蛤蜊科3种贝类16SrRNA基因片段及ITS2核苷酸序列分析

利用PCR技术分别扩增连云港及启东沿海蛤蜊科的西施舌（Coelomactra antiquata）、中国蛤蜊（Mactra chinensis）和四角蛤蜊（Mactra veneriformis）3种双壳贝的16SrRNA基因片段和ITS2核苷酸序列．测序后用DNAstar软件分析了核苷酸差异。结果显示：三种贝类16SrRNA基因片段长度相同，均为306bp（去除引物）．核苷酸存在多态性。共有45个变异位点，54个核苷酸发生了变异。全部为碱基置换。西施舌与中国蛤蜊此片段核苷酸的同源性为88．9％．与四角蛤蜊的同源性为88．6％．中国蛤蜊与四角蛤蜊的同源性为90．6％。三种蛤蜊ITS2序列分别为390bp（西施舌）、441bp（四角蛤蜊）和466bp（中国蛤蜊）。存在长度多态性．ITS2核苷酸差异分析结果显示．西施舌与中国蛤蜊的同源性为70．9％-71．1％，西施舌与四角蛤蜊的为70．5％-71．0％。中国蛤蜊与四角蛤蜊的同源性为88．1％-88．8％。ITS2序列分析结果与16SrRNA基因片段分析结果一致．2种分子分析法均显示中国蛤蜊与四角蛤蜊的亲缘关系近。     

用16S rRNA基因的内切酶图谱快速鉴别几种对虾病原菌

坎普氏弧菌是青岛海洋大学生物系微生物实验室于１９８９－１９９０年自对虾养殖场中国对虾红腿病心脏及血淋巴中分离并鉴定的菌株，副溶血菌和溶藻胶弧菌两菌株于１９９４年９月得自中国科学院微生物研究所，为建立快速、准确的中国对虾病原菌的诊断技术，根据几种细菌的１６ＳｒＲＮＡ基因的序列，设计并合成该基因的多聚酶反应的引物ＰＬ１和ＰＬ２。并用该对引物分别从坎普氏弧菌、副溶血弧菌和溶藻胶弧菌的ＤＮＡ要品中扩增出分     

耳鲍(Haliotis asinina)核糖体小亚基(18S rRNA)编码基因的克隆与序列分析

采用分子生物学的方法,对南海不同海区的两个地理群体耳鲍(Haliotis asinina)18S rRNA基因全长进行了克隆和序列分析,并将耳鲍18S rRNA基因的序列与NCBI数据库中已收录鲍的18SrRNA基因进行了比较。结果发现,南海耳鲍核糖体18S rRNA基因与耳鲍H.asinina isolate H11核糖体18S rRNA基因序列的相同率高达98%;同一地理群体内耳鲍核糖体18S rRNA基因序列完全一致;不同地理群体间耳鲍核糖体18S rRNA基因在碱基组成上的相似率为99%,仅在某些位点处发生了碱基替换,即腺嘌呤(T)被鸟嘌呤(G)替换;同时,将这两个不同群体中耳鲍的18S rRNA基因与泰国耳鲍18S rRNA基因序列进行比较分析发现,它们之间也只是发生了碱基替换。     

3种蛏类线粒体16SrRNA和COI基因片段的序列比较及其系统学初步研究

对3种蛏类大竹蛏（Solen grandis），长竹蛏（Solen strictus）和小刀蛏（Cultellus attenuatus）的线粒体16SrRNA和COI基因片段序列进行了比较并对其系统学进行了初步研究。得到的序列总长度分别为472-481bp（16S）和658bp（COI）。3种蛏序列的碱基组成均显示出较高的A＋T比例（16SrRNA基因62．1％；COI基因62．8％）。对位排序比较表明，16SrRNA片段种内个体间变异较小，3种类间存在128个碱基变异位点（其中包括127个简约信息位点）和5个插入／缺失位点，总共12个碱基长；COI片段有200个碱基存在变异，其中包括191个简约信息位点，不存在任何插入／缺失位点。数据分析结果表明16SrRNA和COI基因片段大竹蛏与长竹蛏两片段的遗传距离分别为0．0856和0．1712，两竹蛏类与小刀蛏的遗传距离分别为0．3054，0．2798和0．2662，0．2933。作者认为小刀蛏与竹蛏之间的遗传距离已达到科之间的水平，结果支持将其提升为刀蛏科的分类观点。3种蛏类线粒体16SrRNA和COI基因在种间明显的多态性，证实了16SrRNA和COI基因序列均普遍适用于蛏类种及以上阶元的系统学分析。     

