稻曲病菌厚垣孢子不同破壁方法的比较

为了探究稻曲病菌[Ustiloginoidea virens(Cooke)Takahashi]厚垣孢子的最佳破壁方法,研究采用4种破壁法对该病菌黄色和黑色厚垣孢子进行破壁,血球计数板计算破壁效果,并用考马斯亮蓝法测定不同破壁方法中厚垣孢子壁内可溶性蛋白含量。结果表明,在普通光学显微镜下观察,破壁后厚垣孢子多数为碎片,少数为孢壁内空圆球。4种破壁方法中液氮研磨-超声破碎法破壁效果最好,黄色和黑色厚垣孢子的破壁率均可达98%以上,用该法破壁测得的黄色和黑色厚垣孢子壁内可溶性蛋白质含量也最高。由此可见,液氮研磨-超声波破碎法是一种稻曲病菌厚垣孢子破壁的有效、简便、适宜在实验室应用的方法。     

稻曲病菌厚垣孢子壁多糖的提取方法优选

探究稻曲病菌Ustiloginoidea virens (Cooke) Takahashi厚垣孢子壁多糖的最佳提取方法,为孢壁多糖含量和组成的研究提供基础.采用5种方法提取该病菌黑色厚垣孢子壁多糖,用苯酚-硫酸法测定多糖含量.经研究比较,最佳提取方法为复合酶-热水浸提-sevag法,最佳提取条件是复合酶量4％,pH 4,浸提温度70℃,浸提时间120 min,物料比1:75(V/V);在优选的方法和条件下,测定稻曲病菌黑色厚垣孢子壁粗多糖相对得率21.2％,多糖含量72 3％;黄色厚垣孢子壁粗多糖相对得率17.5％,多糖含量66.7％,前者明显高于后者.研究表明复合酶-热水浸提-sevag法的工艺简单、可行,适宜稻曲病菌厚垣孢子壁多糖的测定.     

稻曲病菌厚垣孢子壁黑色素提取方法研究

探究稻曲病菌（Ustiloginoidea virens（Cooke） Takahashi）厚垣孢子壁黑色素的最佳提取方法,采用以HCl为提取剂的酸提法和以NaOH为提取剂的碱提法,对该病菌黑色和黄色2种厚垣孢子壁的黑色素进行提取,用3因素3水平进行正交设计试验,结果表明以NaOH作提取剂为佳,其提取黑色素效果最佳的组合条件为3 mol/L NaOH、2 mol/L HC l、水浴温度80℃、水浴时间120 min。     

稻曲病菌厚垣孢子中cAMP提取条件优选

探究稻曲病菌(Ustiloginoidea virens(Cooke.) Takahashi)黑色(休眠)与黄色(非休眠)厚垣孢子中的环磷酸腺苷(cAMP)最佳提取条件,为进一步的研究cAMP功能奠定基础.采用超声-水浴法对cAMP进行浸提,按3因素3水平正交设计,用高效液相色谱法检测cAMP含量;在设定V(甲醇)∶V(0.05 mol/L KH2PO4)=20∶80、流速为0.8 mL/min、检测波长为254 nm、进样量为20 μL的条件下,以黄绿色厚垣孢子为提取样品,其提取cAMP效果最佳组合条件:超声破碎时间10 min(功率400 W、间歇时间2 s),水浴温度80℃,物料比为1∶100,提取的cAMP为6.827 6 μg/mL.在此最佳条件下,测定出黄色厚垣孢子的cAMP为12.805 0±0.533 2μg/mL,黑色厚垣孢子的cAMP为4.171 7±0.097 1μg/mL.此结果表明,由黄色转换为黑色,其厚垣孢子的cAMP含量显著降低.     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

稻绿核菌(稻曲病菌)分离方法的比较研究

作者对不同情况下稻曲病菌的分离方法进行了比较研究。结果表明成熟早期稻曲球上的绝大多数新鲜的厚垣孢子具有萌发能力,及时进行分离培养是病原菌成功分离的关键。随保存时间的延长,厚垣孢子萌发能力急剧下降;消毒处理可杀死大部分的厚垣孢子。菌核可长期保存并保持极高的萌发生长能力,是稻曲病菌分离最为理想的材料。稻曲球中部的致密菌丝组织分离难度较大,只能作为稻曲病菌分离的一种补救方法。     

