11种鲈形目鱼类ITS2特征及系统应用

ITS2 (Internal transcribed spacer 2)是位于核糖体5.8S和28S基因之间的非编码序列。为了探讨该片段的多样性特征以及进化模式,本研究选取了鲈形目(Perciformes) 5科11种鱼类为研究对象,共获得了444条ITS2克隆序列,其长度范围为332～515 bp。比较种内不同序列的长度发现,金带细鲹(Selaroides leptolepis)在种内存在24 bp的差异,剑鱼(Xiphias gladius)在种内存在32 bp的差异,这2种鱼类的差异较为明显;其余9种鱼类的长度相对比较保守,长度差异小于14 bp。依据11种鱼类的保守位点数、变异位点数、简约信息位点数、单倍型数、保守位点比例、单倍型多样性指数、核苷酸多样性等特征分析发现,种内存在着不同程度差异,特别是金带细鲹的ITS2序列存在着Type A、Type B和Type C 3种类型,各类型间差异较大。根据序列的多样性特征推断,金带细鲹和剑鱼的进化方式为非协同进化;蓝圆鲹(Decapterus maruadsi)、大甲鲹(Megalaspis cordyla)、吉打副叶鲹(Alepes djedaba)和日本竹筴鱼(Trachurus japonicas)的长度和变异位点均存在着一定程度的差异,但差异并不明显,视为不严格的协同进化;泰拉鰆鲹(Scomberoides tala)、布氏鲳鲹(Trachinotus blochii)、尖吻鲈(Lates calcarifer)、射水鱼(Toxotes chatareus)和军曹鱼(Rachycentron canadum) 5种鱼类为协同进化;另外,协同和非协同进化状态与分类系统没有相关性。序列比对发现,大甲鲹种内存在着由协同进化方式演变为非严格的协同进化方式的过度序列;在金带细鲹的3个不同个体中,序列间存在着从协同进化、非严格的协同进化演变为非协同进化的3种进化方式。基于ITS2序列构建的11种鱼类的邻接系统树显示,每种鱼类的克隆都分别按种单独聚为一支,鲹科7属鱼类各属也是单独聚支,表明ITS2不仅可以用在种类的分子鉴定,同时也可以作为分子标记应用于鲹科和属级水平的系统关系研究。     

海南4个地区暹罗炭疽菌群体遗传结构分析

对分离自海南4个地区13种寄主植物上暹罗炭疽菌的ITS-CAL-GAPDH 3基因序列进行群体遗传结构分析。95条暹罗炭疽菌多基因序列可定义为16个单倍型,其中,单倍型H5为主要单倍型,分布于所有寄主植物。AMOVA分析显示,遗传变异主要发生在种群内;病菌未经历过大规模的种群扩张过程。研究结果表明,暹罗炭疽菌种群具有丰富的遗传多样性。     

进境美国苜蓿草中截获的菊花滑刃线虫检疫鉴定

2016年,天津口岸检疫人员从美国进境饲用苜蓿草中发现一种滑刃属线虫,通过形态学特征、形态测计值以及核糖体28S r RNA-D2/D3区与ITS区核酸序列比对和系统进化分析,最终确定为我国检疫性有害生物菊花滑刃线虫(Aphelenchoides ritzemabosi)。从进境饲用苜蓿草中截获该线虫为全国口岸首次。     

松材线虫组DNA条形码筛选

本文基于Gen Bank中登录的14种松材线虫组线虫的28S、18S和ITS部分基因DNA序列,利用遗传距离法及分子生物学方法评价其各自作为松材线虫组DNA条形码序列的适用性。结果显示,28S(拟松材线虫不分亚种)、28S(拟松材线虫分亚种)、18S和ITS的基因序列种内成对遗传距离平均值分别为0.0071、0.0030、0.0007、0.0043,种间成对遗传距离平均值分别为0.0476、0.0454、0.0052、0.1556,其中28S、18S基因序列种内及种间距离存在一定程度的重叠,ITS具有一定程度的Barcoding gap。3个基因序列构建的NJ进化树显示,28S、ITS构建的系统进化树具有较高的节点支持率,可以有效将14个松材线虫组线虫分离成独立分支,而且28S可以有效鉴别出拟松材线虫的2个亚种;基于18S基因构建的进化树不能对吉拉尼伞滑刃线虫、日本冷杉伞滑刃线虫、拟松材线虫进行有效区分。因此,28S、ITS区具有一定的遗传距离间隔以及相对较高的物种识别率,可以作为松材线虫组线虫候选条形码基因。     

