用改进的重叠PCR引入血管内皮生长因子基因突变

血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)的PCR产物克隆于T载体上 ,经转化JM1 0 9感受态菌株后 ,随机挑取 8个白斑菌落 ,混合后制成混合模板 .采用 3条引物 ,做两轮重叠PCR反应 ,获得了VEGF的突变基因 ,经PCR鉴定 ,酶切鉴定和测序分析表明所得基因为目的产物 .实践证明这种突变方法简单快速 ,为下一步实验大量引入突变奠定了实验基础

Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum

Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.

An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

Expression profiles of penaeidin from Fenneropenaeus chinensis in response to WSSV and vibrio infection by real-time PCR

Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a "U" Re expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse "U" with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the alpha-, gamma- and delta-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13A degrees N.

栉孔扇贝抗鳗弧菌感染性状候选基因的多态性研究

栉孔扇贝（Chlamys farreri）是我国北方地区主要的养殖贝类之一，曾为沿海各省带来巨大的经济效益。但自1997年以来，陆续爆发的病害问题给扇贝养殖业造成了巨大的经济损失，严重影响了该产业的健康发展。目前认为培育抗病性强的扇贝优良品种是解决病害问题的根本途径。由于传统的育种方法费时费力，无法满足对良种的迫切需求，因此有必要通过分子手段加快抗病品种的培育步伐。标记辅助育种（marker assisted selection，MAS）是成功应用于动物育种中的分子手段之一，但由于缺乏与抗病性状相关的标记，MAS目前还无法在软体动物中得到应用。因此，寻找与抗病性状相关的分子标记是在软体动物中发展MAS的关键。 本研究利用鳗弧菌（Listonella anguillarum）对栉孔扇贝进行攻毒感染实验，初步得到敏感群体和抗病群体后采用PCR、PCR-RFLP、Bi-PASA PCR等方法研究了CfLysG、CfC1qDC和CfLITAF基因多态性及其与栉孔扇贝对鳗弧菌抗性的关系。 研究发现，栉孔扇贝CfLysG的基因序列中共有104个单核苷酸多态性（SNP）位点和29个插入/缺失（I/D）多态性位点。有17个多态性位点位于启动子区域，选择其中的-753 I/D、-391A/G和-284I/D多态性进行检测，发现这三个位点的基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡（P>0.05）。其中-753 ID基因型和-284 ID基因在抗病群体中的频率高于在敏感群体中的频率，但两者之间无显著性差异（P>0.05）。-391 AG基因型在抗病群体中的频率显著高于敏感群体（P=0.007），表明-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。为验证这一相关性，对-391位点不同基因型的扇贝进行攻毒感染实验。统计发现，具有-391 AA基因型的扇贝累计死亡率显著高于具有-391 AG基因型的扇贝（P=0.001），进一步证实了CfLysG基因-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因的外显子共有3处SNP，其中仅第三外显子上的+3473 A/C为非同义突变。统计分析表明，+3473位点不同基因型在敏感群体中的分布频率符合Hardy-Weinberg平衡（P>0.05），而在抗病群体中则偏离Hardy-Weinberg平衡（P<0.01）。+3473 AA基因型在抗病群体中的频率显著高于在敏感群体中的频率（P=0.022），表明+3473 AA基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因第1内含子存在+96 I/D和+487 I/D两处大片段的I/D多态性。统计发现，这两个位点的基因型在敏感群体和抗病群体中的分布频率均符合Hardy-Weinberg 平衡（P>0.05）。其中+96 DD基因型和+487 ID基因型在抗病群体中的频率均略高于在敏感群体中的频率，但两者之间无显著性差异（P>0.05）。表明这两个位点的多态性与栉孔扇贝对鳗弧菌的抗性无显著相关性。对CfLysG基因各多态性位点的统计分析表明，各位点之间存在不同程度的连锁不平衡，提示有单体型的存在。对19种频率>1%的单体型在敏感群体及抗病群体中的频率进行分析，发现-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型在抗病群体中的频率显著高于敏感群体（P=0.044），表明该单体型与栉孔扇贝对鳗弧菌的抗性显著相关。 在栉孔扇贝CfC1qDC基因cDNA序列上共发现14处SNP。对+423 T/C多态性与栉孔扇贝对鳗弧菌抗性的关系进行了分析。统计发现，+423位点各基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡（P>0.05）。+423 TT基因型在抗病群体中的频率显著高于在敏感群体中的频率（P=0.005），表明+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关。 在栉孔扇贝CfLITAF基因cDNA序列中共发现3处SNP及1处I/D多态性。对+145 I/D多态性进行研究，发现所有敏感个体及抗病个体中均同时存在+145 位点所有等位基因，表明+145位点多态性与栉孔扇贝对鳗弧菌的抗性不相关。 以上研究表明，栉孔扇贝CfLysG基因-391 AG基因型、+3473 AA基因型、-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型以及CfC1qDC基因+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关，提示它们可作为与栉孔扇贝抗病相关的候选分子标记应用于贝类抗病育种中，为贝类的标记辅助育种提供参考。此外，抗病相关分子标记的发现还有利于加深对扇贝发病机理的理解，并有助于发掘预防及治疗贝类疾病的新方法。

