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首頁(yè)> 外文學(xué)位 >Characterization of INF56, a gene expressed during infection structure development of the bean rust fungus, Uromyces appendiculatus.
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Characterization of INF56, a gene expressed during infection structure development of the bean rust fungus, Uromyces appendiculatus.

機(jī)譯:INF56的特征,INF56是在豆類(lèi)銹菌烏頭霉菌感染結(jié)構(gòu)發(fā)育過(guò)程中表達(dá)的基因。

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摘要

Expression of the INF56 gene is induced after the initiation of appressorium formation in bean rust germlings, Uromyces appendiculatus. The objectives of the research are to characterize the gene, and to investigate its possible function in infection structure differentiation.; INF 56 was first subcloned as a 2.6kb fragment and sequenced. The location of the 1.0kb transcript was determined by S1 nuclease protection and primer extension. A TATA box is located 38bp upstream and a CAAT box 130bp upstream of the major transcription initiation site. The gene contains two open reading frames, ORF1 and ORF2. ORF1 is predicted to encode a 14.1kD polypeptide. The amino acid sequence is rich in Ser, Gly and Pro, and it has certain sequence similarity to a functional domain of mammalian cytokeratin type II. ORF2 encodes a 10.1kD polypeptide, is rich in Pro, and it has certain sequence similarity to the cell surface recognition region of chicken fibronectin. ORF1 is more biased than ORF2 for pyrimidine in the third position of each codon. The two open reading frames share one 67bp intron. As identified by hybrid-selection and in vitro translation system, the gene codes for two polypeptides with apparent molecular weight of 15.5kD and 23kD on SDS-PAGE. The gene does not share homology with other plant pathogenic Ascomycetes and Basidiomycetes except for cowpea rust, Uromyces vignae, a subspecies of bean rust.; Sequence comparison between INF56 genomic DNA and cDNA revealed some base changes (1%) randomly spread throughout the coding region of the gene. Southern analyses of genomic DNA probed with the cDNA showed that the rust genome contains at least four copies of the gene. Since the uredospore is dikaryotic, this indicated that there may be four alleles in the bean rust genome, two alleles located on each of two loci. Further evidence suggested that the INF56 gene family is relatively homologous with a few restriction site differences. All copies contain the intron.; The INF56 cDNA was fused with TrpE promoter in a pATH expression vector. Two fusion proteins derived from ORF1 and ORF2 of INF56 were expressed in E. coli, isolated and purified individually for induction of specific antibodies. The antibody will be characterized using Western blots and will be used for cytological studies in the future.
機(jī)譯:在豆銹菌的幼蟲(chóng),Uromyces appendiculatus中,發(fā)生了內(nèi)生器官的形成,誘導(dǎo)了INF56基因的表達(dá)。該研究的目的是表征該基因,并研究其在感染結(jié)構(gòu)分化中的可能功能。首先將INF 56亞克隆為2.6kb片段并測(cè)序。通過(guò)S1核酸酶保護(hù)和引物延伸確定1.0kb轉(zhuǎn)錄物的位置。 TATA盒位于主要轉(zhuǎn)錄起始位點(diǎn)上游38bp,CAAT盒位于130bp上游。該基因包含兩個(gè)開(kāi)放閱讀框,ORF1和ORF2。預(yù)測(cè)ORF1編碼14.1kD多肽。該氨基酸序列富含Ser,Gly和Pro,并且與II型哺乳動(dòng)物細(xì)胞角蛋白的功能域具有一定的序列相似性。 ORF2編碼10.1kD多肽,富含Pro,并且與雞纖連蛋白的細(xì)胞表面識(shí)別區(qū)域具有一定的序列相似性。在每個(gè)密碼子的第三個(gè)位置,嘧啶的ORF1比ORF2更偏。兩個(gè)開(kāi)放閱讀框共享一個(gè)67bp的內(nèi)含子。如通過(guò)雜交選擇和體外翻譯系統(tǒng)所鑒定,該基因編碼在SDS-PAGE上具有表觀分子量為15.5kD和23kD的兩種多肽。該基因與其他植物病原體子囊菌和擔(dān)子菌沒(méi)有同源性,除了cow豆銹,豆腐亞種Uromyces vignae。 INF56基因組DNA和cDNA的序列比較顯示一些堿基變化(1%)隨機(jī)分布在基因的整個(gè)編碼區(qū)域。用該cDNA探測(cè)的基因組DNA的Southern分析表明,銹基因組至少包含該