鸡卵清蛋白基因调控序列的克隆与载体构建

卵清蛋白（ovalbumin）基因在鸡基因组中只有一对等位基因,却能每天合成分泌多达2 g的蛋白,占据卵白蛋白质的50%以上,是外源基因载体表达调控构件的首选。该研究从输卵管特异性启动子方面着手,通过对卵清蛋白基因启动子的筛选优化,找出启动子增强因子的位置以及组织特异性区域。将卵清蛋白基因上游-922～-2 073和-2 801～-3 100区域平均分成12个长度约为150 bp的序列,分别插入到-921～+38序列的上游,成功构建12个系列表达载体,为进一步筛选短缩版优化启动子提供材料;卵清蛋白基因第一内含子区域截断成300 bp左右的迷你内含子序列,成功构建8个迷你内含子系列载体,为筛选优化的迷你内含子提供必要的材料;成功分离鸡输卵管上皮细胞并优化电转染条件,通过荧光素酶活性检测初步筛选出具有最强活性重组质粒pGL4 UP 1412和内含子重组质粒pGL4 mini intron 3,同时推断出若干包含增强子序列区域。     

部分茶竿竹亚属竹种亲缘关系的ITS序列分析

为了探讨怀集县铁厘茶竿竹和白水茶竿竹与其他茶竿竹亚属竹种之间在分子水平上的差异以及它们的分类地位,对包括铁厘茶竿竹和白水茶竿竹在内的9个茶竿竹亚属竹种的核糖体基因（nrDNA）的内转录间隔区（ITS）序列进行分析,并构建系统进化树。结果表明,所有供试材料ITS全长在559～648 bp,(G+C)含量变化范围为46.02%～57.65%,其中铁厘茶竿竹和白水茶竿竹ITS全长分别为559 bp和614 bp,(G+C)含量分别为55.64%和57.65%。ITS序列构建的系统进化树显示：正种茶竿竹及其变种聚为一个大支,其中铁厘茶竿竹和白水茶竿竹聚在一起。以上结果佐证了铁厘茶竿竹和白水茶竿竹是正种茶竿竹的2个变种。     

一种新油茶炭疽病原多基因序列鉴定

从湖南、广西油茶叶上分离获得5株能侵染油茶树的病原菌。通过柯赫氏法则证明该病原菌为油茶炭疽病的致病菌。病菌菌落圆形,初期为白色,逐渐变为浅灰色;气生菌丝由白色和灰白色渐变为深灰色绒状;菌落背面中心颜色深,外部颜色浅;菌丝划伤后在PDA培养基上能产生橘黄色的分生孢子堆,分生孢子为单胞、直、圆柱状、无色、光滑、两头钝圆或一端稍尖,分生孢子大小(11~15)μm×(4~5)μm;菌丝体附着胞不规则状,浅褐色,(8~12)μm×(5.5~7)μm。采用形态学结合多基因(核糖体内转录间隔区(ITS)、钙调蛋白-CAL、3-磷酸甘油醛脱氢酶-GAPDH)系统学的方法对它们进行鉴定,结果发现,5个菌株均为暹罗刺盘孢(Colletotrichum siamense)。这是国内油茶上C.siamense的首次报道。     

From the micro-scale to the habitat: Assessment of soil bacterial community structure as shown by soil structure directed sampling

Natural structural units of a luvisol under maize crop were studied to assess if soil structure directed sampling could improve the understanding of arrangements of bacteria in spatially constraint location. Three habitats were defined: (i) soil around fine lateral roots (rhizo-aggregates), (ii) soil close to basal roots (core clods) and (iii) unplanted soil between rows (bare soil clods). These habitats were also investigated with maize plants resulting from Azospirillum lipoferum CRT1 inoculated seeds as a model of enhanced fine root system. Rhizo-aggregates were clearly separated from each other (disconnected habitat) in contrast to micro-samples (fragments) from clods, which belong to cohesive macro-structures. Genetic fingerprints on metagenomic extracts were used to characterize the structure of bacterial communities on 95 micro-samples from the three habitats. For eubacteria, automated RISA (Ribosomal Intergenic Spacer Analysis) of ITS (Internal Transcribed Spacer) profiles were performed. PCR-RFLP on nifH gene were used to describe the N-fixer guilds. Exploratory multivariate analyses (PCA and MDS) revealed bacterial community patterns in the sampled habitats. On the basis of ITS profiles, rhizo-aggregates harboured closely related communities, distant from those of the unplanted soil, and each sampled rhizo-aggregate could therefore be considered as a sub-unit of the whole macro-habitat, comprising all the fine roots. The observed low dissimilarity of disconnected rhizo-aggregates is likely to result from the direct influence of maize root tips on the recruitment of rhizosphere bacteria. Molecular fingerprints of nifH from basal root clods (core) were more similar to bare soil than to rhizo-aggregates, indicating similar ecological conditions without, or with, at least, poor maize exudating root influence. Although our study was performed on a limited number of situations, the distribution of bacteria was revealed to be patterned by soil structure units, which is a first step to improve the modelling of microbial ecology in soils.     

