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Identification of ExpIdentification of expressed Resistance Gene Analogs from Peanut (Arachis hypogaea L.) expressed sequence tags

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Journal of Integrative Plant Biology 2013年第5期55卷 453-461页

作者： Zhanji Liu Suping Feng Manish K. Pandey Xiaoping Chen Albert K. Culbreath Rajeev K. Varshney Baozhu Guo University of Georgia Department of Plant Pathology Shandong Academy of Agricultural Sciences High-Tech Research Center Qiongzhou University College of Bioscience and Biotechnology International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Guangdong Academy of Agricultural Sciences Crops Research Institute US Department of Agriculture Agricultural Research ServiceCrop Protection and Management Research Unit

Low genetic diversity makes peanut （Arachis hypogaea L.） very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat （NBS-LRR） resistance genes （R） have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs （RGAs） from peanut expressed sequence tags （ESTs） and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats （SSRs） were identified from 25 peanut expressed RGAs. One SSR polymorphic marker （RGA121） was identified. Two polymerase chain reaction-based markers （Ahsw-1 and Ahsw-2） developed from RGA013 were homologous to the Tomato Spotted Wilt Virus （TSWV） resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.

关键词： Arachis hypogaea expressed sequence tags resistance gene analogs Tomato Spotted Wilt Virus.

Analysis of expressed sequence tags （ESTs） from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis

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Insect Science 2015年第6期22卷 761-767页

作者： Hui Li Kun-Ling Lang Hai-Bin Fu Chang-Peng Shen Fang-Hao Wan Dong Chu Key Lab of Integrated Crop Pest Management of Shandong Province College of Agronomy and Plant Protection Qingdao AgriculturalUniversity Qingdao Shandong Province China Shenyang Entry-Exit Inspection and Quarantine Bureau Shenyang China StateKey Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences(CAAS) Beijing China

The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags （ESTs） were analyzed from a normalized complementary DNA library of/？. solenopsis. In addition, EST-derived microsatellite loci （also known as simple sequence repeats or SSRs） were isolated and characterized. A total of 1107 high-quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST-based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution.

关键词： expressed sequence tags microsatellite marker normalized cDNA library Phenacoccus solenopsis

Novel expressed sequence tags of an alpine-cold plant species,Gymnadenia conopsea (Orchidaceae)

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Research in Cold and Arid Regions 2010年第2期2卷 162-167页

作者： JuQing Jia JianPing Zhou Tao Zhang ZhengNan Wen GuangRong Li ZuJun Yang School of Life Science and Technology University of Electronic Science and Technology of ChinaChengduSichuan 610054China

Gymnadenia conopsea,an alpine Orchidaceae plant,was one of the widely used Tibetan traditional medicines.In this study,we sequenced total 105 expressed sequence tags (ESTs) from a full-length cDNA expression library constructed by the Oligo-capping technique.The further bioinformatic analyses suggested that the 65 represented unique sequences showed high homology to previously identified genes in other plants:30 sequences matched to other uncharacterized expressed sequence tags (ESTs),and 10 sequences showed no good matches to available sequences in DNA databases.Gene ontology annotation by InterProScan indicated that many of these cDNAs (7 percent) have no known molecular functions and may be unique to G.conopsea.Fifty-five ESTs with matched proteins were involved in a series of diverse functions,in which molecular function such as 'binding' (42.9 percent) and 'catalytic activity' (25.0 percent) were the most frequent functions of the cDNAs.This cDNA library provided a critical basis for further investigation of functional genes expression under cold stress in this alpine species.In addition,13 ESTs-based polymerase chain reaction (PCR) primers were designed and can also be used for genotypic identification and for the genetic diversity analysis of G.conopsea and its closely related species.

关键词： expressed sequence tags Gymnadenia conopsea Alpine-cold plant

Construction of a cDNA library and preliminary analysis of the expressed sequence tags of the earthworm Eisenia fetida(Savigny, 1826)

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Construction of a cDNA library and preliminary analysis of t...

Construction of cDNA library and Analysis on expressed sequence tags of Polygonum multiflorum Thunb.leaf tissue

中国生物化学与分子生物学会2019年全国学术会议暨学会...

作者： Chang Liu Xuan Wang Rui Guo Bao Feng Yu Bo Niu Department of Biochemistry and Molecular Biology Shanxi Medical University Department of Biotechnology Beijing Municipal Key Laboratory of Child Development and Nutriomics Capital Institute of Pediatrics

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5’End of RNA Template technology. Construction of a cDNA library and analysis of expressed sequence tags(ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits(P value sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a high quality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms.

