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Classification, Expression Pattern, and E3 Ligase Activity Assay of Rice U-Box-Containing Proteins

Molecular Plant 2008年第5期1卷 800-815页

作者： Li-Rong Zenga Chan Ho Park R.C. Venu Julian Gough Guo-Liang Wang Department of Plant Pathology Plant Molecular Biology and Biotechnology Program The Ohio State University 2021 Coffey Rd 201 Kottman Hall Columbus OH 43210 USA Present address: Boyce Thompson Institute for Plant Research Tower Rd Ithaca NY 14853 USA Department of Computer Science University of Bristol Woodland Rd Bristol BS8 1UB UK Crop Gene Engineering Key Laboratory of Hunan Province Hunan Agricultural University Changsha Hunan 410128 China

Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification. The U-box is a recently identified, ubiquitin ligase activityrelated protein domain that shows greater presence in plants than in other organisms. In this study, we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms. Most of the U-box protein genes are expressed, as supported by the identification of their corresponding expressed sequence tags （ests）, full-length cDNAs, or massively parallel signature sequencing （MPSS） tags. Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome. The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions. Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations. The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins. Cell death assay using the rice protoplast system suggests that one rice U-box gene, OsPUB51, might act as a negative regulator of cell death signaling. In addition, the selected U-box proteins were found to be functional E3 ubiquitin ligases. The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.

关键词： ests MPSS U-Box-Containing

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Isolation and characterization of a sterile-dwarf mutant in Asian cotton (Gossypium arboreum L.)

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Journal of Genetics and Genomics 2009年第6期36卷 343-353页

作者： Chuntai Wu Baoliang Zhou Tianzhen Zhang National Key Laboratory of Crop Genetics and Germplasm Enhancement Cotton Research Institute Nanjing Agricultural University Nanjing 210095 China

Plant height is an important trait in cotton. To elucidate the molecular mechanisms of the dwarf phenotype, a sterile-dwarf mutant derived from Gossypium arboreum L. cv. Jinhuazhongmian was developed by ^60Co y-ray irradiation. The results demonstrated that the steriledwarf mutant phenotype was controlled by a pair of recessive gene, which was designated sd^a. Plants carrying the sd^a gene contained lower levels of indole-3-acetic acid （IAA） and abscisic acid （ABA） compared with wild-type （WT） plants. The chlorophyll content and net photosynthetic rate in mutant leaves were markedly decreased. However, it was possible that ABA biosynthesis or signaling was involved in governing the sd^a phenotype. Semi-quantitative RT-PCR analysis detected 13 differentially expressed ests, and the steriledwarf mutant exhibited decreased expression levels relative to the WT. The role of nine potential hormone biosynthetic genes in the synthesis of IAA, ABA, polyamines （PAs） and jasmonic acid （JA） were discussed.

关键词： Gossypium arboreum sd^a auxin abscisic acid ests

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Discovery of immune related factors in Fenneropenaeus chinensis by annotation of ests

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Progress in Natural Science:Materials International 2004年第1期14卷 48-55页

作者： SHEN Yaoqing 1,2 , XIANG Jianhai 1*, WANG Bing 1,2 , LI Fuhua 1 and TONG Wei 3(1. Experimental Marine Biology Laboratory, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071,China 2. Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 3. Genomics & Bioinformatics Institute, Chinese Academy of Sciences, Beijing 101300, China) Chinese Acad Sci Inst Oceanol Expt Marine Biol Lab Qingdao 266071 Peoples R China Chinese Acad Sci Grad Sch Beijing 100039 Peoples R China Chinese Acad Sci Geonom & Bioinformat Inst Beijing 101300 Peoples R China

A total of 10446 expressed sequence tags (ests) are obtained by a large scale sequencing of a cDNA library from cephalothorax of adult Fenneropenaeus chinensis . An EST analysis platform was built up based on local computers and bioinformatic techniques were used to annotate these ests in order to promptly find possible functional genes, especially for immune related factors. About 4% of the ests show similarity to the coding sequences of such factors, including lectin, serine protease, serpin, lysozyme, etc. These ests provide a partial profile of the immune system in F. chinensis and useful information for further study on these genes.

