霸王基因组RAPD优化条件的建立

实验以霸王20 d龄无菌籽苗为材料,使用CTAB法提取其基因组DNA,进行RAPD条件优化分析。通过单因子实验分别研究了Mg2+浓度、dNTPS浓度、Taq酶的浓度、引物浓度和模板DNA浓度对RAPD反应的影响,即在25 μL总反应体系中,Mg2+的适宜浓度为1.5~2.5 mmol\5L-1、dNTPs的适宜浓度为0.2~0.3 mmol\5L-1、随机引物的浓度以1.2 μmol\5L-1为宜、Taq酶的用量以2.0 U为佳、模板DNA的最适用量为50 ng。本研究的PCR扩增程序为94℃预变性8 min, 94℃变性1 min, 37℃退火1 min, 72℃延伸2 min,设40个循环,最后72℃保温6 min。     

大菱鲆mIgD重链基因的克隆与表达分析

利用RACE技术克隆获得大菱鲆(Scophthalmus maximus)膜结合型免疫球蛋白D(Membrane-bounded Ig,mIgD)重链基因的cDNA序列全长,该基因全长3 357bp,包含1个2 997bp的开放阅读框,编码999个氨基酸,基因结构为VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM。重链恒定区δ1~δ7氨基酸序列同源比对结果显示,其与庸鲽(78%)、鳜(71%)、牙鲆(68%)具有较高的同源性;多序列比对结果显示,大菱鲆mIgD的每个δ恒定区内均存在色氨酸与半胱氨酸保守位点。利用邻位相连法构建硬骨鱼类免疫球蛋白系统发育树,结果显示,鱼类IgD聚为一大支,大菱鲆IgD与庸鲽及牙鲆聚为一支。利用RT-PCR分析了大菱鲆IgD重链基因的组织差异表达,结果显示,其在外周血白细胞、脾脏、前肾及中肾组织中具有较高的表达。将大菱鲆腹腔注射福尔马林灭活鳗弧菌后,实时荧光定量PCR检测其前肾组织中mIgD重链基因应答表达量,结果显示,mIgD重链基因在注射鳗弧菌后呈显著下调趋势,在注射后8h时降至最低值,其相对表达量约为对照组的1/28,然后呈逐渐恢复上调趋势,在48h时mIgD相对表达量与对照组无显著差异。     

Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene （18S rDNA） libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18S rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.     

Use of species-specific PCR for the identification of 10 sea cucumber species

We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species. Ten reverse species-specific primers designed from the 16S rRNA gene, in combination with one forward universal primer, generated PCR fragments of ca. 270 bp length for each species. The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species. Amplification was observed in specific species only. The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber, and was proven to be a useful, rapid, and low-cost technique to identify the origin of the sea cucumber product.     

Comparative analysis of different uni- and multi-variate methods for estimation of vegetation water content using hyper-spectral measurements

Assessment of vegetation water content is critical for monitoring vegetation condition, detecting plant water stress, assessing the risk of forest fires and evaluating water status for irrigation. The main objective of this study was to investigate the performance of various mono- and multi-variate statistical methods for estimating vegetation water content (VWC) from hyper-spectral data. Hyper-spectral data is influenced by multi-collinearity because of a large number of (independent) spectral bands being modeled by a small number of (dependent) biophysical variables. Therefore, some full spectrum methods that are known to be suitable for analyzing multi-collinear data set were chosen. Canopy spectral reflectance was obtained with a GER 3700 spectro-radiometer (400–2400 nm) in a laboratory setting and VWC was measured by calculating wet/dry weight difference per unit of ground area (g/m²) of each plant canopy (n = 95). Three multivariate statistical methods were applied to estimate VWC: (1) partial least square regression, (2) artificial neural network and (3) principal component regression. They were selected to minimize the problem related to multi-collinearity. For comparison, uni-variate techniques including narrow band ratio water index (RWI), normalized difference water index (NDWI), second soil adjusted vegetation index (SAVI2) and transferred soil adjusted vegetation index (TSAVI) were applied. For each type of vegetation index, all two-band combinations were evaluated to determine the best band combination. Validation of the methods was based on the cross validation procedure and using three statistical indicators: R², RMSE and relative RMSE. The cross-validated results identified PLSR as the regression model providing the most accurate estimates of VWC among the various methods. The result revealed that this model is highly recommended for use with multi-collinear datasets ($R_{\text{CV}}^2=0.94$, RRMSE_CV = 0.23). Principal component regression exhibited the lowest accuracy among the multivariate models ($R_{\text{CV}}^2=0.78$, RRMSE_CV = 0.41).     

