Acanthamoeba castellanii: gene profile of encystation by ESTs analysis and KOG assignment

The trophozoite of Acanthamoeba transforms into a cyst, the resistant form under harmful environments such as starvation, cold and certain chemicals used in medical treatment. To investigate the factors mediating encystation, ESTs of encystation-induced A. castellanii were analysed and compared to those of trophozoites. Each EST was compared by the predicted proteins from the ESTs, to the cyst and the trophozoite by reciprocal BLAST analysis, KOG assignment, and gene annotation. In addition to the genes previously reported to encystation mediate such as cyst specific protein 21, protein kinase C, proteasome and heat shock protein, several genes like cullin 4, autophage protein 8 and ubiquitin-conjugating enzymes were identified to be related to encystation. Five kinds of proteinase genes were detected in cyst ESTs. The information of the genes expressed during encystation may open the way to further study on differentiation and resistance of cyst-forming pathogenic protozoa.     

Differentially expressed genes of Acanthamoeba castellanii during encystation

To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed upregulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

大白菜霜霉菌诱导抑制性消减杂交cDNA文库的构建和分析

以抗霜霉病大白菜双单倍体系（DH）‘T12-19’为材料,构建了霜霉病诱导表达的正向抑制性消减文库,并利用反向Northern斑点杂交技术对768个阳性克隆进行了筛选,共获得57个病原菌诱导上调表达的克隆。测序后得到55条通读表达序列标签（ESTs）,对这些ESTs序列进行聚类和拼接分析,共获得50个unigenes。Blast分析表明,37个unigenes与已知基因高度同源,占全部非重复序列的67.3%。对已知功能基因按MIPS的分类方法进行功能分类,发现这些基因的功能主要涉及物质与能量代谢、转录调控、蛋白质合成与代谢、膜及转运、信号转导、抗病防御等。为了验证文库筛选结果的可靠性,采用实时荧光定量PCR技术分析了其中2个克隆BFCH10和BFIA7的表达谱。结果表明,这2个克隆在接种病菌6h后明显上调表达,与反向Northern斑点杂交结果基本一致。     

热胁迫下中甸角蒿叶片SSH文库的构建及初步分析

高温可能是限制中甸角蒿（Incarvillea zhongdiannensis）向低海拔地区引种驯化的主要因素之一。为了从基因表达水平研究中甸角蒿高温胁迫响应的分子机制,本研究利用SSH技术构建了中甸角蒿对高温（30℃）处理响应的正反向抑制性差减杂交文库。文库质量检测表明抑制性差减杂交效率较高,质量较好。通过对正反向文库中部分EST进行序列测定,获得了60条高质量的表达序列标签,平均长度为537bp。对序列进行BLAST比对及功能注释,50条EST为功能已知的基因,分别参与信号转导与转录、植物抗逆性反应、光合作用、代谢与能量、蛋白质合成与转运、蛋白质命运、细胞结构和细胞生长等过程。6个EST与功能未知基因的同源性较高。获得的4个未匹配的EST推测为新基因,可能在植物热耐受性方面具有重要的作用。     

白桦雌花序抑制性消减文库构建及EST分析

为研究白桦雌花序发育过程中特异基因的表达,以白桦雌花序样品为tester,雄花序样品为driver,利用SMART策略构建了白桦雌花序抑制性消减（SSH）文库。构建SSH文库的重组率为72%,插入片段的平均长度为400 bp左右。随机挑选文库克隆测序,获得150条EST序列,这些序列被GenBank的dbEST数据库收录,收录号为EE284580-EE284681,EE595316-EE595363。通过BlastX对EST进行功能注释,并对其中同源性较高的111条EST按功能进行分类,EST功能涉及了代谢、细胞防御、转录调节、能量代谢及信号传导等途径。发现了多个已知的控制花发育相关的EST,它们占已知功能EST的21%其功能涉及到调控花序形成和花分化、调控花粉与柱头亲和性以及调控花粉管发育等,包括MADS-box、S-locus F-box等基因。这些EST的获得为了解白桦花期基因表达,白桦花发育相关基因克隆和功能解析奠定了基础。     

热胁迫下中旬角蒿叶片SSH文库的构建及初步分析

高温可能是限制中甸角蒿（Incarvillea zhongdiannensis）向低海拔地区引种驯化的主要因素之一。为了从基因表达水平研究中甸角蒿高温胁迫响应的分子机制,本研究利用SSH技术构建了中甸角蒿对高温（30℃）处理响应的正反向抑制性差减杂交文库。文库质量检测表明抑制性差减杂交效率较高,质量较好。通过对正反向文库中部分EST进行序列测定,获得了60条高质量的表达序列标签,平均长度为537bp。对序列进行BLAST比对及功能注释,50条EST为功能已知的基因,分别参与信号转导与转录、植物抗逆性反应、光合作用、代谢与能量、蛋白质合成与转运、蛋白质命运、细胞结构和细胞生长等过程。6个EST与功能未知基因的同源性较高。获得的4个未匹配的EST推测为新基因,可能在植物热耐受性方面具有重要的作用。