嗜热链球菌CRISPR-Cas系统的检测

【目的】CRISPR-Cas系统为嗜热链球菌抵抗噬菌体等外源基因元件提供获得性免疫,分析NCBI中已公开发表全基因组序列的9株嗜热链球菌所含CRISPR-Cas系统的数目和类型,对实验室相应菌株的CRISPR-Cas系统进行检测。【方法】利用生物信息学方法对NCBI中9株已测序嗜热链球菌所含CRISPR-Cas系统进行分析,根据其Cas基因序列设计引物,对实验室嗜热链球菌菌株的Cas基因进行扩增、测序,分析实验室6株嗜热链球菌的CRISPR-Cas系统情况。【结果】9株标准菌株均含不同数目的CRISPR-Cas系统,其类型主要为Ⅱ-A型、Ⅲ-A型和Ⅰ-E型,各类型的标志Cas基因高度保守。6株供试菌中,S4仅含Cas9基因,其它5株均含有Cas9基因、Cas10基因和Cas9*基因,79和KLDS3.0207还含有Cas3基因。【结论】可根据标准菌株高度保守的Cas基因设计引物,预测未知嗜热链球菌所含CRISPRCas系统的数目和类型。S4仅含1个Ⅱ-A型CRISPR-Cas系统,其它5株均含有2个Ⅱ-A型CRISPR-Cas系统和1个Ⅲ-A型CRISPR-Cas系统,此外,79和KLDS3.0207均含有1个Ⅰ-E型CRISPR-Cas系统。     

安徽定远盐矿可培养嗜盐微生物多样性

【背景】嗜盐微生物多生活于高盐环境,具有独特的生理代谢特征,是一类重要的极端环境微生物资源。【目的】为更好地认识我国陆相盐矿的嗜盐微生物多样性组成,更好地开发利用嗜盐微生物资源积累丰富的微生物菌种。【方法】对安徽定远盐矿盐芯样品进行嗜盐微生物的纯培养分离,并对所分离菌株进行基于16SrRNA基因的测序和序列相似性分析,并对所分离菌株进行物种多样性分析。在此基础上,对代表菌株进行菌落形态和耐盐度及酶活测定。【结果】通过纯培养共分离获得了嗜盐微生物264株,其中嗜盐古菌150株,占56.8%;嗜盐细菌114株,占43.2%。嗜盐古菌物种分别来自于Halorubrum、 Halopenitus、 Haloterrigena、 Natrinema、 Natronoarchaeum和Natronomonas等6个属;嗜盐细菌物种分别来自于Pseudomonas、Aliifodinibius、Halobacillus、Halomonas和Halospina等5个属。通过代表菌株的酶活平板检测,发现产胞外蛋白酶菌株1株,酯酶1株,淀粉酶2株;能液化明胶菌株2株。在物种多样性组成方面,发现嗜盐古菌的物种多样性指数高于嗜盐细菌。【结论】本研究对我国安徽定远陆相盐矿的可培养嗜盐微生物多样性进行探究,积累了丰富的嗜盐微生物菌株资源。     

基于比较基因组学分析嗜热链球菌的遗传多样性和防御系统

【目的】嗜热链球菌(Streptococcusthermophilus)是发酵乳制品的基础发酵菌种之一,全基因组水平解析嗜热链球菌的遗传多样性和工业发酵特性对于优良发酵菌株的筛选意义重大。【方法】本研究通过比较基因组学方法对27株嗜热链球菌的遗传多样性和防御系统进行分析。【结果】全基因组分析结果显示嗜热链球菌群体内具有较高的遗传多样性;基于核心基因集构建的系统发育树划分为2个分支,其中分支2菌株缺乏完整的组氨酸合成途径,经验证,分支2菌株在缺乏组氨酸的培养基中不能正常生长。通过对嗜热链球菌不同菌株的防御系统进行分析发现,同类型的CRISPR基因座和限制修饰系统在基因组中出现的位置相对固定。CRISPR-Cas系统(P0.05,r=0.43)和限制修饰系统(P0.01,r=–0.59)的数量与编码转座酶基因的数量均显著相关,表明嗜热链球菌为了阻止外源DNA入侵会进化出多种防御系统来保护自身遗传完整性。此外,分支1菌株的CRISPR-Cas系统数量极显著(P0.001)多于分支2,而限制修饰系统无显著差异,表明分支1菌株在噬菌体抗性方面可能更具优势。【结论】本研究基于核心基因构建的系统发育分析将27株嗜热链球菌分为2个分支,不同分支菌株在组氨酸代谢能力和防御系统方面有一定差异。该研究结果为今后快速筛选优良嗜热链球菌发酵剂提供了新思路。     