关键词： Fenneropenaeus chinensis bioinformatics ests immune factors

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

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Chinese Journal of Oceanology and Limnology 2010年第1期28卷 26-36页

作者：牛建峰胡海岩胡松年王广策彭光孙松 Key Laboratory of Experimental Marine Biology Institute of OceanologyChinese Academy of Sciences Key Laboratory of Genome Sciences and Information Beijing Institute of GenomicsChinese Academy of Sciences Graduate University of Chinese Academy of Sciences College of Marine Science and Engineering Tianjin University of Science and Technology

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.Müller),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 10 072 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).

关键词：表达序列标签绿藻门细胞色素C氧化酶 ests cDNA文库系统发育分析光合作用 EST序列

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Isolation, Cloning, and Identification of Expressed Sequence Tags from the Backfat Tissue in Duroc and Tongcheng Pigs by mRNA Differential Display

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Agricultural Sciences in China 2006年第2期5卷 141-145页

作者： REN Zhu-qing XIONG Yuan-zhu DENG Chang-yan LEI Ming-gang ZUO Bo LI Feng-e ZHENG Rong XU De-quan Key Laboratory of Swine Breeding and Genetics Ministry of Agriculture/Huazhong Agricultural University Wuhan 430070 P.R.China

mRNA differential display technique was performed to discuss the differential expression of genes in fat tissue between introduced European and Chinese indigenous pigs. Four anchor primers in combination with five arbitrary primers （20 sets in total） were used and nearly 300 bands were observed in polyacrylamide gel, among which 29 differential display bands were obtained. Twelve of 29 cDNA fragments were identified using reverse Northern dot blot, and subsequently cloned and sequenced. Eight of 12 cDNAs had no matches in GenBank and were submitted to GenBank, and the other 4 showed similarity to identified genes from GenBank. Three among 8 novel ests were selected to be further identified by semiquantitative RT-PCR. In our experiment, silver staining DDRT-PCR and DIG primer DNA labeling reverse Northern dot blot were used to avoid radioactive pollution. The result showed that the expressions of 5 among 8 novel ests were stronger in the backfat of Tongcheng pigs and the others were weaker than that in Duroc pigs. These novel ests were prepared for selecting genes related to adipose cells.

关键词： ests fat tissue reverse Northern dot blot semi-quantitative RT-PCR mRNA differential display

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滇重楼全长cDNA文库的构建及初步分析

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中药材 2014年第1期37卷 22-25页

作者：赵爽董栩马腾云南中医学院中药学院云南昆明650500

目的:构建滇重楼全长cDNA文库,为进行滇重楼生长发育相关基因及次生代谢产物合成途径相关基因的研究奠定基础。方法:改良Trizol法提取滇重楼根茎总RNA,通过SMART(switching mechanism at 5'end of RNA transcript)构建全长cDNA文库,测... 详细信息

目的:构建滇重楼全长cDNA文库,为进行滇重楼生长发育相关基因及次生代谢产物合成途径相关基因的研究奠定基础。方法:改良Trizol法提取滇重楼根茎总RNA,通过SMART(switching mechanism at 5'end of RNA transcript)构建全长cDNA文库,测定分析文库滴度、重组率及插入片段大小,对文库进行随机测序并通过Blastx分析后在GenBank中进行同源性检索和功能预测。结果:文库的库容为2.5×107cfu/mL,文库重组率98.5%,插入片段大小平均为1.5 kb。随机挑取192个单克隆测序,其中有149条ests(Expressed Sequence Tags),其中9条序列与生长发育有关,5条序列与滇重楼次生代谢产物的合成、运输与代谢有关。结论:利用SMART技术成功构建了滇重楼全长cDNA文库,该文库库容量大、重组率高且插入的cDNA片段较长,可以满足滇重楼功能基因的鉴定、筛选及表达调控研究。

关键词：滇重楼全长cDNA文库 SMART ests

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水稻14-3-3蛋白家族的生物信息学分析

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Acta Genetica Sinica 2005年第7期32卷 726-732页

