no significant effect. In regenerated transgenic banana plants, the ubi1 intron significantly enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35 S promoter and did not appear to affect the tissue specificity of the promoter. Received: 28 July 2000 / Revision received: 21 August 2000 / Accepted: 4 October 2000     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.     

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.     

A maize α-zein promoter drives an endosperm-specific expression of transgene in rice

An alpha-zein promoter isolated from maize containing P-box, E motif sequence TGTAAAGT, opaque-2 box and TATA box was studied for its tissue-specific expression in rice. A 1,098 bp promoter region of alpha-zein gene, fused to the upstream of gusA reporter gene was used for transforming rice immature embryos (ASD 16 or IR 64) via the particle bombardment-mediated method. PCR analysis of putative transformants demonstrated the presence of transgenes (the zein promoter, gusA and hpt). Nineteen out of 37 and two out of five events generated from ASD 16 and IR 64 were found to be GUS-positive. A histological staining analysis performed on sections of mature T₁ seeds revealed that the GUS expression was limited to the endosperm and not to the pericarp or the endothelial region. GUS expression was observed only in the following seed development stages : milky (14–15 DAF), soft dough (17–18 DAF), hard dough (20–23 DAF), and mature stages (28–30 DAF) of zein-gusA transformed (T₀) plants. On the contrary a constitutive expression of GUS was evident in CaMV35S-gusA plants. PCR and Southern blotting analyses on T₁ plants demonstrated a stable integration and inheritance of transgene in the subsequent T₁ generation. GUS assay on T₂ seeds revealed that the expression of gusA gene driven by alpha-zein promoter was stable and tissue-specific over two generations. Results suggest that this alpha-zein promoter could serve as an alternative promoter to drive endosperm-specific expression of transgenes in rice and other cereal transformation experiments.     

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。     

Analyzing the action of evolutionarily conserved modules on HMW-GS 1Ax1 promoter activity

Key message

The specific and high-level expression of 1Ax1 is determined by different promoter regions. HMW-GS synthesis occurs in aleurone layer cells. Heterologous proteins can be stored in protein bodies.

Abstract

High-molecular-weight glutenin subunit (HMW-GS) is highly expressed in the endosperm of wheat and relative species, where their expression level and allelic variation affect the bread-making quality and nutrient quality of flour. However, the mechanism regulating HMW-GS expression remains elusive. In this study, we analyzed the distribution of cis-acting elements in the 2659-bp promoter region of the HMW-GS gene 1Ax1, which can be divided into five element-enriched regions. Fragments derived from progressive 5′ deletions were used to drive GUS gene expression in transgenic wheat, which was confirmed in aleurone layer cells, inner starchy endosperm cells, starchy endosperm transfer cells, and aleurone transfer cells by histochemical staining. The promoter region ranging from ??297 to ??1 was responsible for tissue-specific expression, while fragments from ??1724 to ??618 and from ??618 to ??297 were responsible for high-level expression. Under the control of the 1Ax1 promoter, heterologous protein could be stored in the form of protein bodies in inner starchy endosperm cells, even without a special location signal. Our findings not only deepen our understanding of glutenin expression regulation, trafficking, and accumulation but also provide a strategy for the utilization of wheat endosperm as a bioreactor for the production of nutrients and metabolic products.

     

Characterization of the mannan synthase promoter from guar (Cyamopsis tetragonoloba)

Guar seed gum, consisting primarily of a high molecular weight galactomannan, is the most cost effective natural thickener, having broad applications in the food, cosmetics, paper, pharmaceutical and petroleum industries. The properties of the polymer can potentially be enhanced by genetic modification. Development of suitable endosperm-specific promoters for use in guar is desirable for metabolic engineering of the seed gum. A ~1.6 kb guar mannan synthase (MS) promoter region has been isolated. The MS promoter sequence was fused with the GUS reporter gene and overexpressed in the heterologous species alfalfa (Medicago sativa). The potential strength and specificity of the MS promoter was compared with those of the constitutive 35S promoter and the seed specific β-phaseolin promoter. Quantitative GUS assays revealed that the MS promoter directs GUS expression specifically in endosperm in transgenic alfalfa. Thus, the guar MS promoter could prove generally useful for directing endosperm-specific expression of transgenes in legume species.     

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.     

Exploring the potential of the bacterial carotene desaturase CrtI to increase the beta-carotene content in Golden Rice

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.