Expressed sequence tags (ESTs) analysis of the ripening Vitis amurensis cv. Shuang Hong berry skins

Vitis amurensis is a valuable resource for wine production. Ripening of the grape berry is the key phase which determines the com- position of wine. To better understand the gene expression that manifest in V. amurensis berry skins during the ripening, cDNA library of V. amurensis berry skins was constructed. A total of 935 high quality ex- pressed sequence tags （ESTs） were obtained from the library. These ESTs represent 636 unigenes, including 108 contigs and 528 singletons. The EST analysis was performed and genes were assigned to functional categories according to their primary BLAST match. Of these 25.35% were involved with metabolism, 6.27% with cell rescue and defense, 6.84% energy, 11.68% protein synthesis, 18.8% protein activity regula- tion, 11.11% cell structure, 7.98% transport, 6.27% transcription and the remaining 5.7% were signal transduction. The generated ESTs were characterized by the gene ontology analysis and were categorized ac- cording to its cellular component, molecular function and biological process. In the cDNA library, some genes are relevant to the biosynthesis of anthocyanins, while some genes are related to grape berry maturation.

Generation and analysis of expressed sequence tags from the salt-tolerant eelgrass species, Zostera marina

Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- structed. Single-pass sequencing of the 5＇ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags （ESTs）, which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100-1 500 bp. Basic Local Alignment Search Tool （BLASTX） analysis revealed that 1 664 unigenes had significant homology to known genes in the Na- tional Center for Biotechnology Information （NCBI） non-redundant （nr） database （E-value≤5-10）. Among them, the two most abundant genes encoded metallothionein （157 ESTs） and chlorophyll a/b-binding pro- tein （38 ESTs）, accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes （KEGG）, 1 462 unigenes were assigned to 1 161 pathways （E-value≤5-10）. A total of 938 unigenes were assigned Gene Ontology （GO） terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.

Analysis of expressed sequence tags from the Ulva prolifera (Chlorophyta)

In 2008, a green tide broke out before the sailing competition of the 29th Olympic Games in Qingdao. The causative species was determined to be Enteromorpha prolifera （Ulva prolifera O. F. Miiller）, a familiar green macroalga along the coastline of China. Rapid accumulation of a large biomass of floating U. prolifera prompted research on different aspects of this species. In this study, we constructed a nonnormalized cDNA library from the thalli of U. prolifera and acquired 10072 high-quality expressed sequence tags （ESTs）. These ESTs were assembled into 3 519 nonredundant gene groups, including 1 446 clusters and 2 073 singletons. After annotation with the nr database, a large number of genes were found to be related with chloroplast and ribosomal protein, GO functional classification showed 1 418 ESTs participated in photosynthesis and 1 359 ESTs were responsible for the generation of precursor metabolites and energy. In addition, rather comprehensive carbon fixation pathways were found in U. prolifera using KEGG. Some stress-related and signal transduction-related genes were also found in this study. All the evidences displayed that U. prolifera had substance and energy foundation for the intense photosynthesis and the rapid proliferation. Phylogenetic analysis of cytochrome c oxidase subunit I revealed that this green-tide causative species is most closely affiliated to Pseudendoclonium akinetum （Ulvophyceae）.

Analysis of Expressed Sequence Tags from Liver Tissue in Swine

In order to study the expression of function gene and its effect on metabolic control and other physiological function in liver, 438 expressed sequence tags (ESTs) were determined, which were from a cD-NA library of porcine liver tissue. The results showed that the nucleotide sequences of 186 ESTs have already presented in GenBank database, and 37 ESTs could be found the homology with human and other species, while the others were not identified. 45 full length insertion of the clones randomly isolated from cDNA library were also completely sequenced with different size, and the results showed that 19 of them were function-known genes, 11 had no open reading frame ( ORF )at all and 15 had ORF but the function were not elucidated yet.

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai).Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences,after redundancy elimination.Seventeen polymorphic EST-SSRs were developed.The number of alleles per locus varied from 2-17,with an average of 6.8 alleles per locus.The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922,respectively.Twelve of the 17 loci (70.6%) were successfully amplified in H.diversicolor.Seventeen loci segregated in three families,with three showing the presence of null alleles (17.6%).The adequate level of variability and low frequency of null alleles observed in H.discus hannai,together with the high rate of transportability across Haliotis species,make this set of EST-SSR markers an important tool for comparative mapping,marker-assisted selection,and evolutionary studies,not only in the Pacific abalone,but also in related species.

Analysis of Simple Sequence Repeats Information from Floral Expressed Sequence Tags Resources of Papaya (<i>Carica papaya</i>L.)

