湖羊瘤胃微生物GH9家族葡聚糖酶基因IDSGLUC9-25的表达与功能表征

【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30-50℃下活性较高,在pH 4.0-8.0范围内能够保持较高的稳定性,经pH 4.0-8.0缓冲液处理1 h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73) U/mg;利用薄层色谱法(thin layer chromatography, TLC)和高效液相色谱法(high performance liquid chromatography, HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25 (EC 3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。     

A NEW SPECIES OF GENUS HABROPHLEBIODES ULMER (EPHEMEROPTERA: LEPTOPHLEBIIDAE)

Abstract  The present paper deals with a new species Habrophlebiodes zijinensis sp. nov. collected in Nanjing, Jiangsu Povince, China.     

Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar

Summary We have investigated the fate of the mitochondrial genomes of cybrids derived from donor-recipient protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC₁ generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC₁ progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC₂ generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.     

不同血清型肉毒毒素受体结合域研究进展

肉毒毒素(botulinum neurotoxin, BoNT)是人类已知毒性最强的蛋白质之一,可以引起肌肉松弛麻痹,严重时可导致死亡。肉毒毒素共分为7种血清型(BoNT/A-BoNT/G),根据氨基酸序列差异可进一步分为40多种亚型。肉毒毒素分子结构由3个基本结构域组成：重链羧基端细胞受体结合域、氨基端的易位域和轻链催化域。在运动神经元表面,受体结合域首先与聚唾液酸神经节苷脂结合,随后与突触囊泡蛋白2或突触囊泡结合蛋白结合形成双受体复合物。每种血清型的受体结合域都必须与其相应受体结合才能发挥作用。肉毒毒素的结构功能及其对宿主的作用一直都是研究热点。近年来,因受体结合域可以促进肉毒毒素与运动神经元膜特异性结合,而成为新的研究方向。本综述将概述不同血清型肉毒毒素与受体结合过程中受体结合域结构变化和结合位点差异。通过分析不同血清型及亚型的序列以及受体结合域结构特征,可以更好地了解细胞受体结合域的序列差异和功能,并为肉毒毒素的治疗策略提供新思路。     

南极生物粪对汞的生物放大作用

首次报道了南极地区金图企鹅、阿德利企鹅、黑背鸥和巨海燕 4种海鸟 ,以及象海豹、威德尔海豹、毛皮海狮等 3种海豹新鲜粪便中的全 Hg含量 ,并对比了该区风化土壤的 Hg含量 ,其中 Hg含量水平依次为 :土壤背景值 <黑背鸥粪 <金图企鹅粪 <阿德利企鹅粪 <威德尔海豹粪 <象海豹粪 <巨海燕粪 <毛皮海狮粪 ,可见 ,相对于背景土壤生物粪明显富集 Hg元素。此外 ,结合上述粪便样品的 δ15 N测定结果 ,讨论了南极生物粪对 Hg的生物放大作用。研究表明 ,随着 δ15 N升高 ,即生物营养级的升高 ,其粪便的全 Hg含量有逐步增高的趋势 ,这证实了生物粪和生物肌体一样存在对 Hg的生物放大作用。文中还对南极阿德雷岛一个经过企鹅粪污染的 Y2湖泥芯剖面上 15个样品的 2 6种元素进行了聚类分析 ,结果表明 ,沉积物中的汞和企鹅粪的标型元素具有同源性 ,即沉积物中的汞主要是由企鹅粪带入的 ,由于生物粪对汞的生物富集作用 ,生物粪的混入会造成湖泊沉积物中汞含量的增高     

长期不同养分投入对土壤养分和水稻生产持续性的影响

以中国科学院桃源农业生态试验站15a长期田间定位试验为研究对象,分析了不同养分投入对稻田土壤养分和水稻产量可持续性的影响.结果表明,化肥与系统内循环的有机物料循环的肥力效力和产量效应基本一致,有机物料循环更有利于土壤有机质和氮素的积累;在不同养分投入下,土壤耕层有机质和全氮均呈上升趋势,年均增长率分别为1.5%～5.8%和2.5%～9.4%;与试验前相比,不同养分投入耕层磷素变动幅度在-18.3%到30%之间,钾素养分有所亏缺,下降幅度在8.1%～22.6%之间;通过可持续性指数的分析得出,土壤N素养分的可持续性对化肥的依赖性较大,而P、K养分的可持续性则对有机肥的依赖性更高.稻田生态系统具有良好的自维持能力,系统内有机物循环有利于提高稻谷产量的稳定性和可持续性.     

Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term “stutters.” During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.     

Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis

Small RNAs of 21-25 nucleotides (nt), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), act as guide RNAs to silence target-gene expression in a sequence-specific manner. In addition to a Dicer homolog, DCL1, the biogenesis of miRNAs in Arabidopsis requires another protein, HEN1. miRNAs are reduced in abundance and increased in size in hen1 mutants. We found that HEN1 is a miRNA methyltransferase that adds a methyl group to the 3'-most nucleotide of miRNAs, but the role of miRNA methylation was unknown. Here, we show that siRNAs from sense transgenes, hairpin transgenes, and transposons or repeat sequences, as well as a new class of siRNAs known as trans-acting siRNAs, are also methylated in vivo by HEN1. In addition, we show that the size increase of small RNAs in the hen1-1 mutant is due to the addition of one to five U residues to the 3' ends of the small RNAs. Therefore, a novel uridylation activity targets the 3' ends of unmethylated miRNAs and siRNAs in hen1 mutants. We conclude that 3'-end methylation is a common step in miRNA and siRNA metabolism and likely protects the 3' ends of the small RNAs from the uridylation activity.     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.