Hongyu Pan

Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from Thermococcus kodakarensis KOD1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of d-glyceraldehyde 3-phosphate (d-G3P) to 1,3-diphosphoglycerate using NAD+ as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145xa0kDa; the subunit molecular mass was 37xa0kDa. GAPDH-tk is a thermostable protein with a half-life of 5xa0h at 80–90°C. The apparent Km values for NAD+ and d-G3P were 77.8xa0±xa07.5xa0μM and 49.3xa0±xa03.0xa0μM, respectively, with Vmax values of 45.1xa0±xa00.8xa0U/mg and 59.6xa0±xa01.3xa0U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232xa0kDa and ~669xa0kDa) in response to oxidative stress.

High level expression and characterization of a thermostable lysophospholipase from Thermococcus kodakarensis KOD1

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783xa0bp encodes a 260-amino acid protein with a molecular mass of 29xa0kDa. LysoPL-tk has a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence. LysoPL-tk was expressed in Escherichia coli and purified to homogeneity. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent Km value for p-nitrophenyl butyrate was 607.1xa0μM with Vmax values of 95.5xa0U/mg. The enzyme was active at a broad range of pH (5–8) and temperatures (70–95xa0°C) with the optimum pH and temperature being 8.0 and 85xa0°C, respectively. The high yield, broad substrate range along with its thermo-stability indicates that LysoPL-tk is a potential enzyme in industrial application.

An apoplastic chitinase CpCHT1 isolated from the corolla of wintersweet exhibits both antifreeze and antifungal activities

The shrub Chimonanthus praecox L. (wintersweet) which is native to Chinese montane forests produces its flowers in the midst of winter. This indicates that the floral organs of this species are adapted to growth and development under freezing temperatures. Here, we report the isolation and preliminary characterisation of a 33 kDa apoplastic antifreeze chitinase (CpCHT1) from the petals and its corresponding cDNA. The chitinase activity of CpCHT1 was confirmed by activity staining. Antifreeze activity was validated in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. CpCHT1 was also found to affect the germination of fungal spores of four major plant pathogens. In addition, the gene and protein are expressed constitutively not only in flowers, but also in leaves, bark and root tissues. From these data we hypothesize that this protein is multifunctional and may protect wintersweet from freezing injury and provide nonspecific disease resistance.

An Acyl–acyl Carrier Protein Thioesterase Gene Isolated from Wintersweet ( Chimonanthus praecox ), CpFATB , Enhances Drought Tolerance in Transgenic Tobacco ( Nicotiana tobaccum )

In this study, a full-length complementary deoxyribunucleic acid (cDNA) of a fatty acyl–acyl carrier protein thioesterase (CpFATB) was isolated from a Chimonanthus praecox (wintersweet) cDNA library. Wintersweet is a hardy shrub native to Chinese montane regions and is known to be tolerant to many biotic and abiotic stresses including cold, drought, and a variety of plant pathogens. The cDNA is 1,110 nucleotides in length, of which 1,107xa0bp form a single open-reading frame, predicting a 369 amino acid polypeptide. The deduced amino acid sequence of CpFATB displayed high homology to choroplastic palmitoyl–acyl carrier protein thioesterases from other plants. This gene was subsequently overexpressed in Nicotiana tobaccum using Agrobacterium tumefaciens-mediated transformation. Transcripts of CpFATB were shown to be abundant in leaves of the transformants, but were less highly expressed in roots and least highly expressed in stem tissues. Analysis of the fatty acid composition of transformant leaf tissue showed that the levels of myristic acid (C14:0) in leaf material of transgenic plants was increased by 46% compared with levels observed in wild-type plants. In addition, the level of oleate (C18:1) was reduced by 35%. Of most interest, was the observation that the transformants displayed a far higher degree of drought tolerance than wild type. These data suggest that the CpFATB product may be of utility in the production of transgenic, drought-tolerant crop species.

