为调查我国单星藻属(Coelastrella)物种多样性和分布,研究从我国各地区采集29株单星藻藻株,结合形态学及18S rDNA、ITS和tufA三个分子标记对该属进行了系统分类学研究,系统梳理中国记录的单星藻属植物共10种2变种,并新发现3个中国新记录种,分别为密纹单星藻Coelastrella multistriata、陆生单星藻Coelastrella terrestris和气生单星藻Coelastrella aeroterrestrica.上述物种的发现弥补了中国核心单星藻分支标本缺乏的问题,丰富了中国单星藻属的物种多样性,为该类群进一步的分类修订及资源开发奠定基础.

目的:比较评价靶基因16S rRNA、tufA、femA、rpoB和gap序列对葡萄球菌种水平的鉴定能力.方法:选择60株菌种鉴定明确的葡萄球菌(涵盖18个种),比较评价16S rRNA基因序列及VITEK 2全自动微生物生化鉴定系统对常见葡萄球菌鉴定的准确性;采用PCR方法特异扩增60株菌tufA、femA、rpoB和gap的基因序列,并对阳性扩增产物进行核酸测序,利用Mega 7.0和DNASP 5.0软件分析靶基因序列多样性,通过序列聚类关系分析,进一步评价不同靶基因序列对葡萄球菌种水平的分子鉴定能力.结果:16S rRNA基因序列比对不能有效区分山羊葡萄球菌和头状葡萄球菌,而VITEK方法则将巴氏葡萄球菌错误鉴定为沃氏葡萄球菌.序列多样性分析表明,尽管靶基因tufA、femA、rpoB和gap序列比16S rRNA基因序列多样性更强,但基于tufA、femA、rpoB和gap序列的聚类分析在种水平仍会错误鉴定部分葡萄球菌,如表皮葡萄球菌、头状葡萄球菌、腐生葡萄球菌和科氏葡萄球菌等.进一步评价16S rRNA基因序列分别与靶基因tufA、femA、rpoB和gap的串联序列对葡萄球菌种水平的鉴定能力,结果表明只有靶基因emA与16S rRNA基因的串联组合能够实现对18种葡萄球菌的准确鉴定,而其他靶基因与16S rRNA基因的串联组合对于葡萄球菌种水平的鉴定能力没有明显改善.结论:基于单个靶基因的序列分析会造成部分葡萄球菌种水平的错误鉴定,而基于16S rRNA与femA基因的串联序列分析则能够显著提高对葡萄球菌种水平鉴定的准确性.

本研究对海南沿岸采集到石莼(Ulva sp.)和浒苔(Enteromorpha sp.)的rbcL和tufA基因序列进行PCR扩增并测序,并从GenBank下载16种绿藻的rbcL基因序列和tufA基因序列,排序后的18种绿藻的rbcL基因长度为680 bp,变异位点416个,保守位点为264个.序列中C+G的含量为40.4％,说明浒苔属和石莼属的rbcL序列的(C+G)％值较低.而排序后的18种绿藻的tufA基因长度为582 bp,变异位点122个,保守位点为460个.序列中C+G的含量为33.1％,说明浒苔属和石莼属的tufA序列的(C+G)％值较低.利用NJ法以rbcL和tufA序列构建的系统进化树表明,浒苔属和石莼属的物种并未聚类成各自独立的进化枝,而是存在明显的属间交叉分布现象.

单星藻隶属于绿藻纲环藻目栅藻科,多为单细胞或聚合群体,属内已知种类多发现于气生、亚气生生境,在天然抗氧化剂和食品色素生产等领域具有广阔应用前景.但目前关于该类群的基础分类学研究相对薄弱,阻碍了这些优质藻种资源的进一步开发利用.本研究从重庆市及周口市采集到2株单星藻,经形态学及18S rDNA、ITS和tufA三个分子标记鉴定为单星藻属一新种,命名为重庆单星藻Coelastrella chongqingensis sp.nov..重庆单星藻显著的形态特征是细胞壁近似光滑或仅具细微皱纹,不具有典型单星藻类群纵向分布的肋纹,幼期细胞多呈椭球形或卵形,末端钝圆,细胞大小约(6—9)μm×(5—7)μm.成熟细胞多呈圆球形或椭球形,直径约8—14—(18)μm.系统发育分析结果显示重庆单星藻与另外2种细胞壁近似光滑的藻株液泡单星藻Coelastrella vacuolata和薄壁单星藻Coelastrella tenuitheca具有最近的亲缘关系,三者在基于tufA序列的系统发育分析中形成独立单系分支,代表单星藻属第三种形态型,即细胞壁近似光滑或仅具细微网纹,无单星藻属典型的纵向肋纹.新种的发现扩充了单星藻属第三种形态型的物种组成,为该属进一步的分类学修订打下基础.