关键词： Earthworm Eisenia fetida（Savigny,1826） cDNA library expressed sequence tags GO annotation KEGG pathway

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Construction of cDNA library and Analysis on Expressed Seque...

Identification of microsatellites in cattle unigenes

2010年广东省药师周大会

作者： TAN Xue-Mei1),SHEN Yan-Jing1),YAN Ping2),ZHENG Chuan-jin1),ZHAO Shu-jin1) (1).Department of Pharmacy,Guangzhou General Hospital of Guangzhou Military Command,Guangzhou 510010,China 2).Guangdong Institute for Drug Control,Guangzhou 510180,China)

To construct a cDNA library of mature leaves( Polygonum multiflorum Thunb.),so as to further research the gene regulation of secondary metabolite biosynthesis of offcinal.Total RNA was extracted with improved CTAB method and mRNA was purified by oligo(dT)-cellulose column chromatography,which were synthesized to double strands through reverse transcription。After blunting the cDNA termini,the 5’end of EcoR I adapters phosphorylated were conjoined and then digested by Xho I。cDNA fragments less than 400bp were eliminated by Sepharose CL-2B spin column,and then ligated to Uni-λZAP XR vector.The mixture of ligation was finally packed into lambda virus with packaging extracts.Now the primary library had been finished,0.54×106 recombinations had been obtained,and the length of exogenous insert at about 0.5～2.0kb。The amplified library had 4.25×1011 recombinations and the rate of recombination was 98.5%。16 bacteriophage plaques were randomly selected to identify the size range of inserts by PCR。Continued to pick 12 bacteriophage plaques and extracted pBluescript SKphasmids ,which were inverted by the action of ExAssist helper phage。Phasmids,which extracted by alkaline lysis,digested via EcoR I and Xho I and identified the size of inserts.Respectively used the method of PCR and double enzymes digestion to identify the size of inserts.At present,30 positive clones in vivo excision of the pBluescript phagemid from the UniλZAP XR vector.had been picked and extracted plasmid to carry out to sequence 3’ESTs.The results indicated that the cDNA library of Polygonum multiflorum Thunb.had enough volume for screening desired genes .Totally,it was the first time to construct a cDNA library and sequence 3’-ESTs of Polygonum multiflorum thunb.,it should enrich the plant genome research database and promote the study of gene regulation of secondary metabolite biosynthesis of offcinal,which lays a foundation for functional gene screening and analysis.

关键词： Polygonum multiflorum Thunb. cDNA library functional gene expressed sequence tags

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Journal of Genetics and Genomics 2008年第5期35卷 261-266页

作者： Qiuliang Yan Yinghan Zhang Hongbin Li Caihong Wei Lili Niu Shan Guan Shangang Li Lixin Du College of Animal Science and Technology NorthwestA ＆ F University Yangling 712100 China National Center for Molecular Genetics and Breeding of Animal Institute of Animal Sciences Chinese Academy of Agricultural Sciences Beijing 100094 China Institute of Crop Sciences Chinese Academy of Agricultural Sciences Beijing 100081 China

To identify EST-SSR molecular markers, 41,986 cattle UniGene sequences from NCBI were mined for analyzing SSRs. A total of 1,831 SSRs were identified from 1,666 ESTs, which represented an average density of 19.88 kb per SSR. The frequency of EST-SSRs was 4.0%. The dinucleotide repeat motif was the most abundant SSR, accounting for 54%, followed by 22%, 13%, 7% and 4%, respectively, for tri-, hexa-, penta- and tetra-nucleotide repeats. Depending upon the length of the repeat unit, the length of microsatellites varied from 14 to 86 bp. Among the di- and tri-nucleotide repeats, AC/TG （57%） and AGC （12%） were the most abundant type. Annotation of EST-SSRs was also carried out. Three hundred primer pairs were randomly designed using Prime Premier 5.0 program and Oligo 5.0 for further experimental validation.

