Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus.

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-beta-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

Isolation and functional characterization of a novel stress inducible promoter from pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L)

The present study deals with isolation and characterization of a novel hybrid-proline-rich protein gene (CcHyPRP) promoter from pigeonpea. Real time PCR analysis revealed that CcHyPRP expression was strongly induced by dehydration, salt, Abscisic acid (ABA) and Salicylic acid (SA) treatments. The CcHyPRP promoter, isolated by genome-walking method, contained 1112 bp and showed the presence of various cis -regulatory elements necessary for tissue specific expression and stress responsiveness. Different 5′ deletions of the promoter were generated and were used to drive the expression of β-glucuronidase reporter gene (gusA) in Arabidopsis thaliana. Histochemical and fluorometric assays confirmed that GUS expression driven by the full-length fragment (1112 bp) was higher when compared to different deletion fragments. Under normal conditions, GUS expression was predominantly detected in the roots and hypocotyls of transformants, while under mannitol, NaCl, ABA and SA treatment conditions higher GUS expression levels were observed in the roots and leaves. However, the GUS expression was mostly confined to the roots of transformants carrying 477 and 300 bp promoter regions. The results amply indicate that CcHyPRP promoter is regulated by different stress factors, and as such the promoter can be deployed in genetic engineering of crop plants for enhanced abiotic stress tolerance.     

Regulation of expression of the cucumber isocitrate lyase gene in cotyledons upon seed germination and by sucrose

A 6.5 kb cucumber genomic DNA fragment containing the icl gene was introduced into Nicotiana plumbaginifolia and shown to direct isocitrate lyase (ICL) mRNA synthesis in transgenic seedlings upon germination, in a temporally regulated manner. Two putative icl promoter fragments, of 2900 and 572 bp, were subsequently linked to the GUS reporter gene and introduced into N. plumbaginifolia. Both constructs directed GUS expression after transgenic seed germination, and although the 572 bp fragment gave only 1% of the activity of the 2900 bp fragment, it directed expression in the same cotyledon-specific and temporally regulated pattern. Seedlings were transferred to darkness after 18 days growth in the light, to induce a starvation response. The 2900 bp construct was activated by starvation and repressed by exogenous sucrose, whereas the 572 bp construct was not starvation-responsive. To localize the region of the 2900 bp promoter fragment which is responsible for regulation by sucrose, further deletions were make, linked to GUS, and assayed in a cucumber protoplast transient assay system. Constructs with promoters of 2900, 2142 and 1663 bp were activated by starvation and repressed by sucrose, but promoters of 1142 and 572 bp showed no such response. We conclude that the icl gene promoter contains at least two distinct cis-acting elements, one required for the response to sucrose and the other which participates in expression upon seed germination.     

Analysis of transcriptional activity of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> ornithine-delta-aminotransferase gene promoter

Cloning of the Arabidopsis thaliana genomic DNA fragment presumably corresponding to the promoter region of the ornithine-delta-aminotransferase (OAT) gene is reported. The reporter-gene construct, containing the Escherichia coli beta-glucouronidase gene under control of the OAT gene promoter was generated. The Nicotian tabacum SR1 transformants carrying this construct were obtained. It was demonstrated that under normal conditions, expression of the reporter gene was associated with the meristems and the zones of intensive shoot growth. Possible role of the OAT gene in nitrogen metabolism and shoot development is discussed.     

Analysis of transcriptional activity of the ornithine-delta-aminotransferase gene promoter in Arabidopsis thaliana

Cloning of the Arabidopsis thaliana genomic DNA fragment presumably corresponding to the promoter region of the ornithine-delta-aminotransferase (OAT) gene is reported. The reporter-gene construct, containing the Escherichia coli beta-glucouronidase gene under control of the OAT gene promoter was generated. The Nicotian tabacum SR1 transformants carrying this construct were obtained. It was demonstrated that in normal conditions, expression of the reporter gene was associated with the meristems and the zones of intensive shoot growth. Possible role of the OAT gene in nitrogen metabolism and shoot development is discussed.     

Highly efficient Agrobacterium-mediated transformation of Volvariella volvacea

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to the edible straw mushroom, Volvariella volvacea. Mycelium pellets were transformed to cold stress resistance using the afp gene as both a selective marker and a reporter gene, under the control of a heterologous Lentinula edodes gpd promoter. The efficiency of transformation is over 100 times higher than that previously reported in V. volvacea. Stable integration of the afp gene with 1-4 copy numbers was confirmed in all 10 randomly selected transgenic events by Southern blot analysis. The mitotic stability of the transformants was demonstrated after five successive transfers on PDA medium without selection pressure and the PCR analysis of basidiospores harvested from transformants.     

