基于叶绿体rRNA ITS序列的兰科植物系统发育关系分析

叶绿体DNA(Chloroplast DNA,cpDNA)中的反向重复序列中的非编码区对于陆生植物的进化研究非常有指导意义,被越来越广泛的应用在不同的分类阶元的系统研究中.该研究在叶绿体rRNA ITS 序列的保守区设计引物,PCR扩增得到450 bp左右的叶绿体rRNA从4.5 S到5 S的片段,扩增产物直接测序,进而依据叶绿体rRNA ITS基因序列分析了兰科植物(Orchidaceae)的33个品种材料(12个兰属品种,14个兜兰属品种,7个石斛属品种)间的亲缘关系.通过最简约性分析产生的叶绿体rRNA ITS系统树表明,石斛属和兜兰属为两个自然类群,兰属可能为一多源组.兰属的碧玉兰位于系统树的基部,构成其余各种的姐妹支.兜兰属的白花兜兰、麻栗坡兜兰独立于属内其他种而自成一支.由于兰科植物叶绿体rRNA ITS序列变异率较低,最简约分析产生的分支得不到Bootstrap分析的高度支持,各亚属之间的关系也不明确.对兰科植物系统发育关系的研究尚需要更多的证据.     

In vitro culture, serologic and molecular analysis of Acanthamoeba isolated from the liver of a keel-billed toucan (Ramphastos sulfuratus)

Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ecological niches including soil, fresh and brackish water, dust in air, filters of heating, ventilating, and air conditioning units, swimming pools and hot tubs, etc. Occasionally, they are also known to cause central nervous system infections in humans and other animals. We isolated into culture an amoeba from the liver tissue of a keel-billed toucan and identified it as Acanthamoeba sp. based on culture characteristics and immunofluorescent analysis. Further, we characterized the cultured amoeba and also the amoeba in the liver tissue as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.     

Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm

Ortholinea labracis n. sp. is described and its life cycle is inferred from a Southern Portuguese fish farm, with basis on microscopic and molecular procedures. This myxosporean parasite infects the urinary bladder of the European seabass Dicentrarchus labrax and the intestinal epithelium of a marine oligochaete of the genus Tectidrilus. Myxospores subspherical in valvular view and ellipsoidal in sutural view measuring 7.6 ± 0.3 (6.8–8.7) μm in length, 7.2 ± 0.2 (6.7–7.7) μm in width and 6.5 ± 0.4 (5.8–7.7) μm in thickness. Two polar capsules, 3.0 ± 0.2 (2.6–3.4) μm long and 2.4 ± 0.1 (2.0–2.9) μm wide, located at the same level, but with divergent orientation and opening to opposite sides of the suture line. Sequencing of the SSU rRNA gene revealed a similarity of 100% between the analysed myxospores and triactinomyxon actinospores. The phylogenetic setting of O. labracis n. sp. shows subgrouping in correlation with tissue tropism, but identifies this parasite as another exception to the main division of Myxosporea into the main freshwater and marine lineages.     

Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection

Intestinal microorganisms play important roles in maintaining host health, but their functions in aquatic animal hosts have yet to be fully elucidated. The Chinese mitten crab, Eriocheir sinensis, is one such example. We attempted to identify the shift of gut microbiota that occurred in response to infection of white spot syndrome virus (WSSV), an emerging viral pathogen in the crab aquaculture industry. The microbiota may exert some control over aspects of the viral pathogenesis. We investigated the changes in composition and structure of the crab gut microbiome during various WSSV infection stages of 6 h post‐infection (hpi) and 48 hpi, using a 16S rRNA approach on the MiSeq Illumina sequencing platform. Four phyla (Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes) were most dominant in the gut of E. sinensis regardless of the WSSV infection stages. However, further analysis revealed that over 12 bacterial phyla, 44 orders and 68 families were significantly different in abundance at various states of WSSV infection. Several intriguing aspects of E. sinensis gut bacteria that had not been previously reported were also uncovered, such as class Mollicutes was dominant here, but absent in crabs from Yangtze River estuary and Chongming Islands. Overall, this study provided the first evidence that changes in gut microbiome were closely associated with the severity of WSSV infection and that indicator taxa could be used to evaluate the crab health status.     

两种大眼蟹线粒体12S rRNA基因序列的比较研究

测定了宽身大眼蟹和日本大眼蟹线粒体12S rRNA基因部分片段的序列,前者为577 bp, 后者为572 bp.二者的核苷酸序列A、T、G、C的含量相似,分别为39.0%、35.7%、16.5%、8.8%;39.3%、 36.2%、 15.8%、8.7%;不包括11处插入/缺失位点,两序列间有75个变异位点,核苷酸差异率为13.18%,其中转换42个、颠换33个, 转换与颠换比约为1.3.对国内外4种大眼蟹长度为331 bp的12S rRNA基因同源序列进行分析,A+T的平均含量为76.6%,明显高于G+C的平均含量,且存有变异位点52个,简约信息位点25个.系统发生树的拓扑结构显示,所有的大眼蟹聚为一支,支持大眼蟹类为一单系.     

