海洋沉积物中深古菌的固碳潜能——基于DNA的稳定性同位素示踪研究

深古菌分布广泛、生境多样,是地球丰度最高的微生物之一,被认为在全球碳循环中扮演着重要角色.由于分离培养困难,深古菌的生理代谢功能尚不清楚.前期研究表明,深古菌具有降解木质素的功能;宏基因组分析也表明深古菌包含完整的无机碳固定途径(Wood-Ljungdahl pathway,WL途径),但尚未有直接的功能验证报道.本研究利用木质素与NaH¹³CO₃对海洋沉积物样品进行长期培养,探究深古菌固定无机碳的能力.结果表明:在添加了木质素的培养体系中深古菌持续增长,10个月后其丰度增幅高达15.3倍,并在古菌中占比由30%上升为80%.为查明培养体系内微生物的固碳功能,监测了总有机碳δ¹³C随时间的变化,发现两种添加了NaH¹³CO₃的体系的总有机碳在最初的一个月均有升高,随后在未添加木质素的体系中基本保持不变,而添加了木质素的体系中δ¹³C缓慢而持续地增长.对培养过程中不同处理和不同阶段的样品进行DNA提取,并通过超高速密度梯度离心获得不同浮力密度DNA后,利用Q...     

雨生红球藻β-胡萝卜素酮化酶基因的cDNA和基因组DNA克隆及序列分析

根据已知的β-胡萝卜素酮化酶(β-carotene ketolase,BKT)的cDNA序列设计引物,利用长距离PCR扩增获得了bkt基因的cDNA完整编码片段及基因组DNA片段,并对这些片断进行了序列测定。cDNA和基因组DNA比对结果表明该基因至少包括5个内含子,它们的剪切位点皆符合GU-AG规律。在比对结果中同时还发现一个19bp的短序列重复,该重复序列在bkt的cDNA和基因组DNA上都发生了多次重复,推测在BKT的表达调控中可能具有一定功能。另外,在序列比对过程中还发现本实验所获得的cDNA和基因组DNA序列与前人报道的cDNA序列之间都存在多处单碱基差异和19bp的短序列差异。     

虾夷扇贝TET基因在配子发生和早期发育中的表达模式

DNA甲基化参与调节动物配子发生和胚胎发育过程,TET基因负责DNA主动去甲基化,在基因印迹去除和细胞全能性获得中起重要作用。为了解海洋贝类配子发生和胚胎发育过程中DNA去甲基化如何发生,本研究以虾夷扇贝为研究对象,鉴定了其TET基因,并分析了该基因在性腺和胚胎幼虫发育中的表达变化。结果表明,虾夷扇贝基因组中含有1个TET基因(PyTET),该基因长39127 bp,包含10个外显子,编码1592个氨基酸,其蛋白具有完整的2OGFeDO超家族的加氧酶结构域。在性腺发育过程中,PyTET基因表达峰值出现在休止期精巢和增殖期卵巢,原位杂交结果显示其在精原细胞、精母细胞中均有表达,在卵母细胞中的表达明显高于卵原细胞;在早期发育过程中,其峰值出现在囊胚期。以上结果提示虾夷扇贝配子发生和胚胎发育过程中均发生了DNA主动去甲基化,这将有助于系统了解表观调控在贝类发育中的作用。     

Morphological and molecular evidence supports the first occurrence of two fishes,Siganus sutor(Valenciennes, 1835)and Seriolina nigrofasciata(Rüppell, 1829)(Actinopterygii:Perciformes), from marine waters of Odisha coast,Bay of Bengal,India

Marine ecosystems provide a wide variety of diverse habitats that frequently promote migration and ecological adaptation. The extent to which the geographic distribution of marine organism has reshaped by human activities remains underappreciated. The limitations intrinsic to morphology-based identification systems have engendered an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species,particularly those that are prone to overexploitation and/or market substitution. In the present study, however, an attempt has been taken to identify two locally adapted fish species, Siganus sutor(Valenciennes, 1835) and Seriolina nigrofasciata(Rüppell, 1829) of order Perciformes, which happens to be the first record in Odisha coast,Bay of Bengal. The diagnostic characteristics of Siganus sutor are: dorsal fin XIII-10, anal fin VII-9, pectoral fin 15,pelvic fin II-3, while that of Seriolina nigrofasciata dorsal fin VI-I-35, anal fin I-17, pectoral fin 16, pelvic fin 5. All COI barcodes generated in this study were matched with reference sequences of expected species, according to morphological identification. Bayesian and likelihood phylogenetic trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at the species level. The phylogeographic studies based on haplotype network and migration rates suggest that both the species were not panmitic and the high-frequency population distribution indicates successful migration. The result of this study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within a complex marine ecosystem.     

