植物基因组表达序列标签(EST)计划研究进展

植物表达序列标签(EST)计划是随机挑选cDNA克隆,并对其3′或5′端进行大规模一次性测序,将得到的150～500 bp长度的DNA片段与数据库中的序列进行比较,获得对基因组结构、组织、表达等认识的基因组研究策略.就近年来国际植物EST计划的实施情况、植物EST计划的研究范围、生物信息学在EST研究中的应用、EST数据库及查询、植物EST研究中遇到的问题等方面内容进行了综述.     

白菜的EST标记及其对油菜的通用性

根据白菜的表达序列标签,设计了28对引物。在对引物、dNTP、MgCl2的浓度及退火温度等参数进行测试后,建立了合适的PCR反应体系。在此反应体系下,以构建EST的白菜自交系A的DNA为模板,对设计的引物进行了筛选,发现有18对引物能对白菜DNA扩增出产物。用筛选出来的引物分别对17个白菜类品种进行PCR扩增,用琼脂糖凝胶电泳分析其产物的多态性,发现10对引物有多态性,这占了筛选引物的55.6%。为检测白菜EST标记的通用性,进一步利用设计的引物对不同油菜品种的DNA进行PCR扩增。在检测的28对引物中,共有24对引物能扩增出产物,占引物总数的85.7%,显示多态性的引物为18对,占引物总数的64.3%.。在对白菜DNA能扩增出产物的18对引物中,对油菜完全可用,且有13对引物产生多态性。而在那些对白菜未扩增出产物的10对引物中,也有6对能扩增出产物,其中5对显示多态性。文章研究结果证明,通过EST建立分子标记是可行的,而且这种标记对近缘物种是可通用的。     

白粉病菌诱导的小麦表达序列标签(EST)研究

白粉病是我国小麦的主要病害之一.尝试用表达序列标签(expressed sequence tags, EST)技术,研究了经白粉病菌诱导后的小麦基因表达.从构建的普通cDNA文库中随机挑取约1 500个阳性克隆并进行测序, 获不重复ESTs序列387条.不重复序列均获GenBank的存储号.其中49.4%的序列与已知基因同源,196条序列功能未知, 84条序列为新ESTs.将不重复序列制备成高密度点阵膜,用差示杂交法筛选到几个抗病相关序列.     

白粉病菌诱导的小麦表达序列标签(EST)研究(英)

白粉病是我国小麦的主要病害之一。尝试用表达序列标签 (expressedsequencetags,EST)技术 ,研究了经白粉病菌诱导后的小麦基因表达。从构建的普通cDNA文库中随机挑取约 15 0 0个阳性克隆并进行测序 ,获不重复ESTs序列 387条。不重复序列均获GenBank的存储号。其中 4 9.4 %的序列与已知基因同源 ,196条序列功能未知 ,84条序列为新ESTs。将不重复序列制备成高密度点阵膜 ,用差示杂交法筛选到几个抗病相关序列。     

应用生物信息学技术对植物表达序列标签(EST)的大规模分析

目前 ,一些基因组较小的植物 (如拟南芥 ,水稻等 )的全基因组已经基本完成测序 ,较大基因组的测序工作则主要集中在基因组中表达基因的测序上 ,表达序列标签 (EST)计划由此产生。研究表明 ,对EST进行大规模研究已成为功能基因组学研究的最佳途经之一。本文着重介绍和讨论应用生物信息学技术对植物EST数据的大规模分析。     

Expressed sequence tags and mRNA expression levels of tagged cDNAs from watermelon anthers and developing seeds

To understand the molecular events that occur during reproductive organ development and to provide genetic resources for molecular breeding, we generated 328 expressed sequence tags (ESTs) from randomly selected clones of four watermelon cDNA libraries. These libraries were prepared from young and mature anthers, as well as the seed coat and inner seed tissues. EST clones found in the young anthers and inner seed tissues showed similarity with genes related to development and signal transduction. We could deduce that, especially in the developing inner seed tissues, important morphological processes were associated exclusively with seed and embryo development In addition, seed metabolism was tailored toward the accumulation of economically valuable storage compounds such as lipids. In the seed coat, EST clones showed similarity with genes that influence the transport or conversion of nutrients such as porin, sucrose synthase, L-asparaginase, and arginine decarboxylase. We also selected two cDNA clones from each of the four classes of ESTs for studying expression levels and patterns in the various organs. Among those eight clones, three (An88, Is124, and Sc68) were expressed preferentially in their particular organ.     

