Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Expressed sequence tags of young floral buds and characterization of a bud- preferential lectin- like cDNA fromPharbitis nil

To identify the genes involved in flower development, we analyzed 207 expressed sequence tags (ESTs) from a young floral bud cDNA library ofPharbitis nil. Of these, 87 clones (42%) showed significant homology to known protein sequences in the NCBI database. Four of these had not been reported previously in the plant kingdom, indicating that 1.9% of the ESTs were newly identified in plants. Functional categorization revealed that the genes involved in metabolic pathways, such as glycolysis and photosynthesis, were most abundant Reverse-northern and northern analyses showed that one clone,PnFP161, was expressed preferentially in floral buds. DNA sequence analysis indicated that this clone contained 147 bp of 5′-UTR, 264 bp of -UTR, and an open reading frame of 233 amino acids, thereby sharing 33% identity with a lectin fromCalystegia sepium. The C-terminal regionof PnFPI61 had well-conserved residue with that of the lectins. Southern blot analysis demonstrated thatPnFPI61 exists as a multigene family.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

Expressed sequence tags from the red imported fire ant, Solenopsis invicta: annotation and utilization for discovery of viruses

An expression library was created and 2304 clones sequenced from a monogyne colony of Solenopsis invicta. The primary intention of the project was to utilize homologous gene identification to facilitate discovery of viruses infecting this ant pest that could potentially be used in pest management. Additional genes were identified from the ant host and associated pathogens that serve as an important resource for studying these organisms. After assembly and removal of mitochondrial and poor quality sequences, 1054 unique sequences were yielded and deposited into the GenBank database under Accession Nos. EH412746 through EH413799. At least nine expressed sequence tags (ESTs) were identified as possessing microsatellite motifs and 15 ESTs exhibited significant homology with microsporidian genes. These sequences most likely originated from Thelohania solenopsae, a well-characterized microsporidian that infects S. invicta. Six ESTs exhibited significant homology with single-stranded RNA viruses (3B4, 3F6, 11F1, 12G12, 14D5, and 24C10). Subsequent analysis of these putative viral ESTs revealed that 3B4 was most likely a ribosomal gene of S. invicta, 11F1 was a single-stranded RNA (ssRNA) virus contaminant introduced into the colony from the cricket food source, 12G12 appeared to be a plant-infecting tenuivirus also introduced into the colony as a field contaminant, and 3F6, 14D5, and 24C10 were all from a unique ssRNA virus found to infect S. invicta. The sequencing project illustrates the utility of this method for discovery of viruses and pathogens that may otherwise go undiscovered.     

Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 198–206.Original English Text Copyright © 2005 by Z. Zhang, F. Zhang, Tang, Pi, Zheng.This article was submitted by the authors in English.     

Expressed sequence tags from persimmon at different developmental stages

Persimmon (Diospyros kaki Thunb.) is an important fruit in Asian countries, where it is eaten as a fresh fruit and is also used for many other purposes. To understand the molecular mechanism of fruit development and ripening in persimmon, we generated a total of 9,952 expressed sequence tags (ESTs) from randomly selected clones of two different cDNA libraries. One cDNA library was derived from fruit of “Saijo” persimmon at an early stage of development, and the other from ripening fruit. These ESTs were clustered into 6,700 non-redundant sequences. Of the 6,700 non-redundant sequences evaluated, the deduced amino acid sequences of 4,356 (65%) showed significant homology to known proteins, and 2,344 (35%) showed no significant similarity to any known proteins in Arabidopsis databases. We report comparison of genes identified in the two cDNA libraries and describe some putative genes involved in proanthocyanidin and carotenoid synthesis. This study provides the first global overview of a set of genes that are expressed during fruit development and ripening in persimmon.     