一种共生藻PCR模板DNA的快速制备法

采用Chelex-100树脂法制备了26个物种(分别属于腔肠动物门、海绵动物门和软体动物门)的共生藻以及一株纯培养共生藻的DNA样品。通过PCR扩增叶绿体23S rRNA基因片段获得了26个有效序列,测序比对结果显示获得的序列与扩增目的基因相符。该方法不仅样品消耗量小、操作时间短、设备需求低廉,而且可以避免使用有害化学试剂。这是一种高效、环保的DNA提取方法,可为共生藻分子研究提供技术参考。     

基于线粒体COI和16S rRNA基因的中国沿海相手蟹系统发育研究

相手蟹科的诸多种类因其形态极其相似成为方蟹总科分类中疑问较多的一个类群。通过对中国沿海相手蟹线粒体COI和16S rRNA基因序列进行分子系统发育分析,结果表明14种相手蟹COI和16S rRNA基因序列之间差异分别为5.7%～14.5%和1.5%～12.1%,均达到了种间差异水平。构建的系统发育树显示,14种相手蟹分别为独立有效物种,但分属于拟相手蟹属和近相手蟹属的4种拟相手蟹和3种近相手蟹,没有分别形成2个独立的支系,而是混合聚成一大支系。而属于螳臂相手蟹属的无齿螳臂相手蟹则首先与属于中相手蟹属的中华中相手蟹聚成一支,再与红螯螳臂相手蟹聚为一大支,表现出与形态分类的不一致。错综复杂的分子系统关系预示着相手蟹类为多系起源,也表明它们之间的种间关系乃至于属间关系尚有诸多问题有待进一步厘定。     

Phylogenetic analysis of Pectinidae(Bivalvia) based on the ribosomal DNA internal transcribed spacer region

The ribosomal DNA internal transcribed spacer (ITS) region is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, the molecular phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) was performed using the nucleotide sequences of the nuclear ITS region in nine species of this family. The sequences were obtained from the scallop species Argopecten irradians, Mizuhopecten yessoensis, Amusium pleuronectes and Mimachlamys nobilis, and compared with the published sequences of Aequipecten opercularis, Chlamys farreri, C. distorta, M. varia, Pecten maximus, and an outgroup species Perna viridis. The molecular phylogenetic tree was constructed by the neighbor-joining and maximum parsimony methods. Phylogenetic analysis based on ITS1, ITS2, or their combination always yielded trees of similar topology. The results support the morphological classifications of bivalve and are nearly consistent with classification of two subfamilies (Chlamydinae and Pectininae) formulated by Waller. However, A. irradians, together with A. opercularis made up of genera Amusium, evidences that they may belong to the subfamily Pectinidae. The data are incompatible with the conclusion of Waller who placed them in Chlamydinae by morphological characteristics. These results provide new insights into the evolutionary relationships among scallop species and contribute to the improvement of existing classification systems.     

Methods Comparison for Microsatellite Marker Development： Different Isolation Methods, Different Yield Efficiency

Microsatellite markers have become one kind of the most important molecular tools used in various researches. A large number of microsatellite markers are required for the whole genome survey in the fields of molecular ecology, quantitative genetics and genomics. Therefore, it is extremely necessary to select several versatile, low-cost, efficient and time- and labor-saving methods to develop a large panel of microsatellite markers. In this study, we used Zhikong scallop (Chlamys farreri) as the target species to compare the efficiency of the five methods derived from three strategies for microsatellite marker development. The results showed that the strategy of constructing small insert genomic DNA library resulted in poor efficiency, while the microsatellite-enriched strategy highly improved the isolation efficiency. Although the mining public database strategy is time- and cost-saving, it is difficult to obtain a large number of microsatellite markers, mainly due to the limited sequence data of non-model species deposited in public databases. Based on the results in this study, we recommend two methods, microsatellite-enriched library construction method and FIASCO-colony hybridization method, for large-scale microsatellite marker development. Both methods were derived from the mi-crosatellite-enriched strategy. The experimental results obtained from Zhikong scallop also provide the reference for microsatellite marker development in other species with large genomes.     

人工育珠对三角帆蚌(Hyriopsis cumingii)遗传基因影响的初步研究

采用酚-氯仿和试剂盒两种提取法对三角帆蚌怀珠群与非怀珠群各6个个体进行基因组DNA的提取,然后在优化RAPD(随机扩增多态性DNA)检测条件基础上,从80个随机引物中筛选出12个扩增较好且多态性强的引物进行RAPD扩增,产物通过水平板琼脂糖凝胶电泳和垂直板聚丙烯酰胺凝胶电泳两种方法进行检验并对DNA的多态性进行分析。结果显示在怀珠群和非怀珠群检测到的位点数、多态位点比例、平均Shannon多态性信息指数、平均Nei's基因多样性指数、群体内个体间平均遗传相似率和平均遗传距离分别为100、33%、0.1927、0.1324、0.904、0.096,95、47.37%、0.2711、0.1879、0.861、0.139,群体间的平均遗传距离为0.821,非怀珠群的变异性大于怀珠群;本研究还获得了两群体各自的特异扩增谱带及两群体间表达差异较大谱带,这些位点很可能是由人工育珠所引起。根据MEGA4.0软件的UPGMA和NJ程序构建的分子系统树可直观地将两群体分开。