Comparison of Enterococcus density estimates in marine beach and bay samples by real-time polymerase chain reaction, membrane filtration and defined substrate testing

Currently, densities of Enterococcus in marine bathing beach samples are performed using conventional methods which require 24 h to obtain results. Real-time PCR methods are available which can measure results in as little as 3 h. The purpose of this study was to evaluate a more rapid test method for the determination of bacterial contamination in marine bathing beaches to better protect human health. The geometric mean of Enterococcus densities using Enterolert® defined substrate testing and membrane filtration ranged from 5.2 to 150 MPN or CFU/100 mL and corresponding qPCR results ranged from 6.6 to 1785 CCE/100 mL. The regression analysis of these results showed a positive correlation between qPCR and conventional tests with an overall correlation (r) of 0.71. qPCR was found to provide accurate and sensitive estimate of Enterococcus densities and has the potential to be used as a rapid test method for the quantification of Enterococcus in marine waters.     

Identification of genes expressed in juvenile Haliotis rufescens in response to different copper concentrations in the north of Chile under controlled conditions

This study reports molecular markers potentially associated with resistance or sensitivity to the impact of copper in juvenile red abalone, Haliotis rufescens, in the north of Chile under experimental conditions. Genomic analysis was made applying subtractive hybridization libraries (SSH) to identify genes up-and down regulated during cooper exposure in abalone over periods of 12 and 168 h exposed to 2.5 and 10 μg/L of Cu⁺². Results obtained from the SSH library revealed 368 different sequences regulated by copper, that correspond to eight major physiological functions. The validation of these sequences obtained by SSH as well as their expression kinetics were made by PCR in real time on 14 potential genes regulated by metal stress. This study provides information for the characterization of potential genomic markers that may be used in future environmental monitoring and to investigate new mechanisms of stress to copper in this commercially important marine species.     

南移养殖仿刺参（StichopusjaponicusSelenka）铁蛋白基因的克隆及表达特征分析

采用cDNA文库、RACE、荧光定量PCR和Westernblot技术,克隆了南移养殖仿刺参铁蛋白基因的全长序列,并对其表达特征进行了研究。结果表明,南移养殖仿刺参铁蛋白cDNA全长1222bp,包括187bp的5’UTR;513bp的3’UTR和编码173个氨基酸残基的522bp的开放阅读框。诸如铁结合序列标签和铁调...     

2011年夏季北极王湾细菌群落结构分析及浮游细菌丰度检测

采用PCR-DGGE方法,对2011年夏季北极王湾表层海水及沉积物细菌的群落分析结果表明,沉积物中的细菌多样性指数高于海水。深水站位(S1和S3)的沉积物细菌群落不但与浮游细菌群落存在差异,并且与浅水站位(S5)也存在差异。位于湾口、湾内的浮游细菌群落组成也存在一定差异。测序结果显示,王湾海洋细菌的多样性组成包括α-变形细菌、γ-变形细菌、δ-变形细菌、放线菌、拟杆菌及厚壁菌等类群。基于定量PCR方法的检测结果表明,位于湾口、湾内的浮游细菌丰度相似,但湾内玫瑰杆菌支系的丰度明显低于湾口。研究结果表明,与湾口相比,王湾湾内的细菌群落受陆源性淡水输入的影响明显,不但表现在细菌群落的多样性组成上,也表现在某些特定细菌类群的数量分布上。     

青岛崂山湾近海扇贝养殖区细菌多样性及环境因子分析

为研究日本囊对虾(Marsupenaeusjaponicus)耐高亚硝酸盐的分子调控机制,利用新一代高通量Illumina测序技术,对日本囊对虾在高亚硝酸盐胁迫下的肝胰腺进行转录组测序,通过对高质量序列的拼接组装以及功能基因的注释和分类,发掘与耐高亚硝酸盐相关性状的候选基因。实验结果表明:①10个文库共获得920785608个净读本,数据量为6.48G~7.34G。②Q30>93.07%。利用 Trinity软件组装后获得 46308条单基因簇,N50为1833bp。③与对照组相比,高亚硝酸盐组分别识别出593、606、1089、497和988个差异表达基因。④对 DEGs进行功能注释,得到与免疫和代谢相关的通路和基因。⑤采用 qPCR 对随机选择的9个差异基因(DPD、ABCH、ProPOb、ACADL、CYP2J、PAT4、BHMT、CLEC 和PEPCK)在高亚硝酸盐胁迫下的表达情况进行检测,其表达量与转录组测序技术(RNASequencing,RNA-seq)趋势一致。本研究结果丰富了对虾cDNA 数据库的信息,为日本囊对虾在高亚硝酸盐胁迫下的分子机制研究奠定了基础。