红树林植物桐花树内生真菌主要无孢类群的分子鉴定

1 引言 真菌种类繁多,其形态性状复杂多变且易受环境影响,仅依靠形态、生化等表形特征加以鉴别显得较为困难且易出现偏差,尤其是真菌形态鉴定中占有重要地位的孢子,很多植物内生真菌孢子不能在一般实验室条件下形成,这一点给分类鉴定带来困难.随着分子生物学技术的发展,基于ITS(inter-nal transcribed spacer,ITS)区的多态性,ITS区的序列分析近来已广泛应用于真菌种属水平的分类和系统发育方面的研究[1-4].     

A culture-free method for detection of Vibrio vulnificus from coastal seawater based on loop-mediated isothermal amplification targeting vcgC gene

Vibrio vulnificus is an opportunistic pathogen widely distributed in estuarine and coastal seawaters. In this study, a culture-free method was developed to rapid detection of V. vulnificus in all seasons, based on loop-mediated isothermal amplification (LAMP) targeting virulent-correlated gene (vcg). The new assay method allows differentiation between the virulent and non-virulent strain of V. vulnificus accurately. This method also allows effective detection of the pathogeny in winter when the bacterium lives in the viable but non-culturable (VBNC) state. A total of 30 costal seawater samples collected in all seasons were used for the evaluation of this method. The results show that the method is sensitive, accurate and convenient.     

Cloning and characterization of proliferating cell nuclear antigen gene of Alexandrium catenella (Dinoflagellate) with respect to cell growth

Harmful algal blooms (HABs) have been affecting negatively the shellfish and aquaculture industries around the world. Though a lot of efforts have been made to disclose the changes of environmental factors involved and their effects on the HABs events, the molecular mechanism of this process remains unclear. To address this problem, proliferating cell nuclear antigen gene (pcna) was isolated and characterized from Alexandrium catenella. It showed high homology to those of other dinoflagellates (89% and 91% homology to Pfiesteria piscicid and Pyrocystis lunula, respectively), and also 42%–43% homology to those of plant and animals. The expression level of pcna revealed by quantitative real time PCR was the lowest at the late lagging cell growth phase, increased to the highest at the late exponential phase, and then decreased at the stationary phase. Though the cell growth rate was also changing, no positive correlation between pcna expression level and cell growth rate was displayed throughout the whole cell growth stages (r 2 =0.024 6). However, the pcna expression level had the similar trend with the change of cell growth rate throughout the whole growing process, e.g., from increasing at the earlier cell growth stage to decreasing at the following stages, though slightly lagging to the latter.     

钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析

利用线粒体16SrRNA基因和线粒体细胞色素氧化酶亚基Ⅰ（cvtocllrome oxidase Ⅰ,COI）基因片段初步研究钦州湾牡蛎（Ostxea）的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16SrRNA基因部分长度为413bp,COI基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A＋T比例：16SrRNA基因598％;COI基因605％。对位排序比较表明,16SrRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COI片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎（Crassostrea hongkongensis）16SrRNA和COI序列与白肉牡蛎的遗传距离均为0000,有明巨牡蛎（Crassostrea ariakensis）16SrRNA和COI序列和红肉牡蛎（redmeatostxea）的遗传距离分别为0000和0011,白肉牡蛎16SrRNA和COI序列和红肉牡蛎之间的遗传距离分别为0035和0146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16SrRNA和COI基因在种间存在明显的多态性,证实了16SrRNA和COI基因序列适用于牡蛎的系统学分析。     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China (LCDV-cn) genome

An open reading frame (lcn61) of iymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector.Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.     

Sex determination mechanisms in fish

In fish, sex determination (SD) system shows high variation. The SD mechanisms include environmental and genetic regulation. The research on SD system and related genes in intensively studied fish species was reviewed. Although some genes have been described as sex-related, only DMRTlbY can be considered as a master sex determination gene and none of them has been util-ized in aquaculture. The variation of fish SD system, the importance of sex-related genes in evolution research and the relations be-tween environmental factors and sex-related genes were also discussed. The fish sex determination mechanism remains largely un-known. Further research needs to be done considering the significance of fish SD studies in basic and applied aspects.     