应用PCR-DGGE技术分析长江口低氧区和黄海冷水团的细菌群落组成

利用PCR-DGGE技术对长江口外低氧区海域和黄海冷水团海域的细菌群落组成进行了分析。 长江口外低氧区海域的细菌群落组成分析结果为：对获得的25条DGGE条带进行了克隆、测序，所得到的序列进行了系统进化分析（细菌16S rRNA基因V3区序列），分别归属于4个细菌类群：变形菌门(Proteobacteria)、拟杆菌门(Bacteroides)、厚壁菌门(Firmicutes)和蓝细菌门(Cyanobacteria)。其中有16条分别与变形细菌亚群的γ和δ-Proteobacteria相似。通过时空分析发现，低氧水体的细菌群落组成与非低氧水体的组成是不同的。低氧水体的优势菌群是拟杆菌门(Bacteroides)中的Flavobacteria。 黄海冷水团海域的细菌群落组成和优势菌群分析结果为：细菌16S rDNA V3区特征片段经DGGE分离、条带切割，共得到24条DGGE条带，克隆、测序后，将所得序列进行系统进化分析，分别归属于2个细菌类群：变形细菌门(Proteobacteria)和拟杆菌门(Bacteroides)。在24条序列中有16条分别与变形细菌亚群的γ和δ-Proteobacteria相似，有5条与拟杆菌门相似。通过时空分析发现，10月份（冷水团存在期），冷水团内部水体的细菌群落组成包括γ-Proteobacteria、δ-Proteobacteria和Bacteroides，而冷水团外部的水体的细菌群落组成包括γ-Proteobacteria和Bacteroides。冷水团内部水层的优势菌群为γ-Proteobacteria。4月份虽然冷水团没有形成，但是所调查的海域海水温度都不高，在7℃－12℃范围内，所以4月份所有站位，不管是底层的还是总的的细菌群落组成都与10月份冷水团内部（海水温度低于10℃）水体的相同，与10月份冷水团外部（海水温度大于19℃）的不同。

应用单细胞PCR方法研究胶州湾一株鳍藻的遗传特征

近年来，分子生物学技术与方法被越来越多地应用于有害赤潮研究。其中，单细胞PCR方法是对难以室内培养的有害赤潮藻种进行遗传特征研究的一项重要技术。本实验尝试建立微藻的单细胞PCR方法，并将其应用于鳍藻研究。 应用室内培养的亚历山大藻，研究了微藻的单细胞PCR方法，并对藻细胞的固定和保存方法进行了比较。结果表明，浮游植物研究中常用的甲醛固定方法只能用于短期保存样品（少于5天），而乙醇固定、鲁格氏液固定、或者在-20℃下冷冻保存的样品，在较长的时间后（60天）仍可以得到比较理想的PCR扩增结果。 应用单细胞PCR方法，对青岛近海采集的鳍藻进行了研究，扩增并测定了包括核糖体大亚基（LSU）rDNA的5’端D1-D2区序列，以及5.8S rDNA和ITS区的部分序列信息。通过分析软件对所得到的鳍藻序列信息与基因库中已知的鳍藻序列信息进行了分析与对比。根据ITS和LSU序列信息构建的系统进化树都显示，本文采集的鳍藻藻种与国外报道的圆形鳍藻聚为一支，初步确定采集的鳍藻应为圆形鳍藻，对该藻种形态学特征的观察也支持这一结果。该藻种部分LSU rDNA序列与欧洲同种鳍藻相似度达到99%,与其它等鳍藻遗传距离在18%-20%之间。测定的ITS区序列与同种圆形鳍藻有62个碱基的差异，与LSU rDNA序列相比，ITS序列变异更大。应用LC-MS方法对该鳍藻的进行了DSP毒素分析，结果未检测到OA或DTX1毒素。 这是我国首次应用单细胞PCR方法对鳍藻开展的研究工作，首次报道了我国鳍藻的核糖体部分序列信息。圆形鳍藻在青岛近海海域是初次报道，显示了单细胞PCR方法在鳍藻研究中的重要意义。