23株不同地理来源嗜酸硫杆菌的系统发育及其遗传差异

【目的】研究不同地理来源嗜酸硫杆菌的系统发育及其遗传差异,以及基因指纹图谱技术聚类与嗜酸硫杆菌地理来源的相关性。【方法】采用16S-23S r RNA间隔区(ITS)序列建立系统发育树,并结合ERIC和BOXAIR两种引物进行rep-PCR,以及rus基因扩增,对不同地理来源嗜酸硫杆菌进行分析。【结果】分离自不同样点的23株嗜酸硫杆菌遗传差异显著,依据ITS序列系统发育树被划分为5个大类群,与rep-PCR指纹图谱的分类结果较为接近,其中Acidithiobacillus ferrooxidans在ITS系统发育和BOXAIR-PCR指纹聚类分析中被划分为2个类群,但在ERIC-PCR中归为1个类群,rus基因分组中,在系统发育和聚类分析中处于同一类群的菌株拥有不同类型的rus基因,说明嗜酸硫杆菌的亚铁氧化途径与系统发育类群无明显相关性;ITS基因拥有区分近缘种或亚种的能力,且BOXAIR-PCR的分辨能力较强,非常适于嗜酸硫杆菌的遗传差异分析。     

新疆罗布泊地区可培养嗜盐古菌多样性及其功能酶筛选

【目的】探索新疆罗布泊地区高盐环境可培养嗜盐古菌的多样性及其功能酶应用潜力。【方法】采集罗布泊地区13份土样,用纯培养并结合基于16S rRNA基因系统发育分析的方法来研究样品中嗜盐古菌的多样性。按系统进化树的聚类关系,挑选出一些菌株进行盐度耐受及淀粉酶、蛋白酶、酯酶的酶活检测。【结果】从13份土样中共分离到56株嗜盐古菌,经16S rRNA基因克隆测序,通过MEGA 4.0构建N-J树分析,56株菌分布于嗜盐古菌的10个生效发表属和5个潜在新属。运用Shannon-Wiener方法计算其多样性指数为1.820。挑选17株嗜盐古菌所测试盐浓度实验结果表明这一批嗜盐古菌的大部分生长范围在10%-35%之间,最适盐浓度在20%-25%之间。不同酶活检测结果为:淀粉酶酶活率为70.6%,蛋白酶酶活率为35.3%,酯酶酶活率为82.4%。【结论】新疆罗布泊周边地区由于气候及地理位置的独特性,蕴藏丰富的嗜盐古菌资源。本实验所设计的分离方法对嗜盐古菌的分离是极其有效的,为进一步研究新疆罗布泊及周边地区嗜盐古菌资源提供了技术基础。盐度耐受实验结果验证在低盐环境中分离嗜盐古菌新物种的可行性。同时,嗜盐古菌的酶活比率较高且活性较强为进一步开发利用嗜盐古菌资源提供了理论依据。     

极端酸性矿山环境微生物基于CRISPR系统的适应性免疫机制

【目的】通过对酸性矿山环境中嗜酸硫杆菌属(Acidithiobacillus)、脱硫弧菌属(Desulfovibrio)、钩端螺旋菌属(Leptospirillum)、硫化杆菌属(Sulfobacillus)、酸原体属(Acidiplasma)和铁质菌属(Ferroplasma)的100株冶金微生物基因组中CRISPR-Cas系统的结构特征和同源关系进行生物信息学分析,在基因组水平上解析冶金微生物基于CRISPR系统对极端环境的适应性免疫机制。【方法】从NCBI网站下载基因组序列,采用CRISPR Finder定位基因组中潜在的CRISPR簇。分析CRISPR系统的组成结构与功能:利用Clustal Omega对重复序列(repeat)分类;将间隔序列(spacer)分别与nr数据库、质粒数据库和病毒数据库比对,获得注释信息;根据Cas蛋白的种类和同源性对酸性矿山环境微生物的CRISPR-Cas系统分型。【结果】在100株冶金微生物基因组中共鉴定出415个CRISPR簇,在176个c CRISPR簇中共有80种不同的重复序列和4147条间隔序列。对重复序列分类,发现12类重复序列均能形成典型的RNA二级结构,Cluster10中的重复序列在冶金微生物中最具有代表性。间隔序列注释结果表明,这些微生物曾遭受来自细菌质粒与病毒的攻击,并通过不同的防御机制抵抗外源核酸序列的入侵。冶金微生物细菌的大部分CRISPR-Cas系统属于I-C和I-E亚类型,而古菌的CRISPR-Cas系统多为I-D亚类型,两者基于CRISPR-Cas系统的进化过程中存在显著差异。【结论】酸性矿山环境微生物的CRISPR结构可能采用不同免疫机制介导外源核酸序列与Cas蛋白的相互作用,为进一步揭示极端环境微生物的适应性进化机理奠定了基础。     

