不同性别类型苦瓜基因组DNA提取及RAPD标记初步研究

采用改良的CTAB法成功地提取了不同性别类型苦瓜品种的基因组DNA,作为PCR扩增反应模板进行RAPD分析。结果表明,从200个10bp随机寡核苷酸引物中筛选到2个引物S30和S66,其可以在全雌性品种X-黑-d-d稳定地扩增出特异性条带,为下一步克隆苦瓜性别决定基因、探索苦瓜性别决定机理提供了试验依据。至于该特异性标记是否与苦瓜雌性性别有关,还有待进一步分析验证。     

鸡基因组DNA不同提取方法的比较研究

实验用优化煮沸法提取鸡血液基因组DNA,经核酸蛋白定量仪检测,DNA的OD260/OD280达1.77±0.06,证明所提DNA质量较好,用于PCR扩增,可以得到满意的扩增效果。与酚-氯仿法相比,优化煮沸法避免了用氯仿、酚等剧毒试剂,且节约了时间和成本,是一种较好的提取基因组DNA的方法。     

甘薯卷叶病毒江苏分离物基因组全长序列测定及其外壳蛋白基因在大肠杆菌中的表达

为了制备甘薯卷叶病毒(SPLCV)的特异性抗体,克隆了甘薯卷叶病毒江苏分离物SPLCV(JS∶XZ∶3-2)的全长基因,并对其基因结构及分子变异情况进行分析,同时对外壳蛋白(CP)基因进行了克隆和表达。利用PCR方法扩增并克隆了SPLCV(JS∶XZ∶3-2)的全基因组,测序分析表明,SPLCV(JS∶XZ∶3-2)基因组全长为2 834bp,含有6个开放阅读框架,与已发表的SPLCV其他分离物相比,全基因组核苷酸序列相似性为82.7%～94.4%。其中,CP基因由765个核苷酸组成,共编码254个氨基酸残基。SPLCV CP基因核苷酸序列与其他分离物的相似性在89.2%～90.6%,其中与SPLCV美国分离物(HQ333135)的相似性最高,为90.6%;与日本分离物(AB433788)的相似性最低,为89.2%;与中国大陆分离物(FJ515896)CP基因的核苷酸序列相似性为90.2%。将SPLCV CP基因克隆到原核表达载体pGEX-4T-3上,获得重组质粒,经IPTG诱导表达、SDS-PAGE分析得到了大小约为54.3kD的融合蛋白条带,说明CP基因在大肠杆菌中得到了高效表达。     

阿维链霉菌BAC文库的构建及分析

为进一步阐明阿维菌素的生物合成路径和调控途径,提高阿维菌素的产量,采用BamHⅠ限制性内切酶酶解阿维链霉菌基因组DNA的方法,构建了阿维链霉菌的细菌人工染色体(BAC)文库。该文库共包含2 304个克隆,平均插入片段为101kb,空载率约9%,覆盖了该菌基因组的25.9倍。该文库的成功构建可以为其他微生物尤其是基因组具有磷硫酰化修饰的微生物的文库构建提供参考。     

猪圆环病毒复制与转录模式研究进展

猪圆环病毒(PCV)是一种单股、环状DNA病毒,有两种血清型,其中PCV-2是引起断奶仔猪多系统衰竭综合征的重要病原,给世界养猪业造成了巨大的经济损失.文章主要从PCV-1和PCV-2的基因组结构、复制起始区的结构与功能、复制相关蛋白的结构与功能、转录图谱、各转录物的剪接方式及主要转录物的转录效率等方面进行了综述,同时也对二者的复制与转录模式的异同点进行了比较.这些结果对PCV-1和PCV-2毒力差异的分子机制研究具有一定的借鉴意义,同时可为抗PCV药物的研发提供一定的理论依据.     

