南方鲇GK基因片段的克隆及序列分析

为从南方鲇(Silurus meridionalis)中扩增葡萄糖激酶(GK)基因,根据已报道的GK基因结构中的氨基酸保守区域,设计简并引物;从南方鲇肝胰脏中迅速抽提RNA,将扩增出的产物克隆到pMD18-T载体上并导入到大肠杆菌DH5α中;阳性克隆鉴定后测序.将测序得到的南方鲇GK基因与已知的GK基因进行核苷酸序列比...     

猪骨形态发生蛋白15基因的分子克隆及分析

骨形态发生蛋白15(Bone Morphogenetic Portein-15,BMP15)由卵母细胞特异表达,在卵母细胞发育与成熟过程中起重要调控作用.本文以长白猪为研究模型,克隆BMP15基因cDNA全长并探究该基因组织表达图谱.研究显示,家猪BMP15基因cDNA全长1298bp,编码具402个氨基酸的前体蛋白.依赖RT-PCR技术,家猪Bmp15基因被发现特征性表达于家猪卵巢中.在本研究中,还分离到家猪BMP15基因潜在启动子区(3279bp),针对该启动子区,部分转录因子结合位点获得预测.这些研究结果为探究BMP15基因的表达调控机制及其对家猪卵泡发育影响奠定基础.     

基因克隆和功能验证研究

综述了基因克隆和功能研究的方法,并对研究前景进行了展望.     

葛仙米中SOD基因的克隆及在大肠杆菌中的表达

采用PCR技术从葛仙米（Nostoc sphaeroides）总DNA中克隆了一段基因序列,该序列与基因库中已公布的编码普通念珠藻（Nostoc commnue）超氧化物歧化酶的氨基酸序列同源性为98％,将该基因插入含T7启动子的质粒pET-32a（＋）中构建表达质粒pET-sod,然后将该表达质粒转入大肠杆菌BL21中进行蛋白表达,表达菌株用lmmol／L1PTG诱导表达数小时后,产生较多重组的蛋白,且该蛋白以可溶性蛋白形式存在,SDS-PAGE分析表明：在相对分子量约为22kd的位置有一条明显蛋白质带,将诱导表达后的蛋白通过亲和层析的方法进行蛋白纯化;NBT光还原法测定表达产物的比活力,每毫克纯化蛋白约为2550U。     

中度嗜盐菌GbsB基因的克隆、序列分析及生物信息学分析

为构建耐盐的转基因植物提供材料,根据地衣芽孢杆菌Bacillus licheniformisATCC 14580的GbsB基因的核甘酸序列设计1对特异性引物,通过PCR的方法扩增由分离的中度嗜盐菌Bacillus sp.XJ1-05的GbsB基因,经T/A克隆,插入到pBS-T载体上进行序列测序,采用NCBI-BlastX软件在Genbank数据库中进行同源性检索,结果表明:得到的GbsB基因的开放阅读框(ORF)全长为1209bp,编码1个由402个氨基酸残基组成的蛋白质;其与Bacillus licheni-formisATCC 14580的GbsB基因的氨基酸序列同源性高达96%。生物信息学分析表明:该GbsB基因编码蛋白无信号肽,无跨膜区域,高亲水性,有2个乙醇脱氢酶作用位点。     

Gene association study with SVM, MLP and cross-validation for the diagnosis of diseases

Gene association study is one of the major challenges of biochip technology both for gene diagnosis where only a gene subset is responsible for some diseases, and for the treatment of the curse of dimensionality which occurs especially in DNA microarray datasets where there are more than thousands of genes and only a few number of experiments (samples). This paper presents a gene selection method by training linear support vector machine (SVM)/nonlinear MLP (multilayer perceptron) classifiers and testing them with cross-validation for finding a gene subset which is optimal/suboptimal for the diagnosis of binary/multiple disease types. Genes are selected with linear SVM classifier for the diagnosis of each binary disease types pair and tested by leave-one-out cross-validation; then, genes in the gene subset initialized by the union of them are deleted one by one by removing the gene which brings the greatest decrease of the generalization power, for samples, on the gene subset after removal, where generalization is measured by training MLPs with leave-one-out and leave-four-out cross-validations. The proposed method was tested with experiments on real DNA microarray MIT data and NCI data. The result shows that it outperforms conventional SNR method in the separability of the data with expression levels on selected genes. For real DNA microarray MIT/NCI data, which is composed of 7129/2308 effective genes with only 72/64 labeled samples belonging to 2/4 disease classes, only 11/6 genes are selected to be diagnostic genes. The selected genes are tested by the classification of samples on these genes with SVM/MLP with leave-one-out/both leave-one-out and leave-four-out cross-validations. The result of no misclassification indicates that the selected genes can be really considered as diagnostic genes for the diagnosis of the corresponding diseases.     