牙鲆(Paralichthys olivaceus)秦皇岛弧菌感染症及其病原细菌研究

采用病原分离与鉴定、人工感染试验等方法,在对养殖牙鲆病例进行发病情况、病理变化特征检验的基础上,进行病原学检验。结果表明,此病例属于由弧菌属(VibrioPacini1854)细菌引起的败血感染症。经对12株纯培养菌(HQ010712-1—HQ010712-12)进行形态特征、理化特性等表观分类学指征的检验,选择代表菌株(HQ010712-1株)进行16S rRNA基因的测定与系统发育学分析等,判定为弧菌属的一个新种。将HQ010712-1株送中国典型培养物保藏中心(CCTCC)进行了复核鉴定与分类定名,依据分离地定名为秦皇岛弧菌(Vibrioqinhuangdaorasp.nov.);参考菌株为HQ010712-1。     

Genetic relationship between Neobenedenia girellae and N. melleni inferred from 28S rRNA sequences

The fragments of 350 bp in 28S rRNA from the closely related monogenea of trematoda, Neobenedenia girellae and N. melleni are obtained by polymerase chain reaction (PCR) amplified using a couple of special primers and then sequenced. The results show that the comparison of 28S rRNA sequences, with only a base varying in 337 bp accounting for 0.3% genetic difference, from the relative species N. girellae and N. melleni parasitized on the different fishes in different farms displays that they possess a very high genetic similarity of 99.7%, higher than that of 99.41% for the single species N. melleni sampled in different areas, and the intraspecific divergence of N.melleni is 0.59%. Meanwhile, the interspecific differences between the two Neobenedenia and three Benedenia (i.e., B. lutjani, B. rohdei and B. seriolae) range from 2.08% tol 1.73%. In addition, UPGMA and MP molecular phylogenetic trees are constructed and proved to be consistent with each other. Though the morphological characteristics and the results of genetic diversity for the two Neobenedenia show a high similarity, whether they belong to a single species or not are still undefined, and the more genes of them should be further investigated, in combination with the systematical and detailed morphological study.     

Molecular and morphometric characterisation of the invasive signal crayfish populations in Croatia

The signal crayfish Pacifastacus leniusculus (Dana, 1852) is a native species to North America. It was introduced to Europe and Japan where it rapidly spread as an invasive species. In Croatia, it is recorded in the rivers Mura and Drava, where it spread downstream from Slovenia, and in the Korana River, where it has been recently illegally introduced. In the invaded areas, signal crayfish outcompetes native crayfish species. Since the knowledge on the genetic diversity of this invasive species is limited, microsatellite markers and sequences of mitochondrial gene for 16S rRNA were analysed to explore the genetic relations between the two Croatian populations (Mura and Korana rivers) as well as their relation to other already studied European populations. Moreover, 16S rRNA sequence fragments of Croatian samples were compared with those from the native range in the west North America. Morphometric characteristics were also studied to determine if there are significant differences between studied populations and if these are concordant with the genetic analyses results. Also, morphometric data were used to assign Croatian signal crayfish into subspecies classification according to Miller’s discriminant function formula, and to compare claw surface area among Croatian, Japanese and North American populations. Based on the results of morphometric characteristics Croatian samples showed Pacifastacus leniusculus leniusculus-like morphology. Phylogenetic reconstruction based on 16S rRNA, positioned Croatian samples into P. leniusculus sensu Larson et al. (2012). Results on microsatellite markers showed that the genetic diversity of P. leniusculus in the Mura River population is slightly higher compared to the Korana River population, but these two populations do not form separated genetic clusters. This study contributes to the knowledge on genetic variability and morphometric characteristics of signal crayfish in Europe and further understanding of its success as an invasive species.     

