Construction of an electronic physical map of Magnaporthe oryzae using genomic position-ready SSR markers

Magnaporthe oryzae is a model for plant pathogenic filamentous fungi. We have assembled a simple sequence repeat (SSR)-based physical map of the species, using in silico sequence data. A set of 120 SSR markers was developed from the genomic sequence of the reference isolate 70-15. These markers were readily amplified from the genomic DNA of other isolates, and high levels of allelic variation characterised the parental isolates of the two crosses tested. All the markers were locatable to one of the seven M. oryzae chromosomes. An SSR-based physical in silico map was constructed, and pre-existing SSR and RFLP loci were integrated into the map, along with 23 Avr (avirulence) genes and two other genes of importance to the plant/pathogen interaction. This map provides a platform for population genetics and functional genomics studies in the model pathogen, and even in other evolu- tionally related pathogens.     

全长基因的克隆

新基因全长cDNA序列、全长DNA序列的获得常常是分子生物学工作者面临的难题。本文就克隆全长基因的各种技术如文库筛选法、各种PCR技术及新发展起来的电子克隆法等方法作一简单综述。     

生物信息学研究进展

本文就目前生物信息学研究的热点问题做了简单的概述.对如何利用生物信息技术进行电子克隆、RNA二级结构的预测做了分析,并对现存的生物信息学问题进行了讨论.     

采用基于GSS序列的极大节旋藻(Arthrospira maxima)的基因克隆策略及其应用实例

电子克隆是随着基因组计划的实施而发展起来的利用生物信息学手段进行基因克隆的新方法,已经公布的极大节旋藻GSS序列使得电子克隆极大节旋藻的功能基因成为可能.为建立极大节旋藻功能基因的高效克隆策略,应建立利用GSS序列克隆极大节旋藻功能基因的基本策略并加以应用.具体操作方法：对dbGSS数据库中的605条极大节旋藻GSS序列进行拼接及功能注释,并在分析编码区完整性的基础上设计引物并进行PCR扩增以获得基因全长序列,利用以上策略成功克隆了极大节旋藻功能基因ureaseβ和dUTPase的全长序列.     

猪色素上皮衍生因子的电子克隆及序列分析

用鼠的色素上皮衍生因子cDNA序列在猪的ESTs库进行BLASTn搜索,得到一系列不同大小的ESTs片段,经拼接得到完整cDNA序列.序列分析显示该cDNA长1 425 bp,有一个1 242 bp的开放阅读框,编码413个氨基酸,5’非编码区长53 bp,3’非编码区长130 bp,有一个加尾序列和多聚腺苷酸尾巴.其核苷酸序列与牛、人和鼠的同源性分别为89%、87%和82%,氨基酸的同源性分别为89%、87%和84%,且有保守的糖基化位点、半胱氨酸位点和serp in基序,说明所克隆的cDNA序列为猪的PEDF全长cDNA.     

小麦TaCAT新基因克隆及分子生物学和生化特性分析

小麦组织培养再生潜力与抗氧化胁迫能力具有相关性。为解析小麦TaCATs基因家族的分子生物学及生化特性并为后续试验验证奠定理论基础,利用e-PCR方法进行小麦过氧化氢酶(CAT)新基因克隆,结合in silico技术对小麦TaCATs基因家族的生化特性进行分析和预测。氨基酸序列同源性比对结果表明,克隆的小麦TaCATs基因家族新成员与水稻的CatA和玉米的Cat3具有较高的相似性,分别达89%及81.1%,命名为TaCAT3,基因组DNA和cDNA长度分别为1986和1482bp,编码494个氨基酸的蛋白。亚细胞定位结果表明,TaCAT3可能定位在线粒体中,且所有的功能活性位点在小麦TaCATs家族中具有一致的保守性。系统发生树构建结果表明,小麦TaCATs能形成3个独立的分支。在蛋白质编码序列同源比对的基础上,利用SwissModel的Swiss-PdbViewer 3.7软件包对TaCATs高级结构进行同源模拟,发现所构建的模型能很好地反映TaCATs的高级结构。     

The scorpine family of defensins: gene structure,alternative polyadenylation and fold recognition

Small cationic antimicrobial peptides (SCAMPs) as effectors of animal innate immunity provide the first defense against infectious pathogens. This class of molecules exists widely in invertebrate hemolymph and vertebrate skin secretion, but animal venoms are emerging as a new rich resource. Scorpine is a unique scorpion venom defensin peptide that has an extended amino-terminal sequence similar to cecropins. From the African scorpion Opistophthalmus carinatus venom gland, we isolated and identified several cDNAs encoding four new homologs of scorpine (named opiscorpines 1–4). Importantly, we show for the first time the existence of multiple opiscorpine mRNAs with variable 3 untranslated regions (UTRs) in the venom gland, which may be generated by alternative usage of polyadenylation signals. The complete opiscorpine gene structure including its promoter region is determined by genomic DNA amplification. Two large introns were found to be located within the 5 UTR and at the boundary of the mature peptide-coding region. Such a gene structure is distinct, when compared with other scorpion venom peptide genes. However, a comparative promoter analysis revealed that both opiscorpine and scorpion venom neurotoxins share a similar promoter organization. Sequence analysis and structural modeling allow us to group the scorpines and scorpion long-chain K-channel toxins together into one family that shares a similar fold with two distinct domains. The N-terminal cecropin-like domain displaying a clear antimicrobial activity implies that the scorpine family represents a group of real naturally occurring hybrids. Based on the phylogenetic analysis, a possible cooperative interaction between the N and C domains is elucidated, which provides an evolutionary basis for the design of a new class of anti-infectious drugs.Received 5 April 2004; accepted 17 May 2004