关键词： cattle microsatellite expressed sequence tags EST-SSR

RNA-seq analysis of Paris polyphylla var.yunnanensis roots identified candidate genes for saponin synthesis

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Plant Diversity 2016年第3期38卷 163-170页

作者： Tao Liu Xiaoxian Li Shiqing Xie Ling Wang Shengchao Yang Yunnan Research Center on Good Agricultural Practice for Dominant Chinese Medicinal Materials Yunnan Agricultural University Kunming Institute of Botany Chinese Academy of Sciences

Paris polyphylla Smith var.yunnanensis(Franch.) Hand.-Mazz.is a rhizomatous,herbaceous,perennial plant that has been used for more than a thousand years in traditional Chinese medicine.It is facing extinction due to overharvesting.Steroids are the major therapeutic components in Paris roots,the commercial value of which increases with age.To date,no genomic data on the species have been available.In this study,transcriptome analysis of an 8-year-old root and a 4-year-old root provided insight into the metabolic pathways that generate the steroids.Using Illumina sequencing technology,we generated a high-quality sequence and demonstrated de novo assembly and annotation of genes in the absence of prior genome information.Approximately 87,577 unique sequences,with an average length of 614 bases,were obtained from the root cells.Using bioinformatics methods,we annotated approximately 65.51% of the unique sequences by conducting a similarity search with known genes in the National Center for Biotechnology Information's non-redundant database.The unique transcripts were functionally classified using the Gene Ontology hierarchy and the Kyoto Encyclopedia of Genes and Genomes database.Of 3082 genes that were identified as significantly differentially expressed between roots of different ages,1518(49.25%) were upregulated and 1564(50.75%) were downregulated in the older root.Metabolic pathway analysis predicted that 25 unigenes were responsible for the biosynthesis of the saponins steroids.These data represent a valuable resource for future genomic studies on this endangered species and will be valuable for efforts to genetically engineer P.polyphylla and facilitate saponin-rich plant development.

关键词： P.polyphylla var.yunnanensis Saponin pathway Differential expression Transcriptome analysis expressed sequence tags

EST-based Analysis of Gene Expression in the Porcine Brain

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Genomics, Proteomics & Bioinformatics 2004年第4期2卷 237-244页

作者： BingZhang WUJin YanwuZeng ZhixiSu SongnianHu JunYu JamesD.WatsonInstituteofGenomeSciences ZhejiangUniversity／HangzhouGenomicsInstituteHangzhou310008China TheSecondAffiliatedHospitalofChinaMedicalUniversity Shenyang110004China JamesD.WatsonInstituteofGenomeSciences ZhejiangUniversity／HangzhouGenomicsInstituteHangzhou310008China／／BeijingGenomicsInstituteChineseAcademyofSciencesBeijing101300China

Since pig is an important livestock species worldwide, its gene expressionhas been investigated intensively, but rarely in brain. In order to study gene expression profilesin the pig central nervous system, we sequenced and analyzed 43,122 high-quality 5'' end expressedsequence tags (ESTs) from porcine cerebellum, cortex cerebrum, and brain stem cDNA libraries,involving several different prenatal and postnatal developmental stages. The initial ESTs wereassembled into 16,101 clusters and compared to protein and nucleic acid databases in GenBank. Ofthese sequences, 30.6% clusters matched protein databases and represented function known sequences;75.1% had significant hits to nucleic acid databases and partial represented known function; 73.3%matched known porcine ESTs; and 21.5% had no matches to any known sequences in GenBank. We used thecategories defined by the Gene Ontology to survey gene expression in the porcine brain.

关键词： pig brain expressed sequence tags gene identification

Erwinia carotovora ssp. carotovora Infection Induced ＂Defense Lignin＂ Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage （Brassica rapa L. ssp. pekinensis）

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Journal of Integrative Plant Biology 2007年第7期49卷 993-1002页

作者： Song-He Zhang Qing Yang Rong-Cai Ma College of Life Sciences Nanjing Agricultural University Nanjing 210095 China Beijing AgroBiotechnology Research Center Beijing Academy of Agriculture and Forestry Sciences Beijing 100097 China

Erwinia carotovora subsp, carotovora （Ecc） infects and causes soft rot disease in hundreds of crop species including vegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogen infection in plants. In this work, variations of lignin content and gene expression in the molecular interaction between Chinese cabbage and Ecc were investigated. H2O2 accumulation and peroxidase activity were detected by 3, 3＇- Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Klason lignin content in inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 h after inoculation, respectively. Gas chromatography detected more p-coumaryl （H） and less coniferyl （G） and sinapyl （S） monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced ＂defense lignin＂ were composed of more G and H monolignins, and less S monolignin. After searching the expressed sequence tags （EST） data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis were selected to study their expression. All of these genes could be induced by mock inoculation and Ecc infection, while the gene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our results indicated that ＂defense lignin＂ was different from the developmental lignin in composition; G and S monolignins were significantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated and played a role in plant response to the soft rot Ecc.

关键词： Chinese cabbage Erwinia carotovora ssp. carotovora expressed sequence tags gene expression lignin.