基因与学习记忆调控

近年,有关基因与学习记忆调控的研究引人注目。实验显示：（１）ｃ－ｆｏｓ等即刻早期基因（ＩＥＧｓ）的激活是学习记忆形成的必要条件,长时程增强（ＬＴＰ）诱出的同时也伴有ＩＥＧｓ的激活;（２）应用转基因技术获得α－ＣａＭＫⅡ、Ｆｙｎ和Ｎ－ＣＡＭ等基因突变的小鼠,均表现明显的空间学习记忆障碍,以及ＬＴＰ诱导和维持障碍;（３）果蝇单基因（ｄｎｃ,ｒｕｔ等）突变体的学习记已能力明显下降,其机制与突触可塑性的改     

鸣禽发声学习记忆与即刻早期基因

鸣禽受到声音信号的刺激或自身表现出发声行为时,脑内即刻早期基因(immediate early gene,IEG)能迅速被激活而表达.其中zenk、c-fos和c-jun表达的脑区及水平与鸟在鸣唱时神经元的活动区域及活动程度相一致,暗示IEG在鸣禽发声学习记忆中起重要作用.     

果蝇长时程记忆缺陷型突变体的鉴定

长时程记忆作为依赖蛋白合成的记忆组分,对于了解高等认知活动的分子机制有着重要意义.与此同时,细胞粘连分子作为影响突触可塑性的重要因子在学习与记忆研究领域也日益得到重视.为探索作用于长时程记忆的细胞粘连分子,利用P因子在果蝇基因组随机插入制造突变体,并通过大规模行为筛选得到了一个可能的长时程记忆突变体RUO. 测序结果表明,突变体RUO的P因子位于果蝇中selectin超家族对应的furrowed同源基因功能片段和未知功能的CG1806基因编码片段之间,且更靠近furrowed片段.RT-PCR结果和互补遗传学实验均表明,突变体RUO主要影响furrowed基因的表达.为了进一步确认furrowed基因与长时程记忆的相关性,引入已知的furrowed基因突变体fw1.结果表明,fw1同样具有长时程记忆缺陷,同时具备正常的学习能力.荧光共聚焦扫描成像显示,该基因特异性的表达在果蝇大脑两个对称的未知神经元中.此项工作不仅证明了furrowed基因在果蝇长时程记忆中的重要作用,而且在解剖学上揭示了果蝇神经系统中可能参与长时程记忆形成的新的神经元.     

斑马鱼消化器官发育缺陷突变体的遗传筛选

目的正向遗传筛选斑马鱼肝脏、肠和胆囊发育缺陷突变体。方法 ENU诱变野生型斑马鱼并开展经典的F2代筛选,以lfabp为探针的全胚原位杂交、BES-H2O2-Ac荧光染料分别检测斑马鱼早期胚胎肝脏、肠和胆囊的表型。结果在128个突变基因组中筛选获得了源自14个F2家族的斑马鱼消化器官发育缺陷突变体品系23个,并按表型划分为6类。结论斑马鱼肝脏、肠和胆囊的发育调控机制有相似性和差异性。     

拟南芥耐盐突变体stg2的筛选

采取在高盐平板上萌发的方法,对一个雌激素诱导激活型拟南芥突变体库进行了耐盐突变体的筛选,最终得到了2株稳定的耐盐突变体。本文中对其中的一株耐盐突变体,命名为stg2(salt tolerance during germination 2),进行了研究。遗传实验表明它的耐盐特性是受雌激素诱导的,是功能获得型的耐盐突变体。本实验中还探讨了stg2突变体的筛选过程及耐盐生理特点。     

糜子EMS突变体库构建和突变体筛选北大核心CSCD

糜子(Panicum miliaceum L.)具有抗旱、耐瘠、适应性广、生育期短等特点,是我国北方地区的杂粮作物。糜子高产品种不耐水肥、易倒伏,锄草、间苗费时费工的问题严重影响着农民对糜子生产的积极性和收入。EMS(甲基磺酸乙酯)化学诱变方法成本低,操作简单,诱变率高,是作物育种的重要方法。本试验选用糜子品种伊选大红糜进行EMS化学诱变而构建突变体库,对获得的2333个M2材料进行突变频率和全生育期表型多样性等特征的分析。结果显示,所构建的糜子突变体库材料变异类型非常丰富,共发现104个形态性状突变株系,突变频率为4.5%,包括叶色、叶型、株高、生育期、育性、穗型、种皮颜色等性状,该突变体库的构建将为糜子功能基因组学研究和育种提供丰富的突变体资源和新型种质。     

抗拿捕净晋谷21突变体的筛选和分子鉴定

谷子(Setaria italica(L.)P.Beauv.)是我国重要的杂粮作物,具有抗旱耐贫瘠、水分利用率高、适应性广、营养丰富等特点,但谷子田间除草一直制约着谷子的集约化栽培和有机旱作产业规模化的推广与发展.为快速选育抗除草剂谷子品种,本试验以晋谷21突变体M2群体为试验材料,通过喷施0.33％拿捕净除草剂对晋谷...     