基因定位研究与遗传病防治

介绍了基因定位的意义与基本操作,分析了基因定位方法的研究进展,阐述了定位克隆研究取得的若干重大突破以及我国在基因定位研究领域的优势。     

嗜热菌Thermus thermophilus HB8铁硫簇合成途径中IscA和IscU蛋白的表达、纯化和功能研究

Fe-S簇可参与电子传递和基因调节等过程.实验以嗜热菌Thermus thermophilus HB8基因组为模板,通过PCR扩增获得Fe-S簇ISC系统生物合成途径中的IscA和IscU基因,并克隆连接到表达载体pET-28a（＋）上.重组质粒转化E.coli Rosetta（DE3）进行表达,用亲和层析纯化得到浓度和纯度都较高的IscA和IscU蛋白.SDS-PAGE分析,证实其分子量大小分别为13.7KD和14.8KD.实验确定IscA是单聚体,IscA上能形成铁硫簇,IscU上不能形成铁硫簇.在厌氧条件下,RNA的量能提高1.5倍,但是IscA和IscU的表达量都会降低,说明厌氧对这两个基因有损伤.     

Morphological and physiological analysis of narrow and striped leaf 1 （nsl1） mutant of rice （Oryza sativa L.） and the gene mapping

The shape and color of rice leaves are impor- tant agronomic traits that directly influence the proportion of sunlight energy utilization and ultimately affect the yield and quality. A new mutant exhibiting stable inheritance was identified as derived from ethyl methane sulfonate （EMS）-treated restorer Jinhui 10, tentatively named as narrow and striped leaf 1 （nsll）. The nsll displayed pale white leaves at the seeding stage and then white striped leaves in parallel to the main vein at the jointing stage. Meanwhile, its leaf blades are significantly narrower than the control group of Jinhui 10. The chloroplast structures of cells in the white striped area of the nsll mutant break down, and the photosynthetic pigments are significantly lower than that of the wild type. Moreover, fluorescence parameters, such as Fo, Fv/Fm, ФpsⅡ, qP, and ETR, in the nsll mutant are significantly lower than those of the wild type, and the photosynthetic efficiency is also significantly decreased. These changes in leaf color and shape, together with physiological changes in the nsll, result in smaller plant height and a decrease in the most important agro- nomic traits, such as the number of grains per panicle, grain weight, etc. Genetic analysis shows that the narrow and striped traits of the nsll mutant are controlled by a single recessive nuclear gene, which is located between InDel 16 and InDel 12 in chromosome 3. The physical distance is 204 kb. So far, no similar genes of such leaf color and shape in this area have been reported, This study has laid asolid foundation for the gene cloning and function analysis of NSL 1.     

猪Akt3基因克隆、序列分析及组织表达图谱研究

本研究以长白猪(Landrace)肌肉cDNA为模板,克隆得到长白猪Akt3基因.序列分析结果显示:长白猪Akt3基因cDNA全长1493bp,编码一个含479个氨基酸的前体蛋白,与人、牛、小鼠和大鼠的氨基酸序列一致性分别为100%、100%、99.58%、99.79%,具有高度保守性.氨基酸结构分析显示,长白猪Akt3基因都具有典型的Pleckstrin homologous domain(PH)结构域和丝氨酸/苏氨酸磷酸化激活位点.组织表达图谱分析结果显示:Akt3基因在所有检测的家猪组织中均有表达.此外,本研究还将Akt3基因编码区序列克隆转入真核表达载体pcDNA3.1(+)中,为下一步功能验证做准备.