新疆两盐湖可培养极端嗜盐菌组成及功能多样性研究

【目的】通过分析不同成盐类型盐湖中的极端嗜盐菌群落组成差异,探究可培养极端嗜盐菌的功能特性。【方法】采集新疆硫酸盐型盐湖七角井和碳酸盐型盐湖南湖的土壤样品,通过平板稀释涂布法分离极端嗜盐菌,经过形态学观察、特征分析获取代表菌株,通过耐盐性测定和16S rRNA基因序列测序等对代表菌株进行鉴定,并对极端嗜盐菌的蛋白酶、淀粉酶、纤维素酶和酯酶活性进行筛选,同时检测苯酚降解能力。【结果】本研究共获得1 679株极端嗜盐菌,代表菌株45株,隶属于5门14个属,古菌数量(70.58%)明显多于细菌,最优盐浓度生长范围为18.4%–20.0%。在属水平上,盐湖中优势类群为古菌的Haloterrigena属(32.94%)和Natrialba属(26.03%),以及细菌的Aquisalimonas属(9.85%)和Aliifodinibius属(8.10%)。两盐湖中,盐度较低的南湖物种丰富度高于七角井盐湖,古菌物种组成相似,均以Haloterrigena属为主;细菌群落组成有差异,南湖以Aquisalimonas属为主,而七角井以Aliifodinibius属为主。功能筛选表明,盐湖中80%的嗜盐...     

喜温嗜酸硫杆菌SM-1在不同温度下基因组的可塑性

【目的】驯化得到喜温嗜酸硫杆菌(Acidithiobacillus caldus)SM-1在低于最适生长温度下具有较高生长活力的突变菌株,并认知喜温嗜酸硫杆菌在不同温度下的基因组可塑性。【方法】利用实验室长期进化实验对菌株进行3个温度的驯化:37、40、45°C。运用454测序技术对驯化获得的菌株进行基因组重测序,通过比较基因组分析驯化株基因组单核苷酸位点变化(SNPs),对包含位点变化的基因从功能上进行分类,在此基础上,分析可能与温度适应性相关的基因。【结果】通过不同温度下的长期驯化,得到了在低于最适生长温度下具有较高活力的菌株;重测序结果发现,SM-1基因组具有较好的可塑性,不同温度(37、40、45°C)生长的菌株中,基因组中分别有418、384和347个核苷酸位点发生累计变化,其中3个温度下有20个相同的非同义突变位点,分别分布于编码重金属和毒性抗性系统、DNA甲基化和蛋白乙酰化酶、核酸代谢相关酶类等相关基因上;相比而言,在低于最适生长温度(37、40°C)下生长菌株特有的位点变化涉及能量代谢、信号转导以及DNA/RNA稳定性相关基因;其中,2个低温菌株共同发生位点变化的基因有3个,其中两个编码转座相关的蛋白Atc_1031与Atc_1623,另一个编码假想蛋白Atc_1130,该蛋白分别与外膜蛋白组装因子B和二硫键形成蛋白具有23%和35%的相似性。另外,不同生长温度下相关蛋白中氨基酸的组成也发生变化。【结论】喜温嗜酸硫杆菌SM-1基因组具有较好的可塑性,对于其基因组变化的研究结果为认识微生物温度适应性提供了组学数据。本研究揭示喜温嗜酸硫杆菌(At.caldus)SM-1可能通过多种途径适应向低温过渡生长,既包括微生物通用的环境适应机制,也存在菌株特有的温度适应途径。     

嗜酸乳杆菌NCFM对Caco-2细胞基因表达谱的影响

摘要：【目的】嗜酸乳杆菌NCFM作为一株具有良好益生功能的模式菌株,采用基因芯片技术对其作用后的宿主细胞基因的变化情况进行分析,在生物、食品等领域具有较大研究价值。【方法】将嗜酸乳杆菌NCFM和Caco-2细胞共同培养2h,提取Caco-2细胞的总RNA,并将RNA反转录成cDNA,与Human Genome U133 Plus 2.0 Array基因表达谱芯片杂交,杂交后进行图像扫描和数据分析,并采用Real-time RT PCR方法对差异表达的基因进行验证。【结果】采用基因芯片方法检测了Caco-2细胞经嗜酸乳杆菌NCFM作用2h后的基因表达变化,发现差异表达基因为508个,其中有473个基因上调,35个基因下调,初步推测Caco-2细胞能诱导多个基因的表达,以发挥嗜酸乳杆菌NCFM的益生功能。并且经Real-time RT PCR验证,表达差异显著的3个免疫调节相关基因CCL2、PTX3和TNFRSF9的确在嗜酸乳杆菌NCFM作用期间高表达。【结论】以上这些结果促进了对嗜酸乳杆菌NCFM益生功能的认识,也为揭示该乳酸菌的作用机理提供了理论基础。     

