微生物的ARDRA检测

ARDRA（扩增性rDNA限制性酶切片段分析）是新发展起来的一项生物检测技术，可在原位下获取其有关生物性状。本文阐述了ARDRA技术的原理和方法，介绍了该技术在微生物多样性和系统发育研究中的应用，并对ARDRA技术的应用前景提出展望。

乙草胺和甲胺磷对农田黑土可培养真菌数量及种群结构的影响

应用传统及PCRDGGE方法(denaturinggradientgelelectrophoresis),分别对不同浓度乙草胺、甲胺磷胁迫下黑土中可培养真菌CFU(colonyformingunits)、种群丰富度(richness)及种群结构动态变化规律进行了研究.结果表明,在实验室微域条件下,乙草胺对黑土可培养真菌CFU的影响随处理浓度的增加而抑制作用增强,表现出由低浓度(50mg·kg-1)时的刺激生长到高浓度(250mg·kg-1)时的长期抑制效应;250mg·kg-1甲胺磷在8周处理过程中对土壤可培养真菌生长具有显著的刺激效应,使可培养真菌CFU比对照增加10倍,但50和150mg·kg-1甲胺磷处理对土壤可培养真菌CFU无显著影响.种群丰富度系数分析结果表明,高、中浓度乙草胺处理可使土壤可培养真菌种群丰富度不可逆地降低.土壤真菌rDNA特异PCRDGGE聚类分析结果表明,不同浓度乙草胺、甲胺磷处理均不同程度地对土壤可培养真菌的种群组成和结构造成影响,其中甲胺磷尤为显著.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Purification and characterization of the chitosanase from Aeromonas sp HG08

In this work, the characterization of a chitosanase-producing bacterium isolated from soil was reported and this strain was grouped under the genus Aeromonas by virtue of its morphological, physiological properties and 16S rDNA gene sequences. It is the first report that the genus Aeromonas could produce chitosanase. Aeromonas sp. HG08 could secrete the chitosanase ( named AsChi) with molecular weight of 70 kDa. The optimum pH and temperature of AsChi was 6.0 and 55 degrees C, respectively. The activity of AsChi was markedly enhanced by Mn2+ and inhibited by Fe3+, Cu2+, Ag+ and Hg2+; additionally, the activity of AsChi was increased with the degree of deacetylation ( DDA) of chitosan. Through viscosimetric assay, AsChi probably hydrolyzed chitosan in an endo-type fashion.

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of total genomic DNA from 15 red algal species. It resulted in 0.1 mug high quality DNA from 1 mg fresh algal material, with an A(260)/A(280) ratio of 1.68 - 1.90. Using this rapidly isolated DNA, the 18S ribosomal RNA genes ( rDNA) and the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. The tested DNA was suitable for restriction endonuclease digestion, genetic marker analysis and polymerase chain reaction (PCR) amplification, and may be valid for other genetic manipulation.

Molecular phylogeny and species identification of pufferfish of the genus Takifugu (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships and species identification of pufferfishes of the genus Takifugu were examined by use of randomly amplified polymorphic DNA (RAPD) and sequencing of the amplified partial mitochondrial 16S ribosomal RNA genes. Amplifications with 200 ten-base primers under predetermined optimal reaction conditions yielded 1962 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances between 5 species of Takifugu and Lagocephalus spadiceus as the outgroup were calculated from the presence or absence of the amplified fragments. Approximately 572 bp of the 16S ribosonial RNA gene was amplified, using universal primers, and used to determine the genetic distance values. Topological phylogenic trees for the 5 species of Takifugu and outgroup were generated from neighbor-joining analysis based on the data set of RAPD analysis and sequences of mitochondrial 16S rDNA. The genetic distance between Takifugu rubripes and Takifugu pseudommus was almost the same as that between individuals within cacti species, but much smaller than that between T. rubripes, T. pseudommus, and the other species. The molecular data gathered from both analysis of mitochondria and nuclear DNA strongly indicated that T. rubripes and T. pseudommus should be regarded as the same species. A fragment of approximately 900 bp was amplified from the genome of all 26 T. pseudommus individuals examined and 4 individuals of intermediate varieties between T. rubripes and T. pseudommus. Of the 32 T. rubripes individuals, only 3 had the amplified fragment. These results suggest that this fragment may be useful in distinguishing between T. rubripes and T. pseudommus.