裸燕麦EMS突变体库筛选与分析

燕麦是重要的粮饲兼用作物,构建燕麦EMS突变体库对燕麦功能基因组学研究和遗传改良有重要意义。本试验利用化学诱变剂甲基磺酸乙酯(EMS,ethyl methane sulfonate)处理燕麦品种花早2号,获得了4083株M1材料;对其中2000个单株种植了M2株行,进行全生育期调查,鉴定其表型变化;对2份黄化苗突变材料种植了M3家系,进行相关突变性状的稳定性验证。结果表明,燕麦经EMS处理后代变异巨大,在M2发现表型突变材料196份,变异率为9.8%,变异类型非常丰富,包括幼苗习性、叶片性状、分蘖、株高、穗部形态及成熟期等突变株系。M3证实突变的黄化苗特性可以稳定遗传。本研究建立了燕麦EMS诱变体系,获得的燕麦变异类型丰富,为燕麦功能基因组学研究和燕麦遗传改良奠定了材料基础。     

青饲玉米有益突变体筛选的研究

自60年代以来,由于植物细胞组织培养技术的迅速发展,已可获得玉米的再生植株[1～3],其后又发现再生植株中广泛存在着无性系变异,这种遗传变异的利用被确认为一种新的改良作物的重要手段[3～5]。本研究采用体细胞无性系变异突变体筛选技术,选育出生育期较短、分蘖性强、又能保持紫多114原有优良性状的自交系,组配出新的杂交组合,取得了预期的效果。1　材料和方法1.1　实验材料以多秆多穗玉米(Zeamays)自交系紫多114为材料,取幼穗、幼胚、幼茎、幼叶、叶鞘和胚轴等不同外植体培养并进行了比较研究。幼穗为不同发育时期的雄穗,剥掉外圈的叶片,…     

Analysis of a zebrafish behavioral mutant reveals a dominant mutation in atp2a1/SERCA1

Zebrafish embryos demonstrate robust swimming behavior, which consists of smooth, alternating body bends. In contrast, several motility mutants have been identified that perform sustained, bilateral trunk muscle contractions which result in abnormal body shortening. Unlike most of these mutants, accordion (acc)^dta5 demonstrates a semidominant effect: Heterozygotes exhibit a distinct but less severe phenotype than homozygotes. Using molecular‐genetic mapping and candidate gene analysis, we determined that acc^dta5 mutants harbor a novel mutation in atp2a1, which encodes SERCA1, a calcium pump important for muscle relaxation. Previous studies have shown that eight other acc alleles compromise SERCA1 function, but these alleles were all reported to be recessive. Quantitative behavioral assays, complementation testing, and analysis of molecular models all indicate that the acc^dta5 mutation diminishes SERCA1 function to a greater degree than other acc alleles through either haploinsufficient or dominant‐negative molecular mechanisms. Since mutation of human ATP2A1 results in Brody disease, an exercise‐induced impairment of muscle relaxation, acc^dta5 mutants may provide a particularly sensitive model of this disorder. genesis, 48:354–361, 2010. © 2010 Wiley‐Liss, Inc.     

Methods for reverse genetic screening in zebrafish by resequencing and TILLING

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.     

Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice

Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS), a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.     

Characterization of a weak allele of zebrafish cloche mutant

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche(172) (clo(172)) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo(172) mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo(172) mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo(s5) mutant. In contrast, primitive myeloid cells were totally lost in clo(172) mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo(172) mutant, confirmed by the dramatic decrease of lyc in clo(172)runx1(w84x) double mutant. Collectively, the clo(172) mutant is a weak allele compared to the clo(s5) mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene.     

Developmental toxicity screening in zebrafish

Given the ever-increasing toxic exposure ubiquitously present in our environment as well as emerging evidence that these exposures are hazardous to human health, the current rodent-based regulations are proving inadequate. In the process of overhauling risk assessment methodology, a nonrodent test organism, the zebrafish, is emerging as tractable for medium- and high-throughput assessments, which may help to accelerate the restructuring of standards. Zebrafish have high developmental similarity to mammals in most aspects of embryo development, including early embryonic processes, and on cardiovascular, somite, muscular, skeletal, and neuronal systems. Here, we briefly describe the development of these systems and then chronicle the toxic impacts assessed following chemical exposure. We also compare the available data in zebrafish toxicity assays with two databases containing mammalian toxicity data. Finally, we identify gaps in our collective knowledge that are ripe for future studies.     

pyewacket, a new zebrafish fin pigment pattern mutant

Many mutants that disrupt zebrafish embryonic pigment pattern have been isolated, and subsequent cloning of the mutated genes causing these phenotypes has contributed to our understanding of pigment cell development. However, few mutants have been identified that specifically affect development of the adult pigment pattern. Through a mutant screen for adult pigment pattern phenotypes, we identified pyewacket (pye), a novel zebrafish mutant in which development of the adult caudal fin pigment pattern is aberrant. Specifically, pye mutants have fin melanocyte pigment pattern defects and fewer xanthophores than wild-type fins. We mapped pye to an interval where a single gene, the zebrafish ortholog of the human gene DHRSX, is present. pye will be an informative mutant for understanding how xanthophores and melanocytes interact to form the pigment pattern of the adult zebrafish fin.