Soil biodiversity and DNA barcodes: opportunities and challenges

Soils encompass a huge diversity of organisms which mostly remains to be characterized due to a number of methodological and logistical issues. Nonetheless, remarkable progress has been made in recent years toward developing strategies to characterize and describe soil biodiversity, especially thanks to the development of molecular approaches relying on direct DNA extraction from the soil matrix.Metabarcoding can be applied to DNA from any environment or organism, and is gaining increasing prominence in biodiversity studies. This approach is already commonly used to characterize soil microbial communities and its application is now being extended to other soil organisms, i.e. meso- and macro-fauna.These developments offer unprecedented scientific and operational opportunities in order to better understand soil biodiversity distribution and dynamics, and to propose tools and strategies for biodiversity diagnosis. However, these opportunities also come with challenges that the scientific community must face. Such challenges are related to i) clarification of terminology, (ii) standardisation of methods and further methodological development for additional taxonomic groups, (iii) development of a common database, and (iv) ways to avoid waste of information and data derived from metabarcoding. In order to facilitate common application of metabarcoding in soil biodiversity assessment, we discuss these opportunities and challenges and propose solutions towards a more homogeneous framework.     

BPA不影响卵母细胞减数分裂相关基因Dazl的甲基化

为了探讨环境雌激素BPA对小鼠卵母细胞减数分裂相关基因Dazl甲基化的影响,本研究通过给孕鼠饮用含有BPA的水方式使胎鼠在发育过程中接触BPA,利用重亚硫酸盐测序法,分析了胎鼠生殖嵴卵母细胞不同发育时期Dazl甲基化水平的变化。结果显示:Dazl在减数分裂期间处于低甲基化水平,无论对照组或处理组均低于10%,说明Dazl的低甲基化对维持减数分裂的正常进行有重要作用;对照组与处理组的甲基化水平相当,差异不显著,说明本研究的BPA浓度不影响卵母细胞Dazl的甲基化水平。     

Immunogenicity of multiple-epitope antigen gene of HCV carried by novel biodegradable polymers

In order to develop a promising vaccine candidate utilizing a combined approach to induce both antibody production and T-cell activity, the DNA fragment containing MA of HCV with five conserved epitopes was synthesized. Two types of HCV vaccine candidates (the DNA type and DNA/polymers) were constructed using MA. PLA-PEG-PLA and PLGA-PEG-PLGA were synthesized and used as micelles with encapsulated plasmid pcDNA3.1(+)-MA. The preparation of copolymers, the cloning and analysis of recombinant plasmid DNA, in vitro expression, and immunogenicity in transgenic mice were evaluated in detail. The results indicated that even single immunization and oral immunization with DNA/polymers achieved satisfying immune responses in vivo tests. As biodegradable and nontoxic triblock copolymers, the novel copolymers demonstrated a great advantage, as they made long-term and single-immunizing vaccines possible; in addition, the copolymers showed a better adjuvant effect and scarcely any side effects.     

甜菜DAMD-PCR体系的建立及优化

为了建立甜菜DAMD扩增体系,以期利用DAMD引物应用于甜菜品种指纹图谱的构建及分子标记辅助育种。本实验利用单因素变量的方法对甜菜DAMD体系进行优化。同时选用12个甜菜品种,利用优化的体系对25条DAMD引物进行扩增。获得甜菜的最适DAMD体系:总体积为20μL,包含模板DNA 10~80 ng、0.75 U的DNA聚合酶、0.2μL的d NTPs(2.5 mmol/L each)以及2.0μL的引物(10μmol/L)。同时25条引物均扩增出了清晰条带,除了个别引物多态性较差外,其余引物多态性都非常的丰富,其中引物62H(-)就可以把实验中用到的12个甜菜品种全部区分开。由此可见,DAMD引物的扩增效率很高,并且扩增结果稳定,条带清晰,非常适合甜菜品种指纹图谱的构建及遗传多样性分析。     