Morphological and phylogenetic analysis of a Gymnodinium-like species from the Chinese Coast

A Gymnodinium-like species was studied with light microscopy (LM) and scanning electron microscopy (SEM). Also, the internal transcribed spacers (containing 5.8S rDNA) and large ribosomal subunit DNA (D1-D2) sequences were obtained by PCR amplification, and then sequenced to explore the relationships within our isolate, Gymnodinium and other Gymnodinium-like species, including Karenia, Gyrodinium, Karlodinium and Symbiodinium. The LM observation showed that the species was characterized by moving in a levorotatory direction, visible hypocone, epicone and transverse groove, all of which are typical for Gymnodinium. In addition, two flagella could be found under SEM. The phylogenetic analysis revealed that the isolate grouped with Symbiodium, rather than other relevant dinoflagellates. All results showed our isolate belongs to Symbiodium. The strain was isolated from a red tide water sample, denoting that Symbiodium may be causative species for algal bloom.

Molecular phylogenetic analysis of attached Ulvaceae species and free-floating Enteromorpha from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer (ITS) 1, 5.8 S, and ITS 2, the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007, including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations. The result supported the monophyly of all free-floating Enteromorpha samples, implying the unialgal composition of the free-floating Enteromorpha, and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades, suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season.

Vertical distribution of bacterial and archaeal communities along discrete layers of a deep-sea cold sediment sample at the East Pacific Rise (similar to 13 degrees N)

The community structure and vertical distribution of prokaryotes in a deep-sea (ca. 3,191 m) cold sediment sample (ca. 43 cm long) collected at the East Pacific Rise (EPR) similar to 13 degrees N were studied with 16SrDNA-based molecular analyses. Total community DNA was extracted from each of four discrete layers EPRDS-1, -2, -3 and -4 (from top to bottom) and 16S rDNA were amplified by PCR. Cluster analysis of DGGE profiles revealed that the bacterial communities shifted sharply between EPRDS-1 and EPRDS-2 in similarity coefficient at merely 49%. Twenty-three sequences retrieved from DGGE bands fell into 11 groups based on BLAST and bootstrap analysis. The dominant groups in the bacterial communities were Chloroflexi, Gamma proteobacteria, Actinobacterium and unidentified bacteria, with their corresponding percentages varying along discrete layers. Pairwise Fst (F-statistics) values between the archaeal clone libraries indicated that the archaeal communities changed distinctly between EPRDS-2 and EPRDS-3. Sequences from the archaeal libraries were divided to eight groups. Crenarchaea Marine Group I (MGI) was prevalent in EPRDS-1 at 83%, while Uncultured Crenarchaea group II B (UCII B) abounded in EPRDS-4 at 61%. Our results revealed that the vertically stratified distribution of prokaryotic communities might be in response to the geochemical settings and suggested that the sampling area was influenced by hydrothermalism. The copresence of members related to hydrothermalism and cold deep-sea environments in the microbial community indicated that the area might be a transitional region from hydrothermal vents to cold deep-sea sediments.

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7-8 and at temperature close to 35 degrees C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40-45 degrees C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.

