Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp¹¹² and Asp²⁹⁷ were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a K _m of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.     

哈茨木霉天冬氨酸蛋白酶基因SA76的3＇RACE扩增和序列分析

由EST获得全长cDNA对于结构基因组学和功能基因组学都是至关重要的,cDNA末端快速扩增技术RACE是该领域中的重要研究方法.利用BD SMART RACE技术扩增编码分泌天冬氨酸蛋白酶SA76基因的3＇末端,将其与哈茨木霉cDNA文库中的SA76基因的EST序列进行序列拼接,获得2019bp的全长cDNA序列,其开放读码框长1593bp,5＇非编码区266bp,3＇非编码区201bp,编码530个氨基酸,有信号肽.哈茨木霉天冬氨酸蛋白酶基因与玉蜀黍赤霉、粗糙脉孢菌、球毛壳菌天冬氨酸蛋白酶基因的同源性分别为53%, 37%, 36%.利用BD SMART RACE技术首次从哈茨木霉中克隆天冬氨酸蛋白酶基因,为验证SA76基因的功能奠定基础,为进一步研究蛋白酶的作用机制及生物防治功能提供依据.     

天冬氨酸对脂多糖刺激断奶仔猪生长性能、血细胞分类计数和血液生化指标的影响

本试验旨在研究天冬氨酸(ASP)对脂多糖(LPS)刺激断奶仔猪生长性能、血细胞分类计数和血液生化指标的影响。试验选用24头断奶仔猪,分为对照组、LPS组、LPS+0.5%ASP组(0.5%ASP)、LPS+1.0%ASP组(1.0%ASP)。结果表明:0.5%和1.0%ASP可缓解LPS刺激导致的日增重的降低(P<0.05);注射LPS 4 h后,0.5%或1.0%ASP可显著缓解LPS刺激导致的淋巴细胞比例、血小板数量的降低和嗜酸性粒细胞比例的升高,增加红细胞平均血红蛋白(HGB)浓度(P<0.05);注射LPS 24 h后,0.5%和1.0%ASP可显著缓解嗜中性粒细胞数量、嗜中性粒细胞比例的升高和淋巴细胞比例的降低(P<0.05);0.5%或1.0%ASP可显著缓解LPS刺激引起的谷丙转氨酶、谷草转氨酶、碱性磷酸酶、尿素氮、血糖的升高和谷丙转氨酶/谷草转氨酶的降低(P<0.05)。结果显示,ASP缓解了LPS刺激所导致的生长抑制和应激反应。     

Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.     

中国野生毛葡萄白粉菌诱导响应VqAP基因的克隆及表达分析

【目的】克隆中国野生毛葡萄"商-24"天冬氨酸蛋白酶(AP)基因的cDNA序列,明确其在葡萄抗病防御机制中的作用。【方法】在前期获得的中国野生毛葡萄株系"商-24"AP基因(VqAP)EST序列的基础上,采用RT-PCR克隆AP基因的cDNA序列,对其序列及编码产物进行生物信息学分析,并通过实时定量PCR和半定量PCR,分析VqAP基因在白粉菌诱导不同时间(0,6,12,24,48,72,96和120h),不同激素(100μmol/L水杨酸,50μmol/L乙烯和0.5g/L茉莉酸甲酯)刺激及不同组织(嫩叶、嫩茎、花、果皮、卷须)中的表达情况。【结果】序列分析表明,VqAP基因序列长度为1 377bp,具有开放阅读框,编码458个氨基酸残基,相对分子质量为50.48ku,等电点为8.73。保守结构域分析表明,VqAP编码产物含有2个保守的具有催化活性的天冬氨酸残基,位于Asp-Thr/Ser-Gly(DT/SG)结构域,该基因编码的氨基酸序列属于植物天冬氨酸蛋白酶A1家族,为非典型的天冬氨酸蛋白酶。荧光定量PCR结果表明,接种白粉菌后6h,VqAP基因表达量为0h的6倍多,之后迅速下调;不同激素处理后,该基因也均诱导表达,这可能是由于VqAP基因参与了植物早期的抗病反应,以及由不同激素诱导的发病相关基因的调节作用。半定量PCR结果表明,在葡萄嫩叶、嫩茎、花、果皮、卷须等不同组织中VqAP基因的表达量不同,这可能与它参与了植物不同组织中功能蛋白的合成与降解有关。【结论】VqAP的表达响应病原菌侵染并受生物胁迫相关激素的调控,可能在葡萄防御病原菌侵染的机制中发挥重要作用。     

Identification of potential molecular markers for flower sex determination in Vernicia fordii

Vernicia fordii is cultivated as an important woody oil tree, with a long history in China. It is a monoecious and diclinous species. Altering the sex ratio to increase the number of female flowers would lead to better yields. The mechanism of flower development is, however, not yet clear. In this study, the proteins that are differentially expressed between male and female flowers were isolated using a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). A proteomic analysis led to the identification of 14 proteins, of which two were expressed specifically in male flowers and 10 in female flowers. Among the 10 female-specific proteins, aspartic proteinase, ascorbate peroxidase, and cyclophilin were thought to be involved in stamen abortion and gynoecium formation during the development of female flowers. Further results suggested that these proteins might be the sex-related proteins that are the potential molecular markers for flower sex determination in V. fordii.     