稻曲病菌AFLP反应体系的建立及其初步应用

本研究通过一系列梯度试验建立了最佳的稻曲病菌AFLP(Amplifiedfragmentlengthpolymor-phism)反应体系,并从256对EcoRⅠ和MseⅠ引物组合中选择30对条带清晰、多态性好的引物组合分析了2003年北京昌平基地的40个稻曲病菌株的遗传多样性。     

稻曲病菌PMK1类同源基因克隆及在稻瘟病菌遗传互补中的功能验证

[目的]克隆稻曲病菌PMK1类MAPK(Mitogen-activated protein kinase)同源基因.[方法]根据丝状真菌MAPK蛋白保守性设计简并引物扩增稻曲病菌MAPK基因部分片段,进而利用TAIL-PCR进行染色体步移和RT-PCR获得UVMK1基因全长和cDNA全长.构建互补载体,交叉互补稻瘟病菌APMK1突变体菌株nn78进行功能验证,包括附着胞分化和致病性测定.[结果]UVMK1基因全长1435 bp,包含3个内含子,编码355氨基酸的蛋白.UVMK1推导蛋白与丝状真菌Magnaporthe grisea PMK1,Fusarium oxysporum FMK1,Fusarium solani FSMAPK,Colletotrichumlagenarium CMK1,Botrytis cinerea BMK1,Claviceps purpurea CMPK1等编码蛋白高度同源.转化稻瘟病菌菌株nn78,获得5个转化子.其中选取的转化子恢复了稻瘟病菌正常的附着胞分化和对大麦叶片的致病能力.[结论]本研究成功分离了首个稻曲病菌MAPK基因,而且UVMK1基因是稻瘟病菌PMK1的同源基因.     

稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。     

稻曲病两个白化菌株的分离与生物学特性

为了筛选带有自然标记的稻曲病菌菌株,2010年从浙江省象山县和陕西省勉县采集和分离到2个稻曲病白化菌株,ZJa0201和SXa0101。它们在PSA培养基上的生长速度约为其他稻曲病菌株的3倍,未见产生厚垣孢子;在PS培养基上只能产生少量分生孢子。rDNA-ITS和rDNA-IGS序列分析表明,两个白化菌株也与稻曲病菌已知所有菌株的ITS序列同源性高于99.6%;rDNA-IGS序列也属于最为常见的类型,含有2个77bp的重复单元序列。由此推断,这两个白化菌株属于稻曲病菌产孢退化的突变体。白化菌株在PSA上     

PCR-based Specific Detection of Ustilaginoidea virens and Ephelis japonica

A PCR‐based technique for detection of clavicipitaceous pathogens in rice and related grasses was developed. The target pathogens were Ustilaginoidea virens, which causes rice false smut, and Ephelis japonica, which causes rice udbatta disease and black choke in grasses. To design specific primers, a comparison was made on genetic diversity on the rDNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene of U. virens, Ephelis japonica, as well as some other clavicipitaceous fungi. Each fungus was successfully detected by using a specific primer set with high sensitivity. Species‐specific primers designed here were capable of detecting these pathogens in plant tissues. The PCR detection was consistent with conventional histological observation. This nested PCR assay was sensitive and reliable for the detection of U. virens and E. japonica, and thus can be a used to study disease cycles and early prediction of false smut and udbatta‐disease incidence in fields.     

Postnatal changes in fatty acids composition of brown adipose tissue

It has been demonstrated that thermogenic activity of brown adipose tissue (BAT) is higher during the early postnatal period, decreasing towards a low adult level. The present study examined postnatal changes in the lipid composition of BAT. BAT from pre-weaning rats at 4 and 14 days old showed the following differences in lipid composition compared to that from adults of 12 weeks old. (i) Relative weight of interscapular BAT to body weight was markedly greater. (ii) BAT-triglyceride (TG) level was lower, while BAT-phospholipid (PL)level was higher. (iii) In TG fatty acids (FA) polyunsaturated fatty acids (PU; mol %), arachidonate index (AI), unsaturation index (UI) and PU/saturated FA (SA) were higher; rare FA such as eicosadienoate, bishomo--linolenic acid and lignoceric acid in mol % were also higher. (iv) In PL-FA monounsaturated FA (MU) in mol % was lower; PU mol %, AI and UI were higher. These features in BAT of pre-weaning rats resembled those in the cold-acclimated adults, suggesting a close relationship of the PL-FA profile to high activity of BAT.     