Phylogenetic studies on two strains of Antarctic ice algae based on morphological and molecular characteristics

The taxonomic characterization of two strains of Antarctic ice algae, Chlamydomonas sp. ICE-L and Chlamydomonas sp. ICE-W, were analyzed on the basis of morphological and molecular traits. The results indicate that they are the same species and belong to Chlamydomonas (Chlorophyta). According to I SS rDNA and ITS-I sequences they are very close relatives of Chlamydomonas sp. Antarctic 2E9, if not identified as such. They belong to the 'monadina clade', Cd. monadina and Cm. subdivisa as the sister group, on the basis of 18S rDNA sequence. They occur in 'Chlamydomonas clade' according to rbcL sequencing and are close relatives of Cd. kuwadae. The ITS sequences of ICE-L and ICE-W are 1302 base pairs and 1300 base pairs in length, the longest Volvocales ITS sequences ever reported.

Identification and assessing the cultivars of Laminaria Lamx. (phaeophyceae) with molecular markers

Molecular markers were used to identify and assess cultivars of Laminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956. Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars of Laminaria japonica Aresch. used for breeding in China fell into one cluster. L. japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm. formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including TTS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.

利用荧光原位杂交技术（FISH）对中国明对虾和栉孔扇贝若干重要基因定位的研究

本研究将荧光原位杂交（Fluorescence in situ hybridization, FISH）技术引入中国明对虾和栉孔扇贝这两种重要的海水养殖动物的染色体研究中，建立了相关技术平台，在染色体上定位了部分功能基因，详述如下： 1. 在中国明对虾和栉孔扇贝中建立了FISH平台，利用单色和双色FISH技术在中国明对虾和栉孔扇贝染色体上成功定位了多拷贝基因，发现5S rDNA定位于中国明对虾的一对同源染色体上，栉孔扇贝18S rDNA和组蛋白序列也各自定位于一对同源染色体上，它们均可以作为染色体特异性探针来鉴别染色体。 2. 在栉孔扇贝中发展了BAC-FISH技术，定位了包含热休克蛋白70（HSP70）、丝氨酸蛋白酶（Serine Protease）和脂多糖葡聚糖结合蛋白（LGBP）等免疫相关基因的BAC克隆，发现它们均定位于一对同源染色体的长臂上。利用双色BAC-FISH技术，发现6个包含栉孔扇贝LGBP基因的BAC克隆在间期细胞核上共定位。对栉孔扇贝5个探针进行了同时定位，初步鉴别了栉孔扇贝5条染色体。 3. 在栉孔扇贝热休克蛋白70（HSP70）、丝氨酸蛋白酶（Serine Protease）和脂多糖葡聚糖结合蛋白（LGBP）等免疫相关基因内部发掘了数个潜在的SNP位点，展示了栉孔扇贝SNP位点的丰富性。这些SNP位点可以用于图谱整合。