Udoteaceae is a morphologically diverse family of the order Bryopsidales.Despite being very widespread geographically,this family is little known as compared with the closely related Halimedaceae or Caulerpaceae.Using the most extensive Udoteaceae collection to date and a multilocus genetic data set (tufA,rbcL,and 18S rDNA),we reassessed the species diversity of the family,as well as the phylogenetic relationships,the diagnostic morphoanatomical characters,and evolutionary history of its genera,toward a proposed taxonomic revision.Our approach included a combination of molecular and morphological criteria,including species delimitation methods,phylogenetic reconstruction,and mapping of trait evolution.We successfully delimited 62 species hypotheses,of which 29 were assigned (existing) species names and 13 represent putative new species.Our results also led us to revise the genera Udotea s.s.,Rhipidosiphon s.s.,and Chlorodesmis s.s.,to validate the genus Rhipidodesmis,and to propose three new genera:Glaukea gen.nov.,Ventalia gen.nov.,and Udoteopsis gen.nov.We also identified two large species complexes,which we refer to as the"Penicillus-Rhipidosiphon-Rhipocephalus-Udotea complex" and the "Poropsis-Penicillus-Rhipidodesmis complex".Using a time-calibrated phylogeny,we estimated the origin of the family Udoteaceae at Late Triassic (ca.216 Ma),whereas most of the genera originated during Paleogene.Our morphological inference results indicated that the thallus of the Udoteaceae ancestor was likely entirely corticated and calcified,composed of a creeping axis with a multisiphonous stipe and a pluristromatic flabellate frond.The frond shape,cortication,and calcification are still symplesiomorphies for most extant Udoteaceae genera and represent useful diagnostic characters.

目的 构建2型猪链球菌酪氨酸激酶Cps2C基因过表达株,为cps2C基因功能研究提供实验材料.方法 使用重叠延伸PCR技术将延伸因子TufA(又名EF-Tu,编码基因为SSU05_0530)的启动子(命名为P0530)与cps2C基因连接为P0530cps2C片段,将P0530cps2C酶切后连接至猪链球菌-大肠埃希菌穿梭质粒pSET2,构建过表达质粒pSET2∷P0530cps2C.将pSET2∷P0530cps2C电转化导入S.suis 2野生毒株05ZYH33感受态细胞,抗性及组合PCR筛选得到cps2C过表达株.qRT-PCR检测过表达株cps2C基因的转录水平.结果 重叠延伸PCR成功将P0530启动子片段(300 bp)和cps2C基因片段(696 bp)拼接为P0530cps2C(996 bp),片段及载体经BamH Ⅰ和EcoR Ⅰ酶切后连接,成功构建出过表达质粒pSET2∷P0530cps2C;将pSET2∷P0530cps2C电转化导入S.suis 2 05ZYH33感受态细胞,抗性筛选及组合PCR检测结果显示cps2C过表达株构建成功.提取野生株05ZYH33和cps2C过表达株总RNA后进行qRT-PCR实验,结果显示过表达株cps2C基因的相对表达水平较05ZYH33株上调2.4倍.结论 本研究选用S.suis2 05ZYH33的延伸因子TufA强启动子P0530作为启动子,成功构建过表达质粒pSET2∷P0530cps2C并导入S.suis 2感受态细胞,成功获得cps2C过表达株,为进一步研究酪氨酸激酶Cps2C功能提供实验材料.