Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.     

Establishment of a hyper-protein production system in submerged Aspergillus oryzae culture under tyrosinase-encoding gene (melO) promoter control

UV-mediated mutagenesis generated a high glucoamylase-producing mutant of Aspergillus oryzae exhibiting strong melanization in solid-state culture. Expression of the glucoamylase-encoding gene (glaB), which is specifically expressed in solid-state culture, and the tyrosinase-encoding gene (melO), was analyzed using an E. coli beta-glucuronidase (GUS) reporter assay to investigate this phenomenon. Although no common regulation was found for melO and glaB expression, the former was greatly enhanced in submerged culture. Interestingly, the melO promoter was about four times stronger for GUS production than the powerful promoters amyB, glaA, and modified agdA, previously isolated for industrial heterologous gene expression in A. oryzae. These findings indicated that the melO promoter would be suitable for hyper-production of heterologous protein in Aspergillus. The glaB-type glucoamylase selected as the target protein was produced in a submerged culture of A. oryzae under the control of the melO promoter. The maximum yield was 0.8 g/l broth, and the total extracellular protein purity was 99%. Repeated batch culture, to improve productivity, gave a maximum yield of 3.3 g/l broth. The importance of this work is in the establishment of a both high-level and high-purity protein overproduction system in A. oryzae by use of the melO promoter.     

Deletional analysis of functional regions of complementary sense promoter from cotton leaf curl virus

Complementary sense promoter from cotton leaf curl virus (CLCuV) is a novel plant promoter for genetic engineering that could drive high-level foreign gene expression in plant. To determine the optimal promoter sequence for gene expression, CLCuV promoter was deleted from its 5' end to form promoter fragments with five different lengths, and chimeric gus genes were constructed using the promoterdeletion. These vectors were delivered into Agrobacterium and tobacco (Nicotiana tabacum L cv. Xanthi) plants which were transformed by leaf discs method. GUS activity of transgenic plants was measured. The results showed that GUS activities with the promoter deleted to -287 and -271 from the translation initiation site were respectively about five and three times that of full-length promoter. There exists a c/s-element which is important for the expressing activity in phloem from -271 to -176. Deletion from -176 to -141 resulted in a 20-30-fold reduction in GUS activity in leaves with weak activity in leaves and     

Cloning and identification of the promoter of the tobacco Sar8.2b gene,a gene involved in systemic acquired resistance

Expression of the Sar8.2 gene family is induced by salicylic acid (SA) in tobacco during induction of systemic acquired resistance. Expression of Sar8.2b, one member of this 12-member family, was detected as early as 12 h after treatment with SA and was maximal 36 h after SA treatment. In NahG transgenic tobacco plants, benzothiadiazole and dichloroisonicotinic acid induced expression of Sar8.2b but SA did not, suggesting that expression of the Sar8.2b gene is SA-dependent. Several putative cis-acting elements were found in the Sar8.2b gene promoter region, including an as-1 element and GT-1 and Dof binding sequences. We constructed a series of progressive deletion mutations in the Sar8.2b promoter region linked to the β-glucuronidase (GUS) coding region and analyzed GUS activities by stable expression in transformants of Arabidopsis thaliana. Deletions between −728 and −927 bp or between −351 and −197 bp of the promoter region resulted in a significant reduction in GUS activity induced by SA treatment as shown in stable transformants of A. thaliana. The −197 bp fragment of the promoter region was found to confer a relatively low level of GUS activity induced by SA treatment in stable expression of transformants in A. thaliana. The results suggest that 927 bp of the Sar8.2b gene promoter confers full promoter activity and that cis-acting elements required for high-level SA-inducible expression of the Sar8.2b gene may exist within the regions −728 to −927 bp and −197 to −351 bp.     

Efficient heterologous expression in Aspergillus oryzae of a unique dye-decolorizing peroxidase, DyP, of Geotrichum candidum Dec 1

Efficient expression of the dye-decolorizing peroxidase, DyP, from Geotrichum candidum Dec 1 in Aspergillus oryzae M-2-3 was achieved by fusing mature cDNA encoding dyp with the A. oryzae alpha-amylase promoter (amyB). The activity yield of the purified recombinant DyP (rDyP) was 42-fold compared with that of the purified native DyP from Dec 1. No exogenous heme was necessary for the expression of rDyP in A. oryzae. From the N-terminal amino acid sequence analyses of native DyP and rDyP, the absence of a histidine residue in both DyPs, which was considered to be important for heme binding of DyP, was confirmed. These results suggest that rDyP without a typical heme-binding region produced by A. oryzae exhibits a function similar to that of native DyP.     