Comparison of three markers for the determination of bacterial protein in terminal ileal digesta in the growing pig

The aim of the study was to compare three methods commonly used to determine the concentrations of bacterial protein in digesta collected from the terminal ileum of growing pigs that had been fed a casein‐based diet. The amounts of bacterial protein in terminal ileal digesta were determined using three different markers: 2.6‐diaminopimelic acid (DAPA) and the d ‐amino acids, d ‐aspartic acid (d ‐Asp) and d ‐glutamic acid (d ‐Glu). The effectiveness of each marker was compared against a control based on physical fractionation by centrifugation. The total bacterial protein concentrations derived from the markers d ‐Asp and d ‐Glu were significantly different (p = 0.05) to those calculated from DAPA and the control, but there was no difference between DAPA and the control. The percentage of bacterial nitrogen ranged from 40% to 52% dependent on the marker used. Bacterial protein expressed as a percentage of the total protein, ranged from 48% to 62%, a substantial proportion of which (12–28%) was derived from lysed bacterial cells. Statistical correlations between the estimation methods were low. Such poor correlation between the markers may be the result of random errors such as variance in the epimerization of the two d ‐amino acids during protein hydrolysis. DAPA was accepted as a reliable marker for determining microbial protein in ileal digesta.     

奶牛瘤胃微生物元基因组文库中二肽基肽酶Ⅳ的筛选与分析

【目的】研究奶牛瘤胃内参与蛋白质降解过程中二肽基肽酶Ⅳ（dipeptidyl peptidases Ⅳ,DPP-Ⅳ）的序列特征和酶学性质。【方法】从奶牛瘤胃微生物Fosmid文库中挑选17 664个克隆,利用dpp-Ⅳ简并引物筛选含dpp-Ⅳ的阳性克隆。提取阳性克隆的质粒,用限制性内切酶Hind Ⅲ进行酶切。PCR扩增含dpp-Ⅳ目的片段,并克隆测序。对阳性克隆的Fosmid末端进行测序。提取阳性克隆粗酶液,利用底物Gly-Pro-pNA检测肽酶活性。【结果】筛选后获得10个含有dpp-Ⅳ的阳性克隆（命名为DP1—DP10）。Fosmid末端序列经BLASTX比对后发现78%的序列可以与数据库的已知序列相匹配,但相似度变化较大（44%—94%）。DPP-Ⅳ序列经BioEdit比对后,发现均含有N端保守区域（DWVYEEE）和C端催化区域（GWSYGG）,经BLASTP比对发现阳性克隆的DPP-Ⅳ分别与Cyclobacterium marinum（43%）、Capnocytophaga sp.（63%）、Prevotella ruminicola 23（66%）和Solitalea canadensis（50%）的DPP-Ⅳ相似度高。酶活力检测发现DP7肽酶活力最高,为6.88 U•mg-1。【结论】从瘤胃微生物Fosmid文库中获得了10个含有dpp-Ⅳ的阳性克隆,它们具有不同的dpp-Ⅳ序列特征和肽酶活性。     

Recent advances in the development of innovative chemical methods for determining the nutritional value of fish meals and aquafeeds

Aspartic acid racemization and oxysterol content have been evaluated as indicators of fish meal and aquafeed nutritional value in a series of studies reviewed in the present paper. Kinetic studies and assessment of the d -aspartic acid content of commercial fish meals and fish feeds supported the use of the extent of racemization of this amino acid as a reliable indicator of the thermal history of fish meal. Preliminary results suggest that d -aspartic acid could be a useful indicator of the protein nutritional value for fish, expressed by nitrogen retention or in vivo digestibility. However, species-related differences seem to occur. Therefore, further studies are needed to evaluate the reliability of d -aspartic acid content as an indicator of protein nutritional value affected by processing conditions. With regard to lipid quality, two major oxysterols, namely 7β-hydroxycholesterol and 7-ketocholesterol, have been identified and quantified in commercial fish meals. The measured levels were very low compared with the values reported in the literature for fish products, probably because of antioxidant addition during fish meal processing. An effect of storage time on cholesterol oxidation was also demonstrated in a laboratory-scale experiment. Research needs for the future include a deeper understanding of the chemical reactions affecting the nutritional quality of aquafeeds, development of innovative and reliable chemical methods for raw material and feed quality assessment, and identification of critical control points in the manufacturing process to try to maintain the original nutritional value of raw materials.