织纹螺（Nassarius spp.）体内河豚毒素来源的研究

近几十年来，国内沿海地区频繁发生食用织纹螺中毒事件，并导致数十人死亡，这一问题得到了政府相关部门的高度重视。但是，由于织纹螺毒性变化很大，毒素来源不清楚，因此很难预测食用织纹螺中毒事件的发生，这在很大程度上限制了对食用织纹螺中毒事件的有效监测和管理。目前，对于中国沿海有毒织纹螺体内河豚毒素（tetrodotoxin, TTX）的来源还未见过系统研究。本文选取中国沿海常见的半褶织纹螺（Nassarius semiplicatus）、纵肋织纹螺（N. variciferus）和拟半褶织纹螺（N. semiplicatoides sp. nov.）作为实验对象，从毒素的微生物来源与食物链来源这两个角度分别展开研究，以探讨织纹螺体内 TTX 的可能来源，为提出相应的预防管理措施提供科学依据。 首先，我们先后从曾发生过中毒事件的江苏盐城和连云港采集了织纹螺样品，通过小鼠生物测试法和液-质联用分析技术（LC-MS），对织纹螺的毒性和毒素组成进行了测试和分析，分离培养了织纹螺体内及其生活环境中的细菌，应用河豚毒素单克隆抗体酶联免疫检测方法（ELISA）对细菌的产毒情况进行了测试，并通过 16S 核糖体（rRNA）部分基因序列测定对细菌种类进行了初步的分析。研究发现，采自江苏盐城和连云港的半褶织纹螺的毒性分别约为 2 MU/g 和 200 MU/g 组织，体内的毒素成分是河豚毒素及其同系物。从盐城的半褶织纹螺及其生活环境分离的菌株中随机挑出 14 个菌株中，9 个菌株河豚毒素检测结果呈现阳性。从连云港高毒性半褶织纹螺消化腺中分离到的 45 个菌株中，阳性菌株有 21 个。但是，有毒菌株毒素含量较低，毒素含量范围是 15-184ng/g。通过 16S rDNA 部分序列的测序结果发现，大部分有毒菌株与弧菌属（Vibrio）的细菌在遗传序列信息上比较相近。其余有毒菌株分别与希瓦氏菌属（Shewanella）、海单胞菌属（Marinomonas）、黄杆菌属（Tenacibaculum）、动性菌属（Planococcus）、发光杆菌属 (Photobacterium)和气单胞菌属（Aeromonas）的遗传序列比较相近。其中与海单胞菌属、动性菌属和发光杆菌属亲缘关系较近的产毒细菌是首次报道。这一研究表明织纹螺体内及其生活环境中的存在产河豚毒素的细菌，但由于产毒素的量较低，因此可能在织纹螺体内河豚毒素的产生和累积过程并不发挥主要作用。 织纹螺作为一类腐食性的海洋动物，也有可能通过进食含有河豚毒素的生物而累积河豚毒素。对此，我们开展了高毒性半褶织纹螺的室内培养实验，以及河豚毒素在不同种类织纹螺体内的累积和排出的模拟实验，并定期采样，通过液相色谱与串联质谱联用技术（LC-MS/MS）对织纹螺体内河豚毒素及其同系物的含量变化情况进行了分析。室内培养实验发现，从连云港赣榆县采集的高毒性半褶织纹螺，在实验初期，体内毒素含量呈下降的趋势，但从 7月上旬开始，毒素含量突然快速上升，与连云港赣榆县野外采集的织纹螺的毒素含量表现出相似的变化趋势。河豚毒素在不同种织纹螺体内的累积和排出的模拟实验发现，通过投喂高毒性的河豚鱼肝脏（毒性为5×103 MU/g），纵肋织纹螺在一段时间内能够快速累积少量的河豚毒素。当停止投喂有毒河豚鱼肝脏后，毒素含量会快速下降。而在曾导致中毒事件的拟半褶织纹螺中，投喂有毒河豚鱼的肝脏后，其体内毒素含量只有缓慢增加。但在投喂无毒的河豚鱼肝脏后，其毒性却出现了快速增加的现象，这与该地区野外样品的毒性变动状况类似。这些发现显示高毒性半褶、拟半褶织纹螺体内的河豚毒素应当不是食物链累积的结果，而可能是由其自身产生。并且，毒素含量的变化具有一定的生物节律，有可能与产卵、繁殖等自然节律相关。 通过对半褶、纵肋和拟半褶织纹螺的研究工作可以认为，产河豚毒素的细菌不是织纹螺体内河豚毒素的主要来源，并且毒素也不是来自其摄食的食物，推测可能主要是由织纹螺自身产生。织纹螺所表现出的河豚毒素含量的季节性变化，极有可能与产卵、繁殖等自然节律相关，这些发现为预防和管理食用织纹螺中毒事件提供了科学依据。但是，本研究并未完全阐明织纹螺体内河豚毒素的来源，对于织纹螺体内河豚毒素的确切来源以及河豚毒素的代谢和转化机制，还有待于更加深入地研究工作。