Papaya (Carica papaya L.) is one of the most economically, medicinally and nutritionally important tropical fruit crops. Expressed sequence tags (ESTs) derived simple sequence repeat (SSR) markers are more valuable as they are derived from conserved genic portion. Development of EST-SSRs markers through in silico approach is cheaper, less time consuming and labour-intensive. In this study, we aimed to mine SSRs and developed EST-SSR primers from papaya floral ESTs. A total of 75,846 papaya floral ESTs were downloaded from public database National Centre for Biotechnology Information (NCBI). A total of 26,039 floral unigenes (7961 contigs and 18,078 singletons) were generated after assembly of these ESTs. From these floral unigenes, 433,782 perfect SSRs, 204,968 compound SSRs and 6061 imperfect SSRs were mined, respectively. In perfect SSRs, mononucleotide repeats were most abundant (94.7%) followed by tri- (3.1%) and di-nucleotide repeats (1.7%). The frequencies of tetra-, hexa- and penta-nucleotide repeats accounted for only (0.17%), (0.04%) and (0.03%), respectively. In mononucleotide repeats, the most abundant motif was A/T (69.3%) and in di- and tri-nucleotide repeats were AG/CT (61%) and AAG/CTT (31%), respectively. In imperfect SSRs, mononucleotide repeats (56.5%) were most abundant. 176 different types of motifs were identified. A total of 3807 primer pairs for floral papaya ESTs were successfully designed. These developed EST-SSR primers are being used for the genetic improvement of papaya such as study of cross-transferability across genera/species, evaluation of genetic diversity, and identification of sex-specific markers. These EST derived SSRs can also be used in filling gaps in existing linkage maps in papaya.

Primarily screening and analyzing ESTs differentially expressed in rats' primary liver cancer

Objective： To screen and analyze key express sequence tags （ESTs） which were differentially displayed in every period of SD rats＇ primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Methods： Using diethylnitrosamine （DENA） as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique （DDRT-PCR）. After the fragments were sequenced, bioinformatics were .used to analyze the results. Results： Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments （EST-1, EST-3 and EST-5） had extremely low identity to any genes registered in GENBANK databases. Conclusions： BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-I, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.

Confirmation of Pearl Millet-Napiergrass Hybrids Using EST-Derived Simple Sequence Repeat (SSR) Markers

Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids (“PMN”;Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both “Seeded-yet-Sterile” biofuel PMN feedstocks and additional PMN cultivar-and parental species-specific markers.

基于EST-SSR标记的沙棘品种鉴定及指纹图谱构建

以沙棘(Hippophae rhamnoides)优良品种“实优1号”为材料,对其叶片进行转录组测序,利用微卫星识别软件(microsatellite identification tool,MISA)和Primer 3(version 2.3.4)对获得的序列进行简单重复序列(simple sequence repeat,SSR)位点挖掘和引物设计,以收集的42份沙棘品种为研究材料,开展聚合酶链式反应(PCR)和毛细管电泳检测,旨在开发一套多态性高、稳定性好和通用性强的表达序列标签微卫星(express sequence tags from simple sequence repeat,EST-SSR)引物,构建沙棘指纹图谱,从而实现沙棘品种的快速准确鉴定,并对沙棘品种间亲缘关系进行分析。“实优1号”转录组测序共获得6196个SSR位点,其中,重复基元类型为182种,SSR基序长度主要分布在10~21 bp区间,占全部SSR的81.58%,主要SSR重复类型为单核苷酸重复(48.72%)、二核苷酸重复(22.68%)和三核苷酸重复(18.85%)。利用筛选出的28对引物在42份沙棘品种中共检测出193个等位基因,等位基因数(Na)、有效等位基因数(Ne)、观测杂合度(Ho)、期望杂合度(He)、多态信息含量(PIC)和Shannon信息指数(I)等遗传多样性参数的均值分别为6.964、3.495、0.617、0.671、0.623和1.384。UPGMA聚类分析表明,42份沙棘品种间的遗传相似性系数为0.601~0.990,当遗传相似性系数为0.694时,供试品种可分为2组;当遗传相似性系数约为0.7402时,供试品种可分为3组。优选6对引物构建指纹图谱,可以实现沙棘品种的快速准确鉴定。该研究可为沙棘的良种鉴定、指纹图谱构建以及遗传多样性和亲缘关系分析等提供分子水平的理论基础和数据支撑。

Identification of Differentially Expressed Genes in the Salivary Gand of Rhipicephalus haemaphysaloides by the Suppression Subtractive Hybridization Approach