稻绿核菌无性孢子形成过程及厚垣孢子萌发率测定

对马铃薯蔗糖人工培养基(PSA)上绿核菌(稻曲病菌)不同培养时期的产孢情况进行了系统的扫描电镜观察。研究结果表明,在培养的前期(前20d),菌落表面往往形成集结状菌丝结构,其上开始产生大量分生孢子;一些分散的菌丝上也可产生少量的分生孢子。而在培养的后期,菌落表面往往形成黄色子实体,内部集生大量厚垣孢子。说明绿核菌在人工培养基上前期以形成分生孢子为主,后期则以厚垣孢子为主,且厚垣孢子的量远远大于分生孢子。萌发试验表明,成熟的厚垣孢子会随着保存时间的延长萌发率急剧下降。因此,新鲜的成熟厚垣孢子是最为理想的接种体。     

Application of high-performance liquid chromatography of plasma fatty acids as their phenacyl esters to evaluate splanchnic and renal fatty acid balance in vivo

Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0–100 nmol per injection of nine fatty acids was achieved within 60 min using [²H₃₁]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0±0.3 to 1.0±0.1 μmol/kg min (p<0.01), whereas net renal balance remained neutral (−0.04±0.04 vs. −0.06±0.03 μmol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012±0.005 (myristic acid) to a high of 0.95±0.08 (oleic acid) μmol/kg min across the splanchnic tissues and from 0.005±0.002 (stearic acid) to 0.21±0.1 (oleic acid) μmol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.     

The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine-rich protein SCRE1, which is an essential virulence effector in U. virens. When SCRE1 was heterologously expressed in Magnaporthe oryzae, the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity-associated hypersensitive response in the nonhost plant Nicotiana benthamiana. Induced expression of SCRE1 in rice also inhibits pattern-triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important ‘cysteine-proline-alanine-arginine-serine’ motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full-length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens.     

Pros and cons of fatty acids in bone biology

Despite the growing interest in deciphering the causes and consequences of obesity-related disorders, the mechanisms linking fat intake to bone behaviour remain unclear. Since bone fractures are widely associated with increased morbidity and mortality, most notably in elderly and obese people, bone health has become a major social and economic issue. Consistently, public health system guidelines have encouraged low-fat diets in order to reduce associated complications. However, from a bone point of view, mechanisms linking fat intake to bone alteration remain quite controversial. Thus, after more than a decade of dedicated studies, this timely review offers a comprehensive overview of the relationships between bone and fatty acids. Using clinical evidences as a starting-point to more complex molecular elucidation, this work highlights the complexity of the system and reveals that bone alteration that cannot be solved simply by taking ω-3 pills. Fatty acid effects on bone metabolism can be both direct and indirect and require integrated investigations. Furthermore, even at the level of a single cell, one fatty acid is able to trigger several different independent pathways (receptors, metabolites…) which may all have a say in the final cellular metabolic response.     

Detection of microbial-derived fatty acids in carious dentin by gas chromatography and gas chromatography-mass spectrometry

The aim of the present study was to analyze the fatty acid content of carious and sound human dentin. Gas chromatography and gas chromatography-mass spectrometry revealed the presence of fatty acids of C₁₀-C₁₈ size in the carious dentin, whereas fatty acids of C₁₆ size were present in minute amounts in three samples of the corresponding sound dentine controls. No fatty acids were detected in the other sound dentin control samples. The source of fatty acids was considered to be microorganisms invading the dentin during the progression of the caries lesion. The presence of bacterial fatty acids in carious dentin may serve as a marker for the pathological process and thus contribute to the understanding of the mechanisms involved.