虹鳟杂交胚胎(2n♀×3n♂)的发育状况及其DNA倍性分析

雄性三倍体虹鳟(Oncorhyncus mykiss)可以发育至生理成熟并表现出较强的雄性副性征,性腺发育基本正常；雌性三倍体虹鳟性腺几乎不发育,并且存在着类雄性化的发育趋势.本研究拟通过对虹鳟杂交胚胎(2n♀×3n♂)发育状况及其DNA倍性的分析,进而确认三倍体虹鳟是否具有与二倍体虹鳟类似的生育能力并分析其原因.选取经两个世代家系选育的虹鳟优良品系二倍体雌性虹鳟作为母本,以热休克方法人工诱导的优质雄性三倍体虹鳟为父本,常规人工采集精卵授精,获得三倍体雄性与二倍体雌性杂交胚胎,观察杂交胚胎的发育进程,通过流式细胞仪检测杂交胚胎的DNA含量,进而与二倍体雌雄交配获得的胚胎对照确定杂交胚胎的倍性.结果表明,杂交胚胎的受精率、发眼率和孵化率均显著地低于对照组,杂交胚胎多数在发眼期之前死亡,破膜的胚胎在仔鱼期上浮前全部死亡.杂交胚胎的DNA含量分布范围较大,大致可以分为两个水平,接近84％的胚胎的DNA含量介于二倍体与三倍体之间,约为对照组DNA含量的2.5倍；约有16％的胚胎的DNA含量在三倍体和四倍体之间.本研究认为,染色体非整倍体化可能会引起杂交胚胎细胞核质不相容,进而导致基因表达调控紊乱,从而使杂交胚胎不能正常发育,无法完成由母体卵黄供给的内源营养向外源营养的转换,成为导致杂交胚胎发育过程中大量死亡,以及杂交仔鱼在破膜之后开始进食之前全部死亡的一个重要原因.     

DNA折纸术的研究进展

为了帮助广大研究者在短时间内了解DNA折纸术的最新研究现状,掌握DNA折纸术相关软件的应用,本论文将DNA折纸术的相关内容进行了归纳。首先介绍了DNA折纸术的起源与基本原理;然后综述了近几年国内外的研究进展与取得的成果;接下来对DNA折纸术相关软件-Tiamat和Cadnano的应用和功能进行了简单的介绍,并对不同的软件进行了比较;最后对DNA折纸术的应用前景进行了展望。DNA折纸术虽然取得了一定的研究进展,但其未来的发展和应用前景将更广阔。     

Response of the soil fungal community to multi-factor environmental changes in a temperate forest

Both environmental and climatic changes are known to influence soil microbial biomes in terrestrial ecosystems. However, there are limited data defining the interactive effects of multi-factor environmental disturbances, including N-deposition, precipitation, and air temperature, on soil fungal communities in temperate forests. A 3-year outdoor pot experiment was conducted to examine the temporal shifts of soil fungal communities in a temperate forest following N-addition, precipitation and air temperature changes. The shifts in the structure and composition of soil fungal communities were characterized by denaturing gradient gel electrophoresis and DNA sequencing. N-addition regimen induced significant alterations in the composition of soil fungal communities, and this effect was different at both higher and lower altitudes. The response of the soil fungal community to N-addition was much stronger in precipitation-reduced soils compared to soils experiencing enhanced precipitation. The combined treatment of N-addition and reduced precipitation caused more pronounced changes in the lower altitude versus those in the higher one. Certain fungal species in the subphylum Pezizomycotina and Saccharomycotina distinctively responded to N fertilization and soil water control at both altitudes. Redundancy discrimination analysis showed that changes in environmental factors and soil physicochemical properties explained 43.7% of the total variability in the soil fungal community at this forest ecosystem. Variations in the soil fungal community were significantly related to the altitude, soil temperature, total soil N content (TN) and pH value (P < 0.05). We present evidence for the interactive effects of N-addition, water manipulation and air temperature to reshape soil fungal communities in the temperate forest. Our data could provide new insights into predicting the response of soil micro-ecosystem to climatic changes.     