For the purpose of screening and analyzing the differentially expressed genes from the salivary gland of Rhipicephalus haemaphysaloides, two salivary gland-subtracted cDNA libraries of partially fed female ticks and fed male ticks were constructed using suppression subtractive hybridization （SSH）. A total of 247 female expression sequence tags （ESTs） and 168 male ESTs were obtained from the two SSH cDNA libraries. It is predicted that 25 female ESTs and 44 female ESTs contain the 5＂ and 3＂ ends, respectively, and that 53 male ESTs and 74 male ESTs contain the 5＂ and 3＂ ends, respectively. To identify the subtraction rate of the two SSH cDNA libraries, the RT-PCR method was used to test 24 female ESTs and 21 male ESTs selected randomly but not repeatedly. The results showed that there were 13 upregulated or differentially expressed genes in the partially fed salivary gland of the female R. haemaphysaloides and that the differentially expressed rate was 54%. In addition, they indicated that there were 9 upregulated or differently expressed genes in the fed salivary gland of the male R. haemaphysaloides and that the differentially expressed rate was 43%. Putative translations of 141 （57%） female ESTs and 125 （74%） male ESTs had similarity to GenBank sequences, and 32 （23%） female ESTs and 29 （23%） male ESTs exhibited similarity to tick proteins, which showed that most of the proteins in the libraries were mainly related to the feeding blood physiology of the ticks.

Effect of 4% glycerol and low aeration on result of expression in <i>Escherichia coli</i>of Cin3 and three <i>Venturia inaequalis</i>EST’s recombinant proteins

The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel Venturia inaequalis 5704 (Cin3) and three expressed sequence tags (ESTs);38, 6987, and 4010 are strongly up-regulated in the early stages of infection. The CIN3 and three ESTs using two vectors pMAL-c2 and pET 21 were expressed in Escherichia coli. Recombinant proteins expression, solubility and yields were analyzed. 38, 5704 (Cin3) and 6987 re- combinant proteins were expressed in soluble form and while 4010 was expressed in inclusion bodies. Re- solution on native-PAGE, the recombinant proteins;38, 5704 (Cin3), 6987 were shown to be present in dimmer, tetramer and polymer. A method was de- veloped, consisting of induction of expression at va- rious temperatures, and using enriched broth with 4% glycerol together with slow shaking, led to a decrease in concentration of nascent polypeptide and production of soluble recombinant proteins of;38, 5704 (Cin3), 6987 and 4010. Resolution on native- PAGE, the recombinant proteins were shown to be present as monomer.

Genome Sequence and Genetic Diversity Studies of Melon

Cucumis melo is one of the most important fruit vegetables cultivated in tropical and temperate regions, as it is highly valued for the quality of its fruits. Traditionally, C. melo is considered to be divided into two subspecies according to ovary hairiness, i.e., subspecies melo （long hairs, distributed from India to Europe and in America） and subspecies agrestis （short hairs, in Eastern Asia from India to Japan and in Africa）. Today, the species comprises wild, feral and cultivated varieties, including sweet melons used for dessert and non-sweet ones consumed raw, pickled or cooked. The goal of this paper was to summarize melon genome sequencing and the genetic diversity studies from different references.

从中国对虾ESTs中筛选微卫星标记的研究

利用生物信息学方法在含有10446个中国对虾ESTs的数据库中进行微卫星序列的筛选,共发现微卫星序列229个,占整个ESTs数据库的2.19%,其中含双碱基重复序列146个和3碱基重复序列58个,分别占在ESTs数据库中发现微卫星序列总数的63.76%和25.33%,大部分发现的微卫星序列均为Perfect形式的重复序列。根据筛选得到的微卫星序列设计并合成引物19对进行多态性检测,在有扩增产物的16对引物中,首次筛选得到8个中国对虾微卫星标记,并对这些微卫星标记进行了等位基因频率、观测杂合度、期望杂合度、PIC值等统计学指标的评价。结果表明,在8个微卫星位点上,等位基因的数目从5到15不等,等位基因长度从165～305bp,期望杂合度和多态性信息含量分别为0.59到0.89和0.56到0.88,表明这8个中国对虾微卫星标记完全适合于遗传分析。

杨树EST-SSR标记的开发

利用来自美洲黑杨及欧美杨的20023条EST序列,进行EST-SSR标记开发研究。首先对这些EST序列进行序列拼接,然后在非重复序列中发现由二核苷酸至五核苷酸组成的SSR重复序列。共在10816个非重复序列中发现1604个序列含有SSR,占总非重复序列的14.8%。在发现的1918个SSR重复序列中,二核苷酸重复是最丰富的重复类型,占总类型的62.3%。设计合成48对SSR引物,利用6个不同的杨树品种对其进行验证。结果约90%的引物获得扩增产物,58%的引物在6个不同杨树品种中产生多态性分离。另外,还对重复序列的数量及重复类型进行了讨论。