三种鳜鱼(Siniperca)生长激素基因内含子多态性的比较研究

鳜(Siniperca chuatsi)、大眼鳜(Siniperca kneri)、斑鳜(Siniperca schezeri)为鳜属中3种主要经济鱼类,具有较近的亲缘关系和相似的生活习性,但生长性状差异较大.为探索3种鳜鱼生长差异与基因差异间联系,以野生群体为材料,采用PCR扩增、电泳及测序等方法,对生长发育相关基因生长激素(GH)基因进行了多态性检测与分析.在第一内含子、第二内含子前段、第二内含子中段微卫星序列及第三内含子中均检测到多态性.各区域共检测到25种长度类型,其中4种为鳜与大眼鳜共有长度类型,3种为鳜、大眼鳜与斑鳜共有长度类型,其余18种为各物种特有长度类型.各区域长度类型共组成14种单倍型,无种间共有单倍型.序列长度差异主要由重复序列及DNA片段的插入或缺失形成.在第1内含子3'端-6位与第3内含子5'端+16位各发现一处靠近剪接位点的序列差异.根据多态性检测结果构建系统发育树,鳜与大眼鳜先聚为一枝.本研究可为进一步确认鳜鱼GH基因差异与生长性状间联系,并利用基因差异进行鳜鱼种质资源保护及分子育种奠定基础.     

一株甲胺磷高效降解菌——巴氏葡萄球菌(Staphylococcus pasteuri)的筛选及其分子鉴定

从湘潭南天化工厂的甲胺磷废水和污泥中分离细菌样品,以甲胺磷为唯一碳源和能源,经过定向筛选,得到1株可高效降解甲胺磷的菌株HN003,气相色谱测定其对甲胺磷的平均降解效率在24h、48h和72h分别达到45.8%、88.5%和100%。对其进行常规生理生化测试,结果表明,菌株HN003革兰氏染色为阳性,在10-42℃、pH3.0-pH14.0范围内均能生长,并与巴氏葡萄球菌的表型特征非常相似。为了进一步确定HN003的分类学地位,测定了其16S rRNA基因序列(1541bp),分析了相关细菌相应序列的同源性,构建了分子系统发育树,结果表明,菌株HN003与巴氏葡萄球菌的亲缘关系最近。综合上述结果,菌株HN003可鉴定为巴氏葡萄球菌(Staphylococcus pasteuri)。     

凡纳滨对虾溶菌酶基因在大肠杆菌中的表达和活性检测

将凡纳滨对虾(litopenaeus vannamei)血淋巴细胞中提取的总RNA,经RT-PCR扩增溶菌酶基因(LvLys 基因)的开放阅读框,将其克隆至pMD18-T并测序.用限制性内切酶BamHⅠ和HindⅢ双酶切取目的基因并与表达载体pET-28a(+)连接,构建重组质粒pET-28(a+)/LvLys,转移到BL21(DE3)中诱导表达.经亲和纯化得到凡纳滨对虾重组溶菌酶.抑菌活性检测表明重组溶菌酶对大肠杆菌TOP10有一定抑制作用.     

凡纳滨对虾内脏几丁质酶基因的克隆、序列分析与结构预测

几丁质酶是甲壳动物顺利完成生理性蜕壳的关键功能酶．已有研究表明几丁质酶是一个多基因家族．根据甲壳动物几丁质酶保守序列设计引物,应用反转录PCR方法从凡纳滨对虾（Litopenaeus vannamei）内脏中扩增得到部分几丁质酶编码基因片段,进一步结合RACE法,克隆得到该几丁质酶完整编码序列．生物信息学分析表明其含有信号肽序列、几丁质酶催化中心序列、PEST连接区和几丁质底物结合部位序列．序列比对发现其与中国对虾（ABB85237．1）、斑节对虾（AF157503．1）和日本对虾几丁质酶（BAA12287．1）具有很高的相似度．