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。

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。

A PCR-based RFLP analysis of Sarcocystis cruzi (Protozoa : Sarcocystidae) in Yunnan province, PR china, reveals the water buffalo (Bubalus bubalis) as a natural intermediate host

A polymerase chain reaction-based restriction fragment length polymorphism (RFLP) approach is used to examine Sarcocystis cruzi-like taxa from the atypical intermediate host, water buffalo, in Yunnan, People's Republic of China. The loci examined lie with

Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin. (C) 2004 Elsevier B.V. All rights reserved.

光周敏感核不育水稻phyA基因的RFLP分析

本文用RFLP方法分析农垦58和光敏核不育水稻农垦58S以及其他一些水稻品种如IR36等，得到如下结果： 1．以第5染色体上8个RFLP标记(RG】3、RG】19、RG470、RG573、RG360、RG403、RG229、RG556)作探针对58和58S进行RFLP分析．没有发现多态性。 2． 58和58S光敏色素(phyA)基因内部（或附近）一些CpG岛的甲基化程度不同。 以IR36 phyA 基因的c DNA克隆作探针，用限制性内切酶EcoRV、Dra I、HindⅢ、Xba工、BglⅡ、Sca工、Msp工对58和58S进行RFLP分析，没有发现多态性，但用HpaII现了多态性。 3．根据IR36 ph yA序列，在5’一非编码区设计一对PCR引物，在IR36、58、58S等水稻品种中均扩出了一段DNA，此DNA片段大小在这几个不同品种中都相同。以此DNA片段（称为PCRI）作探针进行RFLP分析．发现在58S和IR36之间具有多态性，根据测出的58S的PCRI序列以及RFLP分析结果，推测58S和IR36之间的差异是由于插入或缺失引起的。

Detection of usual and atypical aldehyde dehydrogenase alleles by mismatch amplification mutation assay

The genotypes of liver mitochondrial high-affinity aldehyde dehydrogenase-2 (ALDH2) are strongly associated with the drinking behavior and the alcohol liver diseases, since the individuals with atypical ALDH(2)(2) allele have higher levels of acetaldehyde in their plasma. The atypical ALDH(2)(2) allele has a nucleotide base transition (G-->A) in its exon 12. Based on this point mutation, we developed a rapid, reliable and inexpensive method, mismatch amplification mutation assay (MAMA), for the determination of human ALDH2 usual and atypical alleles. Two pairs of primers were designed for the amplification of the usual ALDH(2)(1) allele and the atypical ALDH(2)(2) allele, respectively. If the sample for the detection was heterozygous, it could be amplified by both of the primers. The product of polymerase chain reaction (PCR) of ALDH2 exon 12 could be easily screened by electrophoresis on a 2% agarose gel. The results of the MAMA method were further confirmed by sequencing. In the total of fifty samples from unrelated healthy Chinese Han people from Wuhan, China, the frequency of atypical ALDH(2)(2) allele was found to be 12%.

Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip

Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1(+)/2(-), pXO1(-)/2(+) and pXO1(-)/2(-)) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal. (C) 2004 Elsevier B.V. All rights reserved.

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

胶州湾超微型浮游真细菌16s rDNA RFLP分析相关技术的研究

通过生态学与分子生物学相结合的方法，对胶州湾超微型浮游真细菌从16s rDNA的角度分析其多样性，建立了一系列行之有效的方法，对于今后开展特定的超微型生物的研究提供他有益的借鉴。主要结果如下：1.建立了行之有效的从极稀密度的海水中提取浮游细菌总基因组DNA的方法，其得率约为0.3～0.5 μg/L海水，并且提供了DNA浓缩、纯化的一系列步骤，最后所制得的DNA其纯度和含量足以开展后续工作。2.建立了可靠的具有较高转化效率的感受态细胞，并从多个途径来制备，并对各种方法的成败及注意事项进行了探讨。3.对PCR反应的条件进行了深入的探讨，成功在从混合型基因组中扩增出细菌特异性引物(Universal 1406R和Eubacterial 68F)所对应的16s rDNA。并且对PCR的几个控制因子进行了分析，对于今后采用不同引物的高效扩增将提供有益的帮助。4.深入地对TA克隆效率进行了探讨，较好地将PCR混合产物分离开，并成功地导入大肠杆菌DH5α菌株，并对重组质粒进行了初步的RFLP分析，为今后通过序列测定构建系统进化树奠定了基础。5.RFLP分析表明胶州湾海水中至少含有7种亲缘关系较远的真细菌。