鸡脂肪组织TCF21基因启动子区DNA甲基化与其表达的关系

旨在研究鸡脂肪组织中TCF21基因启动子区DNA甲基化水平与其表达的关系。以东北农业大学高、低腹脂双向选择品系（简称高、低脂系）第24世代7周龄肉鸡为试验材料，利用RT-qPCR检测高、低脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平；利用生物信息学和双荧光素酶报告系统分析TCF21基因启动子的结构与功能；利用Sequenom MassARRAY飞行质谱检测高、低脂系肉鸡腹部脂肪组织中TCF21基因启动子区CpG位点的甲基化水平；利用CpG甲基转移酶处理TCF21启动子报告基因质粒，分析DNA甲基化对TCF21基因启动子活性的影响。结果显示，高脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平极显著高于低脂系（P<0.001）；TCF21基因的启动子区存在40个CpG位点，且在启动子的近端和远端均有分布，但不存在CpG岛；将TCF21基因的启动子划分为5个功能区域，分别为R1区域（-2 000~-1 500 bp）、R2区域（-1 500~-1 000 bp）、R3区域（-1 000~-500 bp）、R4区域（-500~-200 bp）和Core区域（-200~-100 bp）；高脂系R2、R3和R2+R3区域的DNA甲基化水平显著或极显著高于低脂系（P<0.05或P<0.001）；R2、R3、R2+R3区域的DNA甲基化水平与TCF21基因mRNA表达水平呈显著正相关（R2区域：r=0.438，P<0.05；R3区域：r=0.371，P<0.05；R2+R3区域：r=0.489，P<0.05）；R2区域的DNA甲基化显著抑制其转录活性（P<0.05）。综上所述，TCF21基因在高、低脂系肉鸡腹部脂肪组织中的表达水平主要与其启动子R2区域的DNA甲基化水平有关。     

一种用于桑RAD-Seq高通量测序的基因组DNA提取方法

[目的]筛选一种能够得到高质量桑树基因组DNA的方法。[方法]采用天根试剂盒法(Plant Genomic DNA Kit离心柱型),并稍加改进,从11个桑种的新鲜嫩叶中提取DNA。[结果]该方法简单快速,不需要复杂仪器,且能有效地除去桑叶中的多糖、多酚、蛋白质等杂质,得到的DNA能够满足RAD-Seq高通量的测序要求。[结论]建立了一种用于桑RAD-Seq高通量测序的基因组DNA提取方法。     

Devastation of sesame (Sesamum indicum L.) crops in different agroclimatic zones of India by genetically diverse subgroups of phytoplasma

The sesame crop is highly susceptible to infection by phytoplasmas, a class of cell wall-less plant pathogenic bacteria (Mollicutes), which is responsible for widespread loss of sesame crops in both North and South India in recent years. Therefore, characterizing the pathogen population is required before the control measures can be devised and implemented. With molecular tools based on nested polymerase chain reaction (PCR) assays, sequencing, restriction profiling, and phylogenetic analysis, phyllody-affected sesame plants collected from nine different states of India were found to be infected by phytoplasmas belonging to two 16Sr groups, namely 16SrI and II. Two subgroups of phytoplasma −16SrI-B and 16SrII-D— were prevalent in symptomatic sesame samples collected from North India, whereas phytoplasma of only the 16SrII group was found in South India. However, the latter samples were diverse, belonging to three different subgroups (16SrII-A, II-C, and II-D). In addition, yearly phyllody-affected sesame samples from Delhi for 4 consecutive years (2007–2010) showed variation in the infecting phytoplasma: the subgroup 16SrII-D was detected in samples collected in 2007, and 16SrI-B was predominantly found in the samples collected in the subsequent years. The study also provides molecular evidence for the association between 16SrI-B phytoplasma and different symptoms in sesame crops such as fasciation, little leaf, and stunting. This is the first study to report the association of the phytoplasma subgroups 16SrII-A and II-D with sesame crops in India. This study provides a baseline for designing specific detection and molecular analysis strategies for quarantine purposes. It also highlights the need for examining the dynamics of seasonal or location-specific variation in vector populations to determine the pattern of infection outbreaks.