基于CRISPR/Cas9基因编辑技术的本科教学实验体系的创建与实践

CRISPR/Cas9是新兴的基因编辑技术,在生命科学研究中发挥着重要的作用。将它引入本科生的实验教学,使本科生了解这项前沿科研技术很有意义。我们创建了一个基于CRISPR/ Cas9技术的本科教学实验体系。该实验体系侧重CRISPR/Cas9技术在哺乳动物细胞中的应用,选用一株基因组上被插入mCherry基因的小鼠胚胎成纤维细胞为实验材料,命名为STO-82。首先设计靶向mCherry的sgRNA,构建CRISPR-Cas9/sgRNA共表达质粒。经测序验证无误后,转染到STO-82细胞。采用流式细胞仪分析检测mCherry阴性和阳性两群细胞,分选出阴性单细胞并扩大培养。最后用测序检验单克隆细胞中靶标DNA序列的编辑情况。结果显示,靶位点有插入或缺失突变,说明体系创建成功。该实验体系将sgRNA设计、CRISPR-Cas9/sgRNA共表达质粒的构建、细胞转染、单细胞分选、单克隆细胞培养、测序序列分析等内容融合为一个综合实验,用于高年级本科生的实验教学。根据实际情况,将教学实践内容分解分块教学,也可以做完整性项目教学。本教学实践采用10人左右的小班分块教学,2人一组,经过3个班（共13组）的实践,绝大部分学生都能完成实验,得到预期结果。通过这个实验,学生加深了对CRISPR/Cas9技术的原理和实验流程的理解,锻炼了实验操作能力和严谨的科研思维,也使学生对该技术的医疗应用风险有了一些认识。     

辽宁省三级保护植物木根黄芩考证及其与京黄芩比较形态学研究

木根黄芩(Scutellaria planipes Nakai et Kitag.)是唇形科黄芩属(Scutellaria L.)多年生草本,目前仅发现在辽宁省凌源市境内有零星分布,被辽宁省列为三级保护植物。该种发表于1934年,但《中国植物志》、Flora of China、《中国生物物种名录》均将其定为京黄芩(Scutellaria pekinensis Maxim.)的异名。为了阐明二者的差异性,本文首先对木根黄芩的原始文献、模式标本、模式产地、历史分布区及现今发现地进行考证,其次对二者进行差异比较研究,认为木根黄芩与京黄芩无论在分布范围和生境上,还是在宏观外部形态和微观花粉粒形态上均存在明显差异,确定木根黄芩为独立种,同时还对木根黄芩的果实进行形态补充描述。本文确定木根黄芩独立种的地位,对保护野生木根黄芩具有重要意义,同时对其药用价值的进一步研究提供了更为清晰的样本。     

Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A -related kinase 5

BACKGROUNDTubulins, building blocks of microtubules, are modified substrates of diverse post-translational modifications including phosphorylation, polyglycylation and polyglutamylation. Polyglutamylation of microtubules, catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family, can regulate interactions with molecular motors and other proteins. Due to the diversity and functional importance of microtubule modifications, strict control of the TTLL enzymes has been suggested.AIMTo characterize the interaction between never in mitosis gene A-related kinase 5 (NEK5) and TTLL4 proteins and the effects of TTLL4 phosphorylation.METHODSThe interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5 (a.a. 260–708) as bait and confirmed by immunoprecipitation. The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced.RESULTSHere, we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity. We further show that NEK5 can also interact with TTLL5 and TTLL7. The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4. The same effects were observed after the expression of the catalytically inactive form of NEK5. This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues.CONCLUSIONOur results demonstrate, for the first time, the regulation of TTLL activity through phosphorylation, pointing to NEK5 as a potential effector kinase. We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.     

Evidence for a synergistic effect of post‐translational modifications and genomic composition of eEF‐1α on the adaptation of Phytophthora infestans

Genetic variation plays a fundamental role in pathogen''s adaptation to environmental stresses. Pathogens with low genetic variation tend to survive and proliferate more poorly due to their lack of genotypic/phenotypic polymorphisms in responding to fluctuating environments. Evolutionary theory hypothesizes that the adaptive disadvantage of genes with low genomic variation can be compensated for structural diversity of proteins through post‐translation modification (PTM) but this theory is rarely tested experimentally and its implication to sustainable disease management is hardly discussed. In this study, we analyzed nucleotide characteristics of eukaryotic translation elongation factor‐1α (eEF‐lα) gene from 165 Phytophthora infestans isolates and the physical and chemical properties of its derived proteins. We found a low sequence variation of eEF‐lα protein, possibly attributable to purifying selection and a lack of intra‐genic recombination rather than reduced mutation. In the only two isoforms detected by the study, the major one accounted for >95% of the pathogen collection and displayed a significantly higher fitness than the minor one. High lysine representation enhances the opportunity of the eEF‐1α protein to be methylated and the absence of disulfide bonds is consistent with the structural prediction showing that many disordered regions are existed in the protein. Methylation, structural disordering, and possibly other PTMs ensure the ability of the protein to modify its functions during biological, cellular and biochemical processes, and compensate for its adaptive disadvantage caused by sequence conservation. Our results indicate that PTMs may function synergistically with nucleotide codes to regulate the adaptive landscape of eEF‐1α, possibly as well as other housekeeping genes, in P. infestans. Compensatory evolution between pre‐ and post‐translational phase in eEF‐1α could enable pathogens quickly adapting to disease management strategies while efficiently maintaining critical roles of the protein playing in biological, cellular, and biochemical activities. Implications of these results to sustainable plant disease management are discussed.     

染色质重塑调控生物温度适应性的研究进展

温度是限制物种适应性分布的重要环境因子,对极端环境温度的耐受性决定生物分布和扩散范围,而表观遗传可以提供快速的响应机制,促使生物快速适应极端环境温度。染色质重塑作为表观遗传的重要组成部分之一,其可以通过调控胁迫相关基因的表达从而促进生物适应不良环境条件。本文主要阐述了染色质重塑复合物的分类、组成和染色质重塑的方式,梳理了染色质重塑在生物温度适应性中的研究进展,提出染色质重塑在生物适应不良环境温度过程中发挥重要作用,并对未来染色质重塑与温度适应性研究提出建议。     

Efficient rescue of threatened biodiversity data using reBiND workflows

Abstract

Biodiversity data generated in the context of research projects often lack a strategy for long-term preservation and availability, and are therefore at risk of becoming outdated and finally lost. The reBiND project aims to develop an efficient and well-documented workflow for rescuing such data sets. The workflow consists of phases for data transformation into contemporary standards, data validation, storage in a native XML database, and data publishing in international biodiversity networks. It has been developed and tested using the example of collection and observational data but is flexible enough to be transferred to other data types and domains.     

A general synthetic method toward uridylylated picornavirus VPg proteins

Small proteins called viral protein genome‐linked (VPg), attached to the 5′‐end of the viral RNA genome are found as common structure in the large family of picornaviruses. The replication of these viruses is primed by this VPg protein linked to a single uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre‐uridylylated tyrosine building block in an on‐line solid phase‐based approach. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.