种属特异性PCR技术在检测鹅肥肝掺假中的应用研究

建立起一种用于检测鹅肥肝是否掺假的方法:种属特异性PCR技术。以鹅、鸭的12SrRNA基因序列设计特异引物,扩增目的片段分别为97bp和196bp。以三类温度处理和0.1%～100%的鸭肥肝掺假比例,分析方法的有效性和灵敏性。结果表明,这种方法的效果是不受样品被长时间加热的影响(高至100℃,10min);同时,即使当掺假比例只有0.1%时,仍能有效扩增出2特异条带。说明本方法适用于快速、准确地检测鹅肥肝掺假的需要。     

基于16S rRNA和Cyt b基因序列探讨2种梅童鱼的遗传分化

比较分析了棘头梅童鱼（Collichthys lucidus）和黑鳃梅童鱼（C.niveatus）的16SrRNA和Cytb基因片段序列差异及遗传分化程度。在长度为526bp的16SrRNA和379bp的Cytb基因片段的核苷酸序列中,2种间共检测到44处核苷酸替代。分析结果表明,2个基因片段的鸟嘌呤（G）含量较低,在Cytb蛋白质编码基因第三密码子位点上表现尤为明显。基于16SrRNA和Cytb基因片段分析结果显示,2种间平均遗传距离分别为0．012和0．111。构建的系统树显示2种梅童鱼在这2个基因片段上存在显著的遗传分化。根据Cytb基因2％／百万年的进化速率推断,棘头梅童鱼与黑鳃梅童鱼的分化时间约为550万年,发生于上新世（Pliocene）早期。     

鲟源致病性鲁氏耶尔森菌的分离、鉴定及药敏研究

2017年,新疆阜康市某鲟鱼养殖场的西伯利亚鲟出现大批死亡,主要症状为生殖孔红肿外突,腹部有点状出血。自患病西伯利亚鲟肝脏、脾脏分离到一株优势菌株XB2,通过革兰氏染色、生理生化试验、药敏试验,并对其16S rRNA基因序列进行测定,用Mega 6.0对序列进行分析。生理生化试验结果显示,菌株XB2为革兰氏阴性短杆菌,单独或成对出现;产酸不产气,V-P反应呈阳性,能利用蔗糖、果糖、葡萄糖、明胶、枸橼酸;在含1%(m/V)氯化钠中能生长,高于3%(m/V)氯化钠中不生长;在4℃不生长,20～30℃均能生长,但在37℃不生长;不利用乳糖、水杨苷、纤维二糖、硫化氢、酒石酸、阿拉伯糖。进化树显示,菌株XB2与鲁氏耶尔森菌KJ812974同源性达99%。通过回感试验确定其半致死密度为2.37×104 cfu/mL。结合生理生化鉴定结果和16S rRNA基因序列分析结果,确定菌株XB2为鲁氏耶尔森菌。药敏结果显示,鲁氏耶尔森菌XB2对氟苯尼考、左氧氟沙星、哌拉西林、阿洛西林等9种药物敏感,对麦迪霉素、大观霉素、链霉素、氨苄西林等15种药物耐药。     

Formulated diet alters gut microbiota compositions in marine fish Nibea coibor and Nibea diacanthus

Nibea, an economical marine fish, is generally fed on trash fish (the low‐value fish), which can cause high feed costs and waste pollutions in high‐density aquaculture. To assess the effect of formulated diet on the gut microbiota in Nibea coibor and Nibea diacanthus, denaturing gradient gel electrophoresis, clone libraries analysis and Illumina sequencing of the 16S ribosomal RNA gene were used in this study. Two Nibea fishes were both dominated by Gammaproteobacteria (especially Photobacterium) and shared a set of gut microbiota, including Bacilli, Mollicutes, Alphaproteobacteria and Fusobacteriia. Statistical analyses revealed that formulated diet led to lower feed conversion ratio (p < 0.001), lower abundance of Vibrio (p = 0.040) and infectious diseases pathways (p = 0.001), higher abundance of polysaccharide‐degrading bacterium Cellvibrio (p = 0.006) in two Nibea species, with higher weight gain rate (p = 0.023) and microbial diversity (Shannon, p = 0.049 and Simpson, p = 0.044) and more carbohydrate metabolism (p = 0.020) observed in N. coibor. The distribution and correlation network of 17 potential short‐chain fatty acid producing bacteria were obtained and visualized in all treatment groups. The results reveal that formulated diet has beneficial effects on the gut microbial ecology in two Nibea fishes, which suggests the possibility of replacing trash fish diet with formulated diet in Nibea aquaculture.     

含油污泥的堆肥处理对微生物群落结构的影响

采用堆肥方法处理含油污泥,评价堆肥处理对含油污泥中石油烃的去除效果,并采用Biolog方法和构建16SrRNA基因克隆文库的方法对处理过程中微生物碳源利用特征和微生物群落结构进行了研究。结果表明,含油污泥经过90d的堆肥处理,石油烃降解率达53.3%±9.5%,显著高于对照处理。堆肥处理可以显著促进石油烃降解,是一种处理含油污泥的有效措施。Biolog分析结果表明,堆肥处理的孔的平均颜色变化率(AWCD)显著高于对照处理,堆肥处理提高了土壤微生物代谢活性。主成分分析结果表明,对照处理和堆肥处理的微生物碳源利用特征明显不同,堆肥处理改变了含油污泥中微生物的代谢功能特征。对照处理和堆肥处理的16SrRNA基因克隆文库之间存在显著差异,对照处理的优势类群是γ-Proteobacteria,堆肥处理的优势类群是Bacteroidetes,堆肥处理显著改变了含油污泥中的微生物群落结构。Marinobacter和Alcanivorax是对照处理中的优势菌,可能与石油烃的自然降解过程有关,而Pusillimonas和Agrobacterium可能对堆肥处理中石油烃的降解起一定作用。