Plant defense gene promoter enhances the reliability of<Emphasis Type="Italic"> shiva-1</Emphasis> gene-induced resistance to soft rot disease in potato

PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.     

Expression of Bacterial Hemoglobin in the Yeast, <Emphasis Type="Italic">Pichia pastoris,</Emphasis> with a Low O<Subscript>2</Subscript>-Induced Promoter

The PsADH2-promoter of Pichia stipitis alcohol dehydrogenase II (ADH II) gene was employed to control the expression of Vitreoscilla hemoglobin (VHb) gene in Pichia pastoris. As in P. stipitis, the promoter was also induced microaerobically in P. pastoris. The expression level of VHb in P. pastoris at low O₂ tension (<5% air saturation) was 16 nmol/g dry cell wt, i.e. about 24-fold higher than that at 60% air saturation. The expressed VHb enhanced growth of P. pastoris under microaerobic conditions. The application of O₂-regulated promoter in P. pastoris revealed that induction of high-level expression of heterologous protein is feasible without addition of supplementary compounds.     

Electron microscopic studies onNosema mesnili Paillot (Microsporidia: Nosematidae) infecting the Malpighian tubules ofPieris canidia larva

Summary An entire coding region of theCDC24/CLS4 gene and its truncated derivatives were overexpressed in yeast cells under the control of theGAL1 promoter. Western blotting analysis of the yeast cell lysates showed that the CDC24/CLS4 protein (Cdc24p) was induced to reach its maximum level after 9 h incubation of the cells in galactose medium. Overexpression of Cdc24p within the cells caused the morphological change, accumulating large spherical unbudded cells which exhibited actin cytoskeleton disturbed, chitin delocalized on the cell surface, and cell viability decreased. Multiple nuclei were observed in these cells, indicating that only budding cycle but not nuclear division cycle is blocked by the overproduction of Cdc24p. In order to identify the region of Cdc24p responsible for the growth inhibition, several truncatedCDC24 genes were expressed. Surprisingly, overexpression of fragments either containing the C-terminal 76 amino acid residues or deleting the same region inhibited cellular growth. This suggests that Cdc24p contains multiple functional domains for its tasks, likely cooperating signals of bud positioning and bud timing.     

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

Summary A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2–3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.     

Role of SulP, a nuclear-encoded chloroplast sulfate permease, in sulfate transport and H2evolution in Chlamydomonas reinhardtii

Antisense technology was applied to the green alga Chlamydomonas reinhardtiito probe the function of a novel nuclear gene encoding a chloroplast-envelope localized sulfate permease (SulP; GenBank Accession Numbers AF467891 and AF481828). Analysis showed that antiSulP transformants are impaired in sulfate uptake, a consequence of repression in the SulP gene expression. Antisense antiSulP transformants exhibited a sulfur-deprivation phenotype, strong induction of arylsulfatase activity, and global induction of sulfate assimilation gene expression. In sealed cultures, opposite to the wild-type control, antiSulP strains photo-evolved H₂, underlining the notion of sulfate uptake limitation by the chloroplast, a slow-down in the rate of oxygen evolution, establishment of anaerobiosis due to internal respiration and spontaneous expression of the [Fe]-hydrogenase in these strains. It is concluded that antiSulP strains are promising as tools to limit the supply of sulfates to the chloroplast, leading to a down-regulation of H₂O-oxidation and O₂-evolution activity, to the constitutive expression of the [Fe]-hydrogenase and continuous H₂-photoproduction in Chlamydomonas reinhardtii.Thus, antisulPstrains might permit a study of the biochemistry of H₂ metabolism in this green alga under constitutive anaerobic oxygenic photosynthesis conditions.     

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.     

Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

Summary We have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. Unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. Furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the absence of virus infection and viral gene expression. In this study, we have expressed a novel, selectable marker gene, puromycin acetyltransferase, under the control of the Drosophila melanogaster hsp70 promoter or under the control of the AcMNPV ie-1 promoter which is active in Spodoptera frugiperda cells in the absence of virus infection. In addition, we have constructed expression vectors which coexpress two genes from separate promoters, the pac gene which confers resistance to puromycin and a baculovirus gene which inhibits apoptosis, derived from Orygia pseudotsugata nuclear polyhedrosis virus. Both genes were expressed in stable populations of S. frugiperda cells in the absence of continuous drug selection.     

Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii

The efficient expression of exogenous gene in mitochondria of photosynthetic organism has been an insurmountable problem. In this study, the pBsLPNCG was constructed by inserting the egfp gene into a site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA of Chlamydomonas reinhardtii CC-124 and introduced into the mitochondria of respiratory deficient dum-1 mutation of C. reinhardtii CC-2654. Sequencing and DNA Southern analyses revealed that egfp gene had been integrated into the mitochondrial genome of transgenic algae as expected and no other copy of egfp existed in their nucleic genome. Both the fluorescence detection and Western blot analysis confirmed the presence of eGFP protein in the transgenic algae; it indicated that the egfp gene was successfully expressed in the mitochondria of C. reinhardtii.     