用改进的酚-氯仿法提取鱼类基因组DNA效果的分析

提取鱼类基因组DNA的研究报道并不多见,效果也各有差异。本文以泰山螭霖鱼、东平湖鲤鱼、东平湖鲫鱼为材料,采用改进的酚-氯仿法提取基因组DNA,并进行了紫外分光光度、琼脂糖凝胶电泳、微卫星PCR扩增等方法的鉴定。结果表明,本方法提取的基因组DNA浓度在0.9μg/μL~3.25μg/μL之间,A260/A280比值在1.79~1.87之间,电泳条带整齐明亮,适合微卫星PCR扩增,且整个操作过程使用试剂较少,简单易行。     

Prediction of response to marker-assisted and genomic selection using selection index theory

Selection index methods can be used for deterministic assessment of the potential benefit of including marker information in genetic improvement programmes using marker-assisted selection (MAS). By specifying estimates of breeding values derived from marker information (M-EBV) as a correlated trait with heritability equal to 1, it was demonstrated that marker information can be incorporated in standard software for selection index predictions of response and rates of inbreeding, which requires specifying phenotypic traits and their genetic parameters. Path coefficient methods were used to derive genetic and phenotypic correlations between M-EBV and the phenotypic data. Methods were extended to multi-trait selection and to the case when M-EBV are based on high-density marker genotype data, as in genomic selection. Methods were applied to several example scenarios, which confirmed previous results that MAS substantially increases response to selection but also demonstrated that MAS can result in substantial reductions in the rates of inbreeding. Although further validation by stochastic simulation is required, the developed methodology provides an easy means of deterministically evaluating the potential benefits of MAS and to optimize selection strategies with availability of marker data.     

Comparison of genomic and traditional BLUP-estimated breeding value accuracy and selection response under alternative trait and genomic parameters

Accuracy of prediction of estimated breeding values based on genome-wide markers (GEBV) and selection based on GEBV as compared with traditional Best Linear Unbiased Prediction (BLUP) was examined for a number of alternatives, including low heritability, number of generations of training, marker density, initial distributions, and effective population size (Ne). Results show that the more the generations of data in which both genotypes and phenotypes were collected, termed training generations (TG), the better the accuracy and persistency of accuracy based on GEBV. GEBV excelled for traits of low heritability regardless of initial equilibrium conditions, as opposed to traditional marker-assisted selection, which is not useful for traits of low heritability. Effective population size is critical for populations starting in Hardy-Weinberg equilibrium but not for populations started from mutation-drift equilibrium. In comparison with traditional BLUP, GEBV can exceed the accuracy of BLUP provided enough TG are included. Unfortunately selection rapidly reduces the accuracy of GEBV. In all cases examined, classic BLUP selection exceeds what was possible for GEBV selection. Even still, GEBV could have an advantage over traditional BLUP in cases such as sex-limited traits, traits that are expensive to measure, or can only be measured on relatives. A combined approach, utilizing a mixed model with a second random effect to account for quantitative trait loci in linkage equilibrium (the polygenic effect) was suggested as a way to capitalize on both methodologies.     

苇状羊茅与多年生黑麦草内生真菌菌丝基因组DNA提取的优化及PCR初步检测

感染内生真菌的禾草在牧草和草坪业上具有重要的生态和经济意义，家畜采食感染Neotyphodium coenophialum和N.lolii的苇状羊茅和多年生黑麦草会发生中毒。本研究收集天津口岸1998年以来进境的部分苇状羊茅和多年生黑麦草种子，对经镜检确认带有内生真菌的种子进行分离培养，对疑似菌株的菌丝用改进的Moiler等方法进行基因组DNA抽提，测定浓度及纯度，对照原方法，DNA的纯度有较大提高，浓度略有上升。Tubulin2基因的引物IS1-IS3扩增结果显示为单一的条带，结合形态学和序列比对，分离培养得到的菌株可以基本确定为N．coenophialum和N。lolii。根据Genbank中N.coenophialum和N.lolii的NC25基因序列设计出引物F1-R1，扩增得到能区分开N.coenophialum和N．lolii的单一条带（相差160bp），建立了N．coenophialum和N．lolii的PCR检测方法，结果准确可靠。     

棕榈藤基因组DNA提取及RAPD反应条件探索

棕榈藤(rattan)是棕榈科(Palmae Juss．)藤本植物，属棕榈科省藤亚科(Calamoideae)省藤族(Calameae)植物，包括13属600余种，主要分布于热带地区。棕榈藤是热带森林宝库中的多用途植物资源，具有很高的经济价值和开发前景，是仅次于木材和竹材的重要林产品，其中黄藤(Daemonorops margaritae(Hance)Seccari)和单叶省藤(Calamus simplicifolius Wei)是我国大面积栽培的2个重要藤种。