Expressed Sequence Tag Analysis of Vanadocytes in a Vanadium-Rich Ascidian, <Emphasis Type="Italic">Ascidia sydneiensis samea</Emphasis>

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10⁷ times higher than that in seawater. In these species signet ring cells, with a single huge vacuole in which vanadium ion is contained, function as vanadium-accumulating cells, vanadocytes. To investigate the mechanism underlying this phenomenon, we performed an expressed sequence tag (EST) analysis of a complementary DNA library from vanadocytes of a vanadium-rich ascidian, Ascidia sydneiensis samea. We determined the nucleotide sequences of 1000 ESTs and performed a BLAST analysis against the SwissProt database. We found 93 clones of metal-related gene homologues, including the ferritin heavy subunit, hemocyanin, and metallothionein. Two ESTs, in particular, exhibited significant similarity to vanabins that have been extracted from A. sydneiensis samea blood cells as low molecular weight vanadium-binding proteins. We have named the genes encoding these ESTs vanabin3 and vanabin4. Immobilized metal ion affinity chromatography revealed that these novel vanabin homologues bind vanadium(IV) ions.     

Genes expressed in zoospores of<Emphasis Type="Italic"> Phytophthora nicotianae</Emphasis>

The genus Phytophthora includes many highly destructive plant pathogens. In many Phytophthora species, pathogen dispersal and initiation of plant infection are achieved by motile, biflagellate zoospores that are chemotactically attracted to suitable infection sites. In order to study gene expression in zoospores, we have constructed a cDNA library using mRNA from zoospores of Phytophthora nicotianae. The library was arrayed and screened using probes derived from mycelium or zoospore mRNA. More than 400 clones representing genes preferentially expressed in zoospores were identified and sequenced from the 5 end of the insert. The expressed sequence tags (ESTs) generated were found to represent 240 genes. The ESTs were compared to sequences in GenBank and in the Phytophthora Genome Consortium database, and classified according to putative function based on homology to known proteins. To further characterize the identified genes, a colony array was created on replicate nylon filters and screened with probes derived from four Phytophthora developmental stages including zoospores, germinating cysts, vegetative mycelium and sporulating hyphae, and from inoculated and uninoculated tobacco seedlings. Data from sequence analysis and colony array screening were compiled into a local database, and searched to identify genes that are preferentially expressed in zoospores for future functional analysis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by C. A. M. J. J. van den Hondel     

Frequency, type, distribution and annotation of simple sequence repeats in Rosaceae ESTs

Genomic resources for peach, a model species for Rosaceae, are being developed to accelerate gene discovery in other Rosaceae species by comparative mapping. Simple sequence repeats (SSRs) are an important tool for comparative mapping because of their high polymorphism and transportability. To accelerate the development of SSR markers, we analyzed publicly available Rosaceae expressed sequence tags (ESTs) for SSRs. A total of 17,284 ESTs from almond, peach and rose were assembled into putatively non-redundant EST sets. For comparison, 179,099 ESTs from Arabidopsis were also used in the analysis. About 4% of the assembled ESTs contained SSRs in Rosaceae, which was higher than the 2.4% found in Arabidopsis. About half of the SSRs were found in the putative UTR, and the estimated average distance between SSRs in the UTR was 5.5 kb in rose, 5.1 kb in almond, 7 kb in peach and 13 kb in Arabidopsis. In the putative coding region, the estimated average distance was two to four times longer than in the UTR. Rosaceae ESTs containing SSRs were functionally annotated using the GenBank nr database and further classified using the gene ontology terms associated with the matching sequences in the SwissProt database. The detailed data including the sequences and annotation results are available from .     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Construction and characterization of a cDNA library from floral organs and fruitlets of <Emphasis Type="Italic">Citrus reticulata</Emphasis>

To explore and isolate genes related to flowering and fruit development, we constructed a cDNA library from floral organs and fruitlets of Ponkan mandarin (Citrus reticulata Blanco). A total of 661 high-quality expressed sequence tags (ESTs) were generated and submitted to GenBank with the accession numbers from GO343532 to GO344192. All these ESTs were assembled into 43 contigs and 296 singletons (totally 339 unigenes). The BLAST2GO software was employed to annotate the unigenes, among which 77 ones had no significant homology with the sequences in NCBI non-redundant proteins database by BLASTX analysis. Additionally, gene ontology (GO) analysis revealed an overview of sequences distribution, which implied some specially expressed genes related to flower and fruit development. Furthermore, some abundantly expressed unigenes involved in several crucial metabolic pathways related to fruit quality were highlighted and three types of homologues of miraculin-like protein2 were analyzed by both semiquantitative RT-PCR and real-time PCR. The results showed different expression profiles of these genes, which meant that they contribute distinctly to fruit development.