Cloning of the promoter region of plasma membrane aquaporin BnPIP1 from Brassica napus and its functional analysis

Aquaporins make water-selective channels in plants, facilitating the permeation of water through membranes and adjusting water fast transport during seed germination, cell elongation, stoma movement, fertilization and responses to environmental stresses. They belong to the MIP (major intrinsic protein) family with molecular weight of 2629 kD and are characterized by six membrane-spanning a-helixes connected by five loops and short N-terminal and C-terminal domains in the cytoplasm[13]. The p…     

Deletion analysis of the superoxide dismutase (sodM) promoter from Aspergillus oryzae

The manganese superoxide dismutase gene (sodM) is very highly expressed in Aspergillus oryzae. To elucidate the basis for this high-level expression, deletion analysis of the promoter was undertaken using β-glucuronidase (GUS) as a reporter. Deletion of a 63-bp sequence from −200 to −138 in the 1,038-bp sodM promoter caused a drastic decrease in GUS activity. In addition, an electrophoretic gel mobility shift assay (EMSA) implicated a 30-bp element from −209 to −178 containing cis-element(s) in the high-level expression. The results of fine structure deletion analysis of this region were consistent with the EMSA results. To confirm these findings, we constructed enhanced sodM promoters by incorporating tandem repeats of this region, which resulted in an approximate twofold increase in expression relative to the native sodM promoter.     

Developmental and Hormonal Regulation of Rice [alpha]-Amylase(RAmy1A)-gusA Fusion Genes in Transgenic Rice Seeds

Transgenic seeds of rice (Oryza sativa L.) were used to investigate temporal, spatial, and hormonal regulation of a rice [alpha]-amylase gene, RAmy1A. Two overlapping segments of the RAmy1A promoter were fused to the coding region of the bacterial reporter gene, gusA. The resulting promoter-gusA fusions, pE4/GUS (-232 to +31) and pH4/GUS (-748 to +31), were used separately to transform rice protoplasts. [beta]-Glucuronidase (GUS) activity was detected in germinated transgenic seeds, although the two constructs showed no significant difference in timing or location of GUS expression. Both constructs first expressed GUS in the scutellar epithelium and then in the aleurone layer. Aleurone expression of GUS activity was strongly induced when embryoless half-seeds were treated with gibberellic acid. GUS expression in the aleurone layer was also suppressed by abscisic acid. These results indicate that the 5[prime] regulatory region from -232 to +31 is sufficient for temporal, spatial, and hormonal regulation of RAmy1A gene expression.     

Analysis of regulatory elements of the promoter and the 3′ untranslated region of the maize<Emphasis Type="Italic"> Hrgp</Emphasis> gene coding for a cell wall protein

Hydroxyproline-rich glycoproteins (HRGP) are structural components of the plant cell wall. Hrgp genes from maize and related species have a conserved 500 bp sequence in the 5'-flanking region, and all Hrgp genes from monocots have an intron located in the 3' untranslated region. To study the role of these conserved regions, several deletions of the Hrgp gene were fused to the beta-glucuronidase ( GUS) gene and used to transform maize tissues by particle bombardment. The overall pattern of GUS activity directed by sequential deletions of the Hrgp promoter was different in embryos and young shoots. In embryos, the activity of the full-length Hrgp promoter was in the same range as that of the p35SI promoter construct, based on the strong 35S promoter, whereas in the fast-growing young shoots it was 20 times higher. A putative silencer element specific for young shoots was found in the -1,076/-700 promoter region. Other major cis elements for Hrgp expression are probably located in the regions spanning -699/-510 and -297/-160. Sequences close to the initial ATG and mRNA leader were also important since deletion of the region -52/+16 caused a 75% reduction in promoter activity. The presence of the Hrgp intron in the 3' untranslated region changed the levels of GUS activity directed by the Hrgp and the 35S promoters. This pattern of activity was complex, and was dependent on the promoter and cell type analysed.     

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。     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.