海鞘（Ciona savignyi）性腺组织细胞、血细胞和生殖干样细胞的体外培养

随着对海洋无脊椎动物免疫、发育以及细胞生物学等方面研究的需要，海洋无脊椎动物细胞培养日益受到关注。然而，由于海洋无脊椎动物的细胞代谢途径以及生长特性与陆生哺乳动物有很大差异，细胞培养难度较大,至今尚未有连续的细胞系建立。海鞘（Ciona savignyi）属于尾索动物亚门(Urochordata)，是典型的被囊类动物。作为无脊椎动物中进化地位较高等的一类动物，海鞘在免疫、胚胎发育、细胞学等各个领域对研究脊椎动物的系统发生都有着非常重要的作用。 本文分别以海鞘的性腺组织细胞和血细胞为材料，建立和优化海鞘细胞体外培养方法和条件；另外，还识别了成体海鞘性腺内的生殖样干细胞，并对其进行了体外培养和鉴定，为进一步开展海鞘细胞体外培养最终建立细胞系积累了资料。 首先比较了4种基础培养基L-15、M199、DMEM和RPMIl640 与各种培养添加物组合、不同温度和PH值对海鞘性腺组织细胞和血细胞的体外生长的影响。结果表明，20℃，pH6.8，M199基础培养基添加10%胎牛血清最适合海鞘性腺组织细胞生长。同时对海鞘性腺的分离方法即机械解离细胞法和酶学解离细胞法进行了比较，发现机械解离细胞法最适合海鞘性腺组织细胞的分离，分离得到的细胞贴壁率和成活率高。对于海鞘血细胞的培养，20℃，pH6.8条件下，L-15基础培养基并添加10%胎牛血清对血细胞的体外存活和生长效果较好。另外，还成功的利用原代培养的血细胞检测了海鞘肿瘤坏死因子配体家族成员（CsTL）基因的表达变化。 论文还研究了海鞘细胞培养中细菌污染的鉴定和控制方法。对于培养过程中的细菌污染，通过细菌分离、培养和纯培养发现两类菌株检出率较高，均为革兰氏阴性菌。经PCR 扩增16S rDNA 基因序列片断，结果显示这两类菌株分别属于弧菌属和施万氏菌属。药敏试验结果表明，亚胺培南和氯霉素等对受检施万氏菌的敏感度较高；而受检弧菌对氯霉素和环丙沙星的敏感度高。为控制培养中的微生物污染，比较了几种抗生素组合的使用效果，其中氯霉素和亚胺培南与双抗的抗生素组合有较好的抑菌效果并对培养细胞的贴壁和生长没有影响。然而，海鞘性腺组织细胞和血细胞在体外的传代培养并未取得成功，本论文对来源于成体海鞘性腺的生殖样干细胞进行了体外培养和鉴定。结果表明，成体海鞘性腺内存在生殖样干细胞，且在体外可以生长，繁殖并且可能具有分化潜能。体外培养的过程中，生长的细胞克隆明显具有类似胚胎干细胞的形态和基因表达特点。 本研究克隆了海鞘肿瘤坏死因子配体家族成员（CsTL）基因。CsTL全长995个核苷酸编码281个氨基酸。组织表达结果显示，CsTL在性腺组织的表达水平相对比较高，提示CsTL可能对海鞘性腺的发育或分化等起着一定的作用。利用昆虫杆状病毒表达系统表达纯化了CsTL蛋白。结果显示，重组CsTL对L929细胞显示了明显的细胞毒性作用，说明CsTL是具有生物学活性的重组蛋白。但是尝试用获得的重组CsTL蛋白作为培养添加物培养海鞘性腺组织细胞，但并未检测到CsTL对海鞘性腺组织细胞的生长或凋亡有任何影响。 总之，本文筛选到了适合海鞘性腺组织细胞和血细胞生长的培养基，并成功的将这两类细胞在体外进行了原代培养，并且虽然细胞传代未获成功，但为今后继续深入开展海鞘细胞培养研究奠定了基础，另外，海鞘生殖样干细胞的识别和培养也将为海洋无脊椎动物的细胞培养提供一条新途径。