作者：金谷雷汪旭升朱军浙江大学生物信息学研究所杭州310029

通过隐马尔柯夫模型(HiddenMarkovModel,HMM),对粳稻(OryzasativaL.ssp.japonica)基因组的蛋白质数据库进行搜索,结果获得8个1433蛋白的同源序列,其中发现4个新基因。通过对所有粳稻的1433蛋白的DNA序列与各种表达序列标签(ExpressionSe... 详细信息

通过隐马尔柯夫模型(HiddenMarkovModel,HMM),对粳稻(OryzasativaL.ssp.japonica)基因组的蛋白质数据库进行搜索,结果获得8个1433蛋白的同源序列,其中发现4个新基因。通过对所有粳稻的1433蛋白的DNA序列与各种表达序列标签(ExpressionSequenceTags,ests)进一步比对,为1433蛋白找到了ests的证据。结果说明这些基因在水稻不同的处理和不同的部位都有所表达,而且不同成员之间的表达模式存在较大的差异。蛋白质多序列联配分析结果表明,存在可能的功能多态位点。通过基因结构和染色体定位的分析,确认了水稻基因组中存在ε样和非ε样两类1433蛋白。此外,对目前植物中的1433家族作了初步的进化分析。

关键词： 14-3-3蛋白生物信息学分析家族隐马尔柯夫模型 Markov 蛋白质数据库表达序列标签 Model DNA序列染色体定位水稻基因组同源序列 ests 表达模式分析结果多态位点基因结构进化分析行搜索 sp. 新基因粳稻植物

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甜菜无融合生殖系花期差异表达基因cDNA文库的构建

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高技术通讯 2006年第9期16卷 954-958页

作者：于冰李海英马春泉张绍军李荣田郭德栋黑龙江大学生命科学学院哈尔滨150080

为了克隆M14（M14是栽培甜菜与白花甜菜杂交产生的单体附加系,由于附加的白花甜菜第9号染色体上存在无融合生殖基因,其传递率可高达97.5%）品系中无融合生殖相关基因,应用抑制消减杂交方法,在甜菜开花的关键时期减数分裂期取样,建立了... 详细信息

为了克隆M14（M14是栽培甜菜与白花甜菜杂交产生的单体附加系,由于附加的白花甜菜第9号染色体上存在无融合生殖基因,其传递率可高达97.5%）品系中无融合生殖相关基因,应用抑制消减杂交方法,在甜菜开花的关键时期减数分裂期取样,建立了甜菜无融合生殖系M14与能够进行正常有性生殖的二倍体甜菜A2Y之间差异表达基因的cDNA文库,并对295个克隆进行了测序,共测得179个表达序列标签（Expressed Sequence Tags,ests）,通过对生物信息学数据库资料的查询,共得到了89个与已知基因同源的ests,90个新的ests,选择其中2个EST进行了RT-PCR鉴定,证明在M14中特异表达.以上结果为以后克隆与无融合生殖相关的全长基因打下了基础.

关键词：甜菜无融合生殖抑制消减杂交 ests

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A Comparative Study of ests Induced under Drought and Salinity Stress in Hyacinth Bean (<i>Lablab purpureus</i>)

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American Journal of Plant Sciences 2021年第5期12卷 840-857页

作者： S. Kokila Varadahally R. Devaraj Department of Biochemistry Central college campus Bengaluru City University Bengaluru India

Abiotic stressors like drought and salinity are major causes for loss of agricultural productivity. Comparison of expressed sesquence tags (ests) under different abiotic stresses provides insight into underlying mecha... 详细信息

Abiotic stressors like drought and salinity are major causes for loss of agricultural productivity. Comparison of expressed sesquence tags (ESTs) under different abiotic stresses provides insight into underlying mechanism of stress response, and candidate genes to improve tolerance to abiotic and biotic stresses via breading and transgenic methods. In order to identify and compare stress-specific ESTs from drought and salinity stressed Hyacinth Bean, ten days old seedlings were subjected to respective stresses and RNA was extracted from control and stressed leaves for EST identification. 31 and 12 ESTs, respectively, were characterized from leaves of drought and salinity stressed seedlings of Hyacinth Bean, Lablab purpureus by differential display RT-PCR using identical combinations of 48 primers and validated using quantitative RT-PCR. Relative fold expression was higher under salt stress than drought stress. Whereas 19 EST overexpressed under drought, 12 EST were down regulated. Of the 12 EST under salinity, 9 EST were downregulated and 3 EST upregulated. Putative functions predicted from sequence homology indicated that 11 drought specific EST corresponded to metabolic functions, and 4 of them corresponded to transcription regulation. Under salinity, 4 and 2 EST, respectively, corresponded to metabolic and RNA associated functions. Under both stresses, there were ests associated with unknown functions, whose characterization may throw light on the regulatory mechanism. Differing number of ests differentially expressed under drought and salt stress, and their predicted functionalities, suggested distinct set of response genes involved under these two stresses, despite a good number of physiological players being common. From the predicted functions of ests, the paper attempts to explain the possible mechanism of