宁波沿海陆源排污口放线菌(Actinomycetales sp.)的分布特点

利用454高通量测序技术对宁波沿海10个陆源排污口20个站位的放线菌的时空分布及5个工业排污口的放线菌的种类作了整体分析。成功鉴定出了83个属,84个种。研究结果显示:放线菌在陆源排污口的分布呈现季节性分布,从3月份到10月份,放线菌数量呈现先升高后下降的变化,5月份和8月份数量居高,在3月份和10月份偏低;在综合排污口(S4,S6,S8和S9)检测频次较高,在工业排污口(S1,S3,S5,S7和S10)检测频次居中,在市政排污口(S2)检出频次最低。不同类型的排污口,氨氮浓度的排出量不同,放线菌的种类和数量也不同。在5个工业排污口中,S7和S10检出的共同菌最多;在S7独自检出短双歧杆菌(Bifidobacterium breve)、长双歧杆菌(B.longum)和两岐双岐杆菌(B.bifidum),表明存在粪源污染物;在S5检出皱孢链霉菌(Streptomyces scabrisporus)和硫藤黄链霉菌(S.thioluteus),显示有石油降解物和重金属的污染。总体上看,放线菌数量在距排污口外50m处略高于排污口处。排放指标越相似,菌的种类越接近。     

基于第二代测序技术的条斑紫菜(Pyropia yezoensis)贝壳丝状体附生菌群研究

条斑紫菜(Pyropia yezoensis)贝壳丝状体黄斑病给育苗产业造成危害,但目前对健康状态下的菌群结构及其发病后变化仍缺乏认识。本研究基于第二代测序技术和16S r RNA序列分析,对健康和患病丝状体的细菌组成与变化进行比较。测序共产生1.6Gb配对末端序列,获得了第一个高通量的条斑紫菜贝壳丝状体细菌群落数据集。通过数据分析共识别出7,833种可操作分类单元(operational taxonomic units,OTUs),比对确定为18个细菌门。相对于健康样本,黄斑病丝状体样本的微生物多样性显著增加。主坐标分析和非加权组平均法(unweighted pair group method using arithmetic average,UPGMA)聚类均将健康和黄斑病丝状体分成不同的组,且健康组样本间的距离明显小于黄斑病组。上述结果说明,健康贝壳丝状体保持着相似的微生物群落结构,而患病后的丝状体则发生了不同的变化;健康样本和黄斑病样本组间差异显著的细菌属有39个,其中29个在健康样本中的相对丰度明显高于病患组样本。本研究从菌群生态角度分析了健康贝壳丝状体微生物菌群结构、发病后菌群的变化及其对丝状体生长的潜在作用,为丝状体病害的检测和预防提供有价值的信息。     

用于海洋沉积物中厌氧氨氧化细菌分子生态学研究的引物比较

为比较厌氧氨氧化细菌16SrRNA基因的2对特异性引物(Amx368F/Amx820R、Brod541F/Amx820R)在其分子生态学研究方面的优点与不足,本研究采用高通量测序和qPCR技术对长江口及其邻近海域沉积物中厌氧氨氧化细菌细菌的群落组成、多样性和丰度进行比较分析,结果表明,引物Amx368F/Amx820R特异性较高,能够较为完整地反映沉积物中厌氧氨氧化细菌的多样性信息,且其覆盖的厌氧氨氧化细菌丰度与功能基因hzo丰度正相关(P<0.01);而引物Brod541F/Amx820R特异性较低,覆盖部分厌氧氨氧化细菌及其他多个门的细菌,其量化的厌氧氨氧化细菌与功能基因hzo丰度无明显相关关系(P>0.05)。因此,引物Amx368F/Amx820R能够更加真实地反映厌氧氨氧化细菌在海洋沉积物中的群落特征,在其分子生态学研究中更具优势。