黄海近岸海洋趋磁细菌的多样性研究

海洋趋磁细菌在黄海、东海的近岸海域沉积物中分布广泛，形态上以球菌为主，部分地区有杆菌、螺旋菌。在青岛汇泉湾发现大量的海洋趋磁细菌，趋磁球菌占优势，最大丰度可达105 cells/cm3。 透射电镜观察潮间带趋磁细菌以球菌或卵球菌占绝对优势；潮下带菌体形态多样，有球形或卵球形、长短杆状、弧状和螺旋状，其中球形或卵球形趋磁细菌占优势。电镜观察还发现磁小体的排列方式多样化，大多数呈链状排列，有单链、双链及多链，还有的呈环状或者成簇排列。磁小体的形态也多种多样，有正方体、棱柱体、立方八面体、子弹头状、片状和齿状。 PCR-RFLP分析RT收集潮间带得到的三个菌株同属于α-变形菌亚纲中的未培养的趋磁球菌，三者之间相似性都在98%以上，可能都属于同一个属。潮下带RT收集，测序分析得到10个菌株。发现9个属于α-变形菌亚纲，1个属于γ-变形菌亚纲，共有8个不同的属，优势种是MRT-81和MRT-82。目前尚未获得这些细菌的纯培养。 结合电镜观察的结果发现，潮间带形态单一，属于同一个属；潮下带形态多样，属于8个不同的属。电镜结果跟RFLP的结果一致。结合区域特点我们分析潮间带水深大约0.5-1 m，在大潮最低潮时可能暴露于空气中，且受潮汐的影响，物化环境变化较大。潮下带水深常年>2 m，物化环境比较稳定。这可能是造成两个区域多样性差别的主要原因。 我们得到的所有的序列与未培养和已纯培养的海洋趋磁球菌的16S rDNA相似性都不高于94%，两优势菌群与纯培养的MC-1相似性都不高于88%，可能为新发现的海洋趋磁细菌资源。

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.

Identification of Crassostrea ariakensis and related oysters by multiplex species-specific PCR

Genetic markers are needed for rapid and reliable identification of oysters. In this study, we developed multiplex genus- and species-specific PCR markers for the identification of oysters from China. We used the mitochondrial cytochrome oxidase I (COI) and nuclear 28S ribosomal RNA genes for marker development. DNA sequences from different species were obtained from GenBank or by direct sequencing. Sequences were aligned, and genus- and species-specific nucleotides were identified. Primers were designed for genus/species-specific amplification to generate fragments of different sizes. A multiplex set of genus- and species-specific primers from the 28S gene was able to separate C. ariakensis and C. hongkongensis from other species and assign oysters to four genera. A set of species-specific COI primers provided positive identification of all five Crassostrea species from China, C. ariakensis, C. hongkongensis, C. angulata, C. gigas, and C. sikamea in a single PCR. The multiplex PCR assays do not require fluorescence-labeling or post-PCR enzyme digestion, providing a simple, fast and reliable method for the identification of oysters from China.

基因芯片PCR温度模糊自整定PID控制算法的研究

在核酸扩增反应仪中,基因芯片核酸扩增反应过程要求实现温度高精度快速跟踪控制,常规温控方案和算法难以实现。将模糊推理系统与常规PID控制方式相结合,采用模糊自整定PID控制算法实现了温度快速跟踪控制。实验结果表明:模糊自整定PID控制算法比常规PID算法具有更强的鲁棒性,能够克服控制对象热惯性参数时变性的影响,降低了输出温度最大超调量,提高了稳态精度。

硫酸盐还原菌类群分离培养和PCR分析鉴定

探讨分离培养和聚合酶链反应（PCR）在硫酸盐还原菌类群分析鉴定中的应用。［方法］采用分离培养和PCR 对贵州阿哈湖沉积物中硫酸盐还原菌类群进行分析，并对分离纯化的一株硫酸盐还原菌进行鉴定。 ［结果］ 贵州阿哈湖沉积物硫酸盐还原菌类群为脱硫肠菌属、脱硫叶菌属和脱硫球菌- 脱硫线菌- 脱硫八叠菌属；纯化菌株经PCR 扩增初步鉴定为脱硫叶菌属,菌株形态与鉴定结果相符。 ［结论］ 采用分离培养结合PCR，可以对硫酸盐还原菌类群进行分析和鉴定。