嗜盐古菌中Ⅰ-B亚型CRISPR-Cas系统的分子机制和应用进展

嗜盐古菌是一类应用前景广阔的微生物资源,但由于缺乏高效灵活的遗传操作工具,限制了这类生物在科学研究和工业生产方面的应用。CRISPR-Cas系统存在于绝大多数古菌中,应用其内源性CRISPR-Cas系统进行遗传操作是一种兼具可行性和简便性的策略。近年来,人们对Ⅰ-B亚型CRISPR-Cas系统的研究已从模式古菌沃氏富盐菌(Haloferax volcanii)拓展到其他嗜盐古菌中。本文总结了Ⅰ-B亚型CRISPR-Cas系统的分子机制,并介绍了利用这种内源性CRISPR-Cas系统在嗜盐古菌中进行遗传操作的研究进展。     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

New species and new combinations in Peruvian Orchidaceae

seventeen new species and combinations are proposed in the generaChondrorhyncha, Cischweinfia, Cochlioda, Eloyella, Encyclia, Kefersteinia, Koellensteinia, Macroclinium, Rodriguezia, Solenidiopsis, andStenia. All new species are illustrated. A key is provided for 2-flowered species ofMacroclinium, PeruvianSigmatostalix, and PeruvianStenia. Solenidium (Solenidiopsis) peruvianum Schltr. is lectotypified.     

安徽牯牛降的丝孢菌Ⅰ

本文报导作者采自安徽枯牛降自然保护区的18种丝孢菌,分属于5个属,其中有3个新种:牛皮冻生尾孢(Cercospora paederiicola),山鸡椒假尾饱(Pseudocercospora litseae-cubebae),鸡血藤生假尾孢(P. millettiicola)和2个中国新纪录。文中对新种进行了描述及绘图,新记录种作了简要说明。研究的标本保存在中国科学院微生物研究所真菌标本室(HMAS)。     

Changes of dung beetle communities from rainforests towards agroforestry systems and annual cultures in Sulawesi (Indonesia)

Little is known about how tropical land-use systems contribute to the conservation of functionally important insect groups, including dung beetles. In a study at the margin of Lore Lindu National Park (a biodiversity hotspot in Central Sulawesi, Indonesia) dung-beetle communities were sampled in natural forest, young secondary forest, agroforestry systems (cacao plantations with shade trees) and annual cultures (maize fields), each with four replicates (n = 16 sites). At each site we used 10 pitfall traps, baited with cattle dung, along a 100 m transect for six 3-day periods. The number of trapped specimens and species richness at the natural forest sites was higher than in all land-use systems, which did not significantly differ. Each land-use system contained, on average, 75% of the species richness of the natural forest, thereby indicating their importance for conservation. However, a two-dimensional scaling plot based on NESS indices (m = 6) indicated distinct dung beetle communities for both forest types, while agroforestry systems and annual cultures exhibited a pronounced overlap. Mean body size of dung beetles was not significantly influenced by land-use intensity. Five of the six most abundant dung beetle species were recorded in all habitats, whereas the abundance of five other species was significantly related to habitat type. Mean local abundance and number of occupied sites were closely correlated, further indicating little habitat specialisation. The low dung beetle diversity (total of 18 recorded species) may be due to the absence of larger mammals in Sulawesi during historical times, even though Sulawesi is the largest island of Wallacea. In conclusion, the dung beetle fauna of the lower montane forest zone in Central Sulawesi appears to be relatively robust to man-made habitat changes and the majority of species did not exhibit strong habitat preferences.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Die GattungKarschia — Bindeglied zwischen bitunicaten Ascomyceten und lecanoralen Flechtenpilzen?

The genusKarschia, in the earlier sense, including saprophytes and parasites on lichens, has been thought to be a non-lichenized parallel genus of the lichen genusBuellia. Modern workers included it on the one hand inBuellia, on the other hand combined it with bitunicate ascomycetes. It is now proved thatKarschia is heterogeneous and contains but superficially similar members both of the genusBuellia of theLecanorales and of typical or masked bitunicateAscomycetes. Therefore, it can not be regarded as a link betweenLecanorales andDothideales. The type species ofKarschia belongs to theDothideales.