A combination of Lactobacillus buchneri and Pediococcus pentosaceus extended the aerobic stability of conventional and brown midrib mutants–corn hybrids ensiled at low dry matter concentrations by causing a major shift in their bacterial and fungal community

We evaluated the effects of applying a combination inoculant to four corn hybrids harvested at high moisture on their nutritive value and microbial populations. The treatment design was the factorial combination of corn hybrids ensiled with (INO) and without (CON) inoculant. The hybrids were TMF2R737 (MCN), F2F817 (MBR), P2089YHR (PCN), and PI144XR (PBR), ensiled at dry matter (DM) concentrations of 30.5%, 26.3%, 31.1%, and 31.5%, respectively; MBR and PBR were brown midrib mutants (BMR). The inoculant contained Lactobacillus buchneri and Pediococcus pentosaceus (4 × 10⁵ and 1 × 10⁵ cfu/g of fresh corn). The experiment had a complete randomized design with treatments replicated six times. Corn was treated or not with inoculant, packed into 7.6 L bucket silos, and stored for 100 d. At d 0, the relative abundance (RA, %) of Enterobacteriaceae was lower in PBR vs. the other hybrids [51.3 vs. x¯ = (average of) 58.4] and in the case of fungi, incertae sedis (i.s.) Tremellales and Mucoraceae were more and less abundant, respectively, in conventional hybrids vs. BMRs (x¯= 25.8 vs. x¯ = 13.9 and x¯ = 3.64 vs. x¯ = 7.52; P < 0.04). After ensiling, INO had higher LAB (9.3 vs. 7.1 log cfu/g of fresh corn) and acetic acid (3.44% vs. 1.32% of DM) and lower yeast (3.1 vs. 4.6) and molds (1.5 vs. 3.0), and also extended the aerobic stability (582 vs. 111 h) but decreased DM recovery (95.6% vs. 97.4%) vs. CON (P < 0.02). Inoculation reduced bacterial phylogenetic diversity (6.75 vs. 14.4) but increased fungal observed taxonomical units (46 vs. 20) vs. CON (P < 0.01). Also, a higher relative abundance (RA) for Lactobacillaceae (99.2% vs. 75.7%) and lower for Enterobacteriaceae (0.28 vs. 9.93) was observed due to inoculation (P < 0.001). For fungi, INO had a lower RA compared to CON for Monascaceae (12.6 vs. 44.7) and increased i.s. Tremellales (8.0 vs. 1.2) and i.s. Saccharomycetales (6.4% vs. 0.3%; P < 0.006). Inoculation changed the diverse bacterial community found in the phyllosphere across hybrids to a taxonomically uneven one dominated by Lactobacillaceae. In the case of fungi, INO application increased the fungal diversity at d 100 mainly by reducing the dominance of Monascaceae vs. CON. In conclusion, the INO treatment overwhelmed the disparate microbial populations found across BMR and conventional hybrids ensiled at low DM concentrations and ensured a significant concentration of acetic acid that modified fungal populations and in turn extended the aerobic stability of all hybrids.     

两株水牛伊氏锥虫内转录间隔区基因序列的测定与系统进化关系

为研究从水牛分离到的伊氏锥虫广西株和湖北株的分类学地位,对2株伊氏锥虫的核糖体基因内转录间隔区(ITS1-5.8S-ITS2)基因进行了克隆和测序分析.用CLUSTALXl.83软件多重比对分析了序列差异,应用MEGA4.0软件绘制系统进化树.结果表明,这2株水牛伊氏锥虫的ITS1-5.8S-ITS2 rRNA基因扩增产物分别为1 102 bp和1 095 bp,序列存在多态性,其中广西株的ITS-1序列长为340 bp,ITS-2序列长为582 bp,湖北株的ITS-1序列长为339bp,ITS-2序列长为587 bp.系统进化分析显示,这2株伊氏锥虫分布在同一大枝的不同亚群中.