绍鸭卵巢卵泡发育相关新EST的分离与表达

采用银染mRNA差异显示方法从绍鸭卵巢等级卵泡中分离并筛选到 3个差异表达序列标签 (ExpressedSequenceTags ,ESTs)SXDF0 2 0 1(2 71bp)、SXDF0 2 0 2 (2 0 0bp)和SXDF0 2 0 3(173bp) ,通过测序和BLAST检索 ,发现SXDF0 2 0 1与GenBank中登录的所有物种的所有序列均无同源性 ,是在绍鸭卵巢卵泡发现的新EST ,现已登录GenBank(GenBank登录号 :CB0 72 6 2 9) ,而SXDF0 2 0 2和SXDF0 2 0 3则分别与GenBank公布的鸡的EST和肌胃肌球蛋白重链高度同源。用 5′ RACE将SXDF0 2 0 1延伸至 5 4 4bp ,经过BLAST再次检索 ,仍然未发现中度同源序列采用相对定量RT PCR方法进一步研究SXDF0 2 0 1和SXDF0 2 0 2在绍鸭组织中的时空特异性表达 ,发现这两个EST在产蛋高峰期绍兴鸭的下丘脑、垂体、肌肉、肝脏、脂肪等组织都有表达 ;SXDF0 2 0 1在 30日龄卵巢的表达水平显著高于 6 0 (P <0 0 5 )和 90 (P =0 0 15 )日龄 ;而SXDF0 2 0 2在卵巢发育的不同阶段的表达水平没有变化 ;SXDF0 2 0 1在卵巢卵泡颗粒层中的表达总体高于膜层 ,SXDF0 2 0 2在颗粒层中的表达F3 >F5>Fw(P <0 0 1) ,而在F1卵泡降至最低 (P <0 0 1) ,膜层中以Fw 卵泡表达水平最高 (P <0 0 1)。     

Moss (Physcomitrella patens) Expressed Sequence Tags Include Several Sequences which are Novel for Plants*

The moss Physcomitrella patens (Hedw.) B.S.G. is the first land plant in which gene disruption by homologous recombination Is directly accessible. In order to obtain cloned sequences which may be used in such an approach, complementary DNAs (cDNAs) have been isolated by subtractlve hybridisation of representative cDNA libraries from cytoklnin-treated tissue. Sequencing of these clones from both ends yielded over 35 kb of non-redundant sequence Information, of which 20 kb results from clones which appear to be novel to plants. Database comparisons have revealed that 39 of the expressed sequence tags (ESTs) generated show significant homology to identified sequences. Analysis of these ESTs shows a high degree of conservation between Physcomitrella and seed plant sequences, and codon usage is found to be very similar to that In dicotyledonous species. Furthermore, 43 sequences showing no significant homology to sequences in the databases represent previously unidentified expressed genes.     

A Genomics Approach Using Expressed Sequence Tags and Microarrays in Ripening Apple Fruit (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Apple (Malus domestica Borkh.), an important horticultural crop, produces human health-promoting metabolites during fruit ripening. Because that process, which involves complex biochemical and physiological changes, is genetically programmed, molecular and genetic approaches have been taken to understand the associated cellular mechanisms. The release of 151,687 apple expressed sequence tags (ESTs) into a public database has made possible large-scale studies of expression. Analysis of apple ESTs allows for the identification and characterization of genes with potential roles in fruit development, particularly those related to aroma production and protein degradation during ripening. Apple cDNA and oligonucleotide microarrays have been generated for more comprehensive examinations. Such tools are powerful means for elucidating the molecular events involved in metabolite biosynthesis and physiological changes and will also enable researchers to understand how to control that ripening process.     

Identification and Validation of Single Nucleotide Polymorphisms in Poplar Using Publicly Expressed Sequence Tags

By using assembled expressed sequence tags （ESTs） from 14 different eDNA libraries that contain 84 132 sequences reads, 556 Populus candidate single nucleotide polymorphisms （SNPs） were identified. Because traces were not available from dbEST （http：//www.ncbi.nlm.nih.gov/dbEST/index.html）, stringent filters were used to identify reliable candidate SNPs. Sequences analysis indicated that the main types of substitutions among candidate SNPs were A/G and T/C transitions, which accounted for 22.0% and 30.8%, respectively. One hundred and ten candidate SNPs were tested. As a result, 38 candidate SNPs were confirmed by directed sequencing of PCR products amplified from six different individuals. Thirteen new SNPs in intron regions were found and multiple SNPs were found to be located in both intron and exon regions of four contigs. Heterozygosis was found in all 47 candidate sites and five SNP sites were heterozygous in all six samples. This is the first report of SNP identification in a tree species which reveals that assembled ESTs from multiple libraries of the public database may provide a rich source of comparative sequences for an SNP search in the poplar genome.     