磷和葡萄糖及细胞分裂素对拟南芥SEN1基因表达的影响

拟南芥SEN1基因受衰老诱导.将该基因启动子融合报告基因萄聚糖酶(glucuronidase,GUS)基因转入拟南芥,通过染色并测定GUS活性发现,缺氮、缺磷、缺钾诱导叶中SEN1表达,而只有缺磷能导根中SEN1表达.缺磷对根叶中SEN1的诱导被3%葡萄糖和细胞分裂素抑制.3%葡萄糖胺在根和叶中均诱导SEN1表达,外源细胞分裂素不能抑制这种效应.结果表明:SEN1基因可受缺磷信号特异调控,并受糖信号和细胞分裂素负调控;葡萄糖胺能大大促进根和叶中SEN1表达,且不受细胞分裂素的负调控.     

Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-β-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

Optimization of biolistic bombardment parameters for <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17 calluses using GFP and GUS as the reporter system

Genetic transformation system of Dendrobium Sonia 17 was optimized using green fluorescent protein (GFP) and -glucuronidase (GUS) gene as the reporter systems. The 35S-sgfp-TYG-nos (p35S) and pSMDFR, carrying sgfp and gusA gene, respectively, were co-bombarded into the calluses. Parameters optimized were acceleration pressure, target distance, gold particle size, pre-bombardment cultured time, plasmid DNA precipitation, total plasmid DNA and the ratio of the plasmids co-bombarded. Both reporter systems responded similarly to the bombardment parameters investigated. Based on the GUS/GFP spot counts, the GFP expression rate was higher than that for GUS under the control of the same promoter, CaMV 35S. GFP could be used as the reporter system for the co-bombardment as it was rapid and non-destructive system to monitor the transformed tissues. A combination of GFP and antibiotic resistance gene was used to select stable putative transformants.     

Introns control expression of sucrose transporter LeSUT1 in trichomes,companion cells and in guard cells

In solanaceous plants such as tomato and tobacco, the sucrose transporter SUT1 is crucial for phloem loading. Using GUS as a reporter, the promoter and other regulatory cis elements required for the tomato LeSUT1 expression were analyzed by heterologous expression of translational chimeric constructs in tobacco. Although LeSUT1 is highly expressed at the RNA level, GUS expression under the control of a 1.8 kb LeSUT1 promoter resulted in few plants expressing GUS. In GUS-positive transformants, expression levels were low and limited to leaf phloem. Increasing or decreasing the length of LeSUT1 promoter did not lead to a significant increase in positive transformants or higher expression levels. Translational fusion of GUS to the LeSUT1 C-terminus in a construct containing all exons and introns and the 3'-UTR led to a higher number of positive transformants and many plants with high GUS activity. LeSUT1 expression was detected in ab- and adaxial phloem companion cells, trichomes and guard cells. The role of individual introns in LeSUT1 expression was further analyzed by placing each LeSUT1 intron into the 5'-UTR within the 2.3 kb LeSUT1 promoter construct. Results showed remarkable functions for the three introns for SUT1 expression in trichomes, guard cells and phloem cells. Intron 3 is responsible for expression in trichomes, whereas intron 2 is necessary for expression in companion cells and guard cells. The combination of all introns is required for the full expression pattern in phloem, guard cells and trichomes.     

The Aspergillus fumigatus cellobiohydrolase B (cbhB) promoter is tightly regulated and can be exploited for controlled protein expression and RNAi

The utility of the Aspergillus fumigatus cellobiohydrolase cbhB promoter for controlled gene expression has been investigated. cbhB message was present at high levels in the presence of carboxymethylcellulose and undetected in the presence of glucose. A reporter construct using the cbhB promoter showed similar behaviour and gave lower message levels than the Aspergillus nidulans alcA promoter under repressing conditions. An RNAi construct driven by the cbhB promoter was used to down-regulate the alb1 gene; transformants showed low alb1 message levels and a loss-of-function phenotype with carboxymethylcellulose, while both wild-type message levels and phenotype were seen with glucose. The cbhB promoter is therefore tightly controlled and can be exploited for the study of A. fumigatus.