关键词： Hyacinth Bean Drought Salinity DDRT-PCR ests

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环形泰勒虫感染细胞抑制性消减文库的构建及ests分析

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中国农业科学 2015年第22期48卷 4551-4563页

作者：赵洪喜刘军龙李有全杨聪山赵帅阳刘娟刘爱红谢俊仁田占成刘志杰刘光远殷宏关贵全罗建勋中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室江苏省动物重要疫病与人兽共患病防控协同创新中心宁夏大学农学院

【目的】环形泰勒虫(Theileria annulata)可以感染牛B淋巴细胞、树突状细胞和巨噬细胞,并可使被感染细胞发生转化,体外培养时可获得类似肿瘤样细胞的无限增殖能力。本研究通过构建环形泰勒虫转化和非转化宿主细胞抑制性消减文库,筛选环... 详细信息

【目的】环形泰勒虫(Theileria annulata)可以感染牛B淋巴细胞、树突状细胞和巨噬细胞,并可使被感染细胞发生转化,体外培养时可获得类似肿瘤样细胞的无限增殖能力。本研究通过构建环形泰勒虫转化和非转化宿主细胞抑制性消减文库,筛选环形泰勒虫裂殖体基因及转化细胞与宿主正常细胞之间的差异表达基因。【方法】以环形泰勒虫裂殖体转化细胞和非感染牛外周血单个核细胞(即非转化宿主细胞)为试验材料,提取总RNA和m RNA,反转录合成c DNA;然后分别作为tester和driver,进行抑制性消减杂交(SSH),构建环形泰勒虫转化和非转化宿主细胞抑制性消减文库;对随机挑取的阳性克隆进行测序及生物信息学分析,并利用实时荧光定量PCR对文库部分差异基因进行验证。【结果】通过对文库454个阳性克隆进行测序,共获得了有效表达序列标签(ests)364条,大小主要分布在350—1 200 bp之间;将这些ests序列在Gen Bank中进行BLASTn网上序列比对,其匹配于192条基因序列;其中包括环形泰勒虫序列60条(11条为虫体蛋白酶基因序列、10条为膜蛋白基因序列、7条为假设蛋白基因序列、5条为凋亡相关虫体蛋白基因序列、5条为核糖体蛋白基因序列、4条为细胞周期蛋白基因序列、4条为虫体抗原基因序列和虫体其它基因序列14条)、牛基因序列131条(其中19条为肿瘤相关蛋白基因序列)和1条未知序列;其中已有功能注释的ests 285条,再将已知功能的ests通过WEGO软件从生物学过程(biological process)、细胞组成(cellular component)和分子功能(molecular function)3个部分进行GO聚类分析,结果表明,宿主细胞差异基因主要集中在细胞、细胞进程、结合、催化、代谢进程、生物调节等功能方面。另外,通过对部分环形泰勒虫裂殖体转化宿主细胞差异基因进行荧光定量PCR分析,结果表明,肿瘤相关基因HCLS1和SENP5在转化宿主细胞中转录水平分别是非感染牛PBMCs(非转化宿主细胞)的2.06和1.32倍。【结论】利用SSH技术,成功构建了环形泰勒虫感染宿主细胞的抑制性消减文库,筛选获得了转化宿主细胞和环形泰勒虫裂殖体的表达基因,并通过荧光定量PCR分析获得了2个可能参与宿主细胞转化的基因,为进步探索环形泰勒虫与宿主细胞相互作用的分子机制奠定了基础。

关键词：环形泰勒虫抑制性消减杂交 ests

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