一个鼻咽癌相关EST的鉴定及其全长cDNA序列分析

鼻咽癌是我国南方及东南亚地区常见的恶性肿瘤之一.通过对鼻咽癌染色体高频率杂合性丢失区域3p21的表达序列标签(expressedsequencetag,EST)进行同源性比较分析,运用逆转录聚合酶链式反应的方法,筛选到一个在41.18%(14/34)的鼻咽癌活检组织及20.0%(1/5)的鼻咽癌细胞系中表达下调的ESTBG772301;并用Northern杂交方法,检测了该EST在多种正常成人组织中的表达状况及其所代表基因的转录本大小.在此基础上,对该EST来源的cDNA克隆(IMAGE:4839190)进行直接测序,获得了一个全长为2377bp的新cDNA序列;经生物信息学分析,发现它与已知基因序列无明显同源性,属于一个新基因,定位于染色体3p21.3,被命名为鼻咽癌表达下调基因(NPCEDRG,GenBank登录号:AF538150).其编码的蛋白质含169个氨基酸,与一个已报道的在进化上相对保守、功能未知的人类蛋白Nicolin1(简称NICN1)N端170个氨基酸残基的序列同源性为97%,但缺少NICN1蛋白C端43个氨基酸残基,可能是nicolin1基因不同剪接本的编码产物.     

Analysis of Expressed Sequence Tags from Skeletal Muscle-specific cDNA Library of Chinese Native Xiang Pig

A Longissimus Dorsi muscle cDNA library of Xiang Pig was constructed, and 131 randomly isolated clones were sequenced in this study. The results of bioinformatics analysis showed that 131 ESTs represented 109 unique clones sequences, of which 99 showed homology to previously identified genes in humans or other mammals, 3 matched other uncharacterized expressed sequence tags (ESTs), and 7 showed no significant matches to sequences already present in DNA databases. No protein matches were found for 10 ESTs. Functional analysis of the ESTs showed that a considerable proportion of them encoded proteins involved in gene/protein expression (45.46%). Other classes included genes involved in metabolism (10.10%), cell structure/motility (10.10%), cell/organism defense (5.05%), cell signaling/communication (2.02%), and cell division (0.0%). Unclassified genes constituted the remaining 27.27%. This study reported the results of the first gene expression profile analysis of Chinese native Xiang Pig skeletal muscle cells, thereby greatly facilitating the functional study of candidate genes involved in muscle growth as well as in the improvement of meat quality in domestic pigs.     

中国地方品种香猪的肌肉特异组织表达序列标签（ESTs）的分析

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列。在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs。对这10个已知、未知ESTs进行开放阅读框预测并进行BlastX分析,没有找到高度同源的氨基酸序列。对上述EST所对应的基因功能分析结果表明,除去27．27％的EST未能分类外,克隆到的EST大多来自与基因／蛋白的表达调控相关的基因（占45．46％）。来自具有其他功能的基因的EST依次是细胞代谢占10．10％、细胞结构／迁移占10．10％、细胞／机体防御占5．05％和细胞信号／传导占2．02％。没有发现和细胞分裂相关的已知功能基因。本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础。     

中国地方品种香猪的肌肉特异组织表达序列标签(ESTs)的

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列.在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs.对这10个已知、未知ESTs进行开放阅读框预测并进行B1ast分析,没有找到高度同源的氨基酸序列.对上述EST所对应的基因功能分析结果表明,除去27.27%的EST未能分类外,克隆到的EST大多来自与基因/蛋白的表达调控相关的基因(占45.46%).来自具有其他功能的基因的EST依次是细胞代谢占10.10%、细胞结构/迁移占10.10%、细胞/机体防御占5.05%和细胞信号/传导占2.02%.没有发现和细胞分裂相关的已知功能基因.本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础.     

Evaluation of cDNA Libraries from Different Developmental Stages of Schistosoma mansoni for Production of Expressed Sequence Tags (ESTs)

A comparative study of the gene expression profile in differentdevelopmental stages of Schistosoma mansoni has been initiatedbased on the expressed sequence tag(EST) approach. A total of1401 ESTs were generated from seven different cDNA librariesconstructed from four distinct stages of the parasite life cycle.The libraries were first evaluated for their quality for a large-scalecDNA sequencing program. Most of them were shown to have lessthan 20% useless clones and more than 50% new genes. The redundancyof each library was also analyzed, showing that one adult wormcDNA library was composed of a small number of highly frequentgenes. When comparing ESTs from distinct libraries, we coulddetect that most genes were present only in a single library,but others were expressed in more than one developmental stageand may represent housekeeping genes in the parasite. When consideringonly once the genes present in more than one library, a totalof 466 unique genes were obtained, corresponding to 427 newS. mansoni genes. From the total of unique genes, 20.2% wereidentified based on homology with genes from other organisms,8.3% matched S. mansoni characterized genes and 71.5% representunknown genes.