Expression of lexA targeted ribozyme in Escherichia coli BL-21 (DE3) cells

Coding sequences for a hammerhead ribozyme designed to cleave lexA mRNA in a targeted manner was cloned under phage T7 promoter and expressed in E. coli strain BL-21 (DE3) expressing T7 RNA polymerase under the control of IPTG-inducible lac UV-5 promoter. Ribozyme expression in vivo was demonstrated by RNase protection assay. Also, total RNA extracted from these transformed cells following induction by IPTG, displays site-specific cleavage of labeled lexA RNA in an In vitro reaction. The result demonstrates the active ribozyme in extracts of cell transformed with a recombinant cassette and goes beyond the earlier demonstration of the stability of In vitro synthesized ribozyme in cell extracts. The observed rise in lexA mRNA rules out any role for protease activity or resulting fragments of lexA protein in de-repression of RNA. (Mol Cell Biochem 271: 197–203, 2005)     

Characterization of the <Emphasis Type="Italic">AXDH</Emphasis> gene and the encoded xylitol dehydrogenase from the dimorphic yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD⁺ or NADH as coenzymes.     

Chloroplast sulfate transport in green algae – genes,proteins and effects

This review summarizes evidence at the molecular genetic, protein and regulatory levels concerning the existence and function of a putative ABC-type chloroplast envelope-localized sulfate transporter in the model unicellular green alga Chlamydomonas reinhardtii. From the four nuclear genes encoding this sulfate permease holocomplex, two are coding for chloroplast envelope-targeted transmembrane proteins (SulP and SulP2), a chloroplast stroma-targeted ATP-binding protein (Sabc) and a substrate (sulfate)-binding protein (Sbp) that is localized on the cytosolic side of the chloroplast envelope. The sulfate permease holocomplex is postulated to consist of a SulP–SulP2 chloroplast envelope transmembrane heterodimer, flanked by the Sabc and the Sbp proteins on the stroma side and the cytosolic side of the inner envelope, respectively. The mature SulP and SulP2 proteins contain seven transmembrane domains and one or two large hydrophilic loops, which are oriented toward the cytosol. The corresponding prokaryotic-origin genes (SulP and SulP2) probably migrated from the chloroplast to the nuclear genome during the evolution of Chlamydomonas reinhardtii. These genes, or any of its homologues, have not been retained in vascular plants, e.g. Arabidopsis thaliana, although they are encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the Photosystem II D1 reaction center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in Chlamydomonas reinhardtii is discussed along with its impact on the repair of Photosystem II from a frequently occurring photo-oxidative damage and H₂-evolution related metabolism in this green alga.     

The Escherichia coli dam gene is expressed as a distal gene of a new operon

Summary DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650–2100 bp and the other beyon 2400 bp upstream of the dam gene. No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression. The nucleotide sequence upstream of dam has been determined. An open reading frame (ORF) is present between the nearest promoter region and the dam gene. Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa. A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis. No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases. This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E. coli chromosome. Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene. This activity is relatively weak (about 15% of that of the E. coli gal operon promoter). The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene. Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene. Expression through the site adjacent to the dam gene is enhanced 2-to 4-fold in dnaA mutants at 38°C. Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E. coli ponA (mrcA) gene resides about 6 kb upstream of aroB.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation

The cell wall–lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the Tn₅ transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10⁶ mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10³ Hyg^R transformants per 10⁶ recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation ofCh. reinhardtii with heterologous genes are discussed.     

莱茵衣藻E2泛素结合酶CrUBC23调控油脂积累

【目的】为研究莱茵衣藻(Chlamydomonas reinhardtii)泛素结合酶(ubiquitin-conjugating enzymes,E2)CrUBC23在莱茵衣藻油脂代谢中的作用,为高产油微藻基因工程改良和揭示藻类油脂合成及代谢调控机理奠定基础。【方法】qRT-PCR分析莱茵衣藻在低氮、低磷胁迫下泛素结合酶CrUBC23表达情况;克隆CrUBC23同源基因干涉片段和全长基因,构建RNAi干涉载体和过量表达载体,转化莱茵衣藻并检测生物量和油脂含量;构建CrUBC23-GFP融合表达载体,用农杆菌浸染洋葱表皮细胞进行亚细胞定位。【结果】莱茵衣藻在低氮、低磷胁迫下CrUBC23基因表达量显著增加,增加幅度分别为正常培养的4.98–5.80倍和1.85–5.20倍。RNAi干扰结果显示,转基因藻细胞中性脂含量降低5.5%,总脂含量降低3.16%–17.6%。过量表达结果显示,转基因藻细胞中性脂含量增加8.8%,总脂含量增加4.51%–14.03%。【结论】CrUBC23正向调控莱茵衣藻油脂代谢,该基因定位于细胞核。