Characterization and Biocontrol Ability of Fusion Chitinase in Escherichia coli Carrying Chitinase cDNA from Trichothecium roseum

The antifungal mechanism of mycoparasitic fungi involves fungal cell wall degrading enzymes such as chitinases. Trichothecium roseum is an important mycoparasitic fungus with significant antifungal ability, but studies on chitinases of T. roseum were poor. Here, we report a novel chitinase cDNA isolated from T. roseum by PCR amplification based on conserved chitinase sequences. Southern blot analysis suggested that a single copy of the gene exists in the genome of T. roseum. The deduced open reading frame of 1,143 nucleotides encodes a protein of 380 amino acids with a calculated molecular weight of 41.6 kDa. The fusion chitinase expressed in Escherichia coli has been purified by single-step chromatography. It has a pI of pH 5.4 and expresses a thermal stability, but is insensitive to pH in a broad pH range. According to expectation, E. coli efficiently yielded a high amount of active chitinase. Remarkably, the fusion chitinase offered high antifungal activity.

Transgenic Rice Plants Expressing a Novel Antifreeze Glycopeptide Possess Resistance to Cold and Disease

Freezing injury and disease are both restrictive factors in crop production. In order to improve the tolerance ability to these stresses, a better way is to carry out genetic engineering by transferring dualfunctional genes. A predicted rice antifreeze glycopeptide gene was purposefully selected from rice blast-induced cDNA library. Northern blot demonstrated that the gene is expressed not only in blast-infected rice leaves, but also in low temperature-treated rice. In addition, the expressed protein in Escherichia coli exhibits strong antifreeze activities. The gene was overexpressed in rice plants transformed via Agrobacterium tumefacient EHA105. Overall 112 T0 transformants were obtained in this research. Cold tolerance and disease resistance of T1 transformants were, respectively, investigated. The results showed that plants containing overexpressed transgene can withstand D1 ∞C for 24 h without severe chilling injury after thawed, and that disease symptoms of the parallel transformants are highly reduced in response to blast infection, when compared with controls. The relationship of the gene and several pathogenesis-related protein genes to be chosen was analyzed and discussed. All these results confirmed the dual role of the cloned gene, and implied that genetic engineering using this kind of gene is a promising method to reduce biotic and abiotic stresses

Wintersweet accumulates apoplastic chitinase with a dual role in petals

Most monocotyledons like cereals accumulate antifreeze proteins in the apoplast during cold acclimation, but it is still uncertain whether dicotyledons do. Here we report the isolation and characterisation of a 33-kD apoplastic chitinase extracted from the corolla of wintersweet (Chinmonanthus praecox communis L.), which was purified using successive column chromatography on Phenyl-Sepharose, DEAE-Sepharose, and CM-Sepharose. Antifreezing activity of chitinase was confirmed in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. Interestingly, chitinase was also found to affect germination of fungal spores of four major plant pathogens. From these data, we hypothesize that, under natural conditions, wintersweet as one of the overwintering dicotyledons also accumulates apoplastic antifreeze proteins like monocotyledons. To our knowledge, this is the first report on the isolation of dicotyledon apoplastic chitinase with high-level antifreeze and antifungal activities.

Selective methylation of one of the two promoters residing in T-DNA inserted in a retroelement of rice

The upstream sequence of pinb previously isolated from rice and confirmed to be a wound-inducible promoter by detecting GUS in T0 transgenic rice transformed via Agrobacterium tumefaciens-mediated procedures. In a transgenic line (pinb-16), the selectable marker hptII driven by CaMV35S promoter was completely silenced in T2 sublines; but the uidA gene driven by pinb promoter was expressed without being affected, though it, together with hptII, exists in the same T-DNA insertion. Analyses of methylation patterns using bisulphite-sequencing in the homozygous T1 and T2 sublines showed that cytosines in CaMV35S were gradually methylated in T1 plants and almost completely methylated in T2 plants. Interestingly, the process of methylation was accompanied by the occurrence of lesion mimic phenotype in rice leaves. The activity of hygromycin-resistance could be reestablished by treatment with 5-azacytidine. Genomic Southern and isolation of the T-DNA flanking sequences indicated that T-DNA was inserted in a retroelement of rice. These results revealed that methylation shows preference for the heterogeneity promoter fragment in the transgenic rice line and may be induced by the retroelement.