刺参(Apostichopus japonicus)EST序列中微卫星分布分析及其标记的筛选

利用微卫星查找软件对现已公布的6632条刺参ESTs的数据库中2—6个碱基重复单元组成的简单序列重复进行筛选,进而对其微卫星的丰度和分布进行比较研究。共发现微卫星序列416个,占整个ESTs数据库的6.48%;其中含双碱基重复序列146个,数量最多,占ESTs数据库中发现微卫星序列总数的65.86%;三、四、五、六碱基重复序列分别占微卫星序列总数的26.68%、1.68%、0.24%、5.53%。对含有SSR位点符合微卫星引物设计的EST序列利用Primer软件结合人工方法设计合成引物21对,其中13对有扩增产物且均为多态位点。扩增产物变性聚丙烯酰胺胶电泳,统计每个微卫星标记等位基因数目,计算等位基因频率、观测杂合度、期望杂合度等统计学指标的评价。结果表明,在13个微卫星位点上,等位基因的数目从3到8不等,等位基因长度从175—382bp,观测杂合度和期望杂合度分别为0.158—0.650和0.198—0.841,所筛选微卫星标记可于遗传分析。

基于EST信息的百合SSR标记的建立

依据已知的百合EST(expres sedsequence tags)序列信息,开发新的SSR(simple sequence re-peats)标记,在NCBI的EST数据库1688条EST中检索到98条含有101个SSR的序列,SSR的检出率为5.98%,其中三核苷酸重复类型占主导地位,出现频率为2.84%。EST-SSR的重复基元共搜索到47种,其中三核苷酸重复基元类型最为丰富,大约占重复基元类型总数的一半。利用部分EST-SSRs序列共设计23对SSR引物,以铁炮百合‘Snow Queen’DNA为模板,对引物进行筛选,其中18对引物有扩增产物,占所设计引物总数的78.26%;进一步用这些引物在5个杂种系列13个百合品种进行多态性测试,显示多态性的引物占可扩增引物的66.7%。本研究结果证明了基于百合EST信息建立SSR标记是一种有效而又可行的方法。

缢蛏(Sinonovacula constricta)EST-SSR分布特征及引物开发利用

采用CAP3软件对NCBI上的5296条缢蛏ESTs序列进行了微卫星特征分析。结果表明,经拼接、去冗得到非冗余EST序列3453条,含SSR位点的EST序列267条,共307个SSR位点,检出率为8.89%,平均每6.83kb出现1个SSR位点。设计了40对EST-SSR引物并进行PCR扩增,29对引物能扩增出理想的PCR产物,其中多态性引物14对。利用14对多态性引物分析了乐清湾缢蛏遗传多样性,共检测到等位基因数(Na)61个,每个位点的等位基因数为2—12个。二核苷酸、三核苷酸和四核苷酸重复是最主要的重复类型,分别占15.96%、37.13%和35.50%。乐清湾缢蛏群体观测杂合度(Ho)、期望杂合度(He)和多态信息含量(PIC)分别为0.569、0.490和0.449,表明乐清湾缢蛏遗传多样性较丰富。

鼻咽癌中染色体3p21区域一个表达下调的EST的鉴定

背景与目的:研究显示鼻咽癌细胞3p14-25存在高频率杂合性丢失位点。本研究拟寻找与筛选染色体3p21区域与鼻咽癌相关的表达序列标签(expressedsequencetag,EST),为定位候选克隆鼻咽癌相关新基因奠定基础。方法:充分利用网上的生物信息资源,采用定位查找ESTs,对ESTs进行同源性比较分析、筛选;运用逆转录PCR(reversetranscription-PCR,RT-PCR)方法,检测ESTs在鼻咽癌和正常鼻咽组织中的表达;并用Northernblot杂交方法,检测EST在人其他正常组织及肿瘤细胞系的表达状况。结果:在3p21区域筛选到一个在鼻咽癌中表达下调的EST(N31985),在60.00%(3/5)的鼻咽癌细胞株及47.06%(16/34)的鼻咽癌活检组织检测到有EST(N31985)表达下调,与正常鼻咽上皮组织相比较,差异有统计学意义(P<0.05)。结论:染色体3p21区域EST(N31985)在鼻咽癌中表达下调,提示其可能参与鼻咽癌癌变过程。

植物EST-SSR技术及其应用

EST是指从EST cDNA文库中随机挑取克隆进行单轮自动测序所获得的短的、常常是未加编译的cDNA序列。目前,大约有200种植物共超过1500多万条的EST的序列,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。分析和比较EST序列就可以得到关于基因的表达、功能及进化信息,利用大量的EST序列已经成为发现新基因及了解植物新陈代谢一种简便有效的方法。通过介绍基于植物表达序列标签(EST)的微卫星(SSR)标记的研究现状,并对一些植物中利用EST-SSR标记在遗传图谱构建、遗传多样性、通用性等方面的应用进行了综合评述。同时也提出利用EST存在的一些不足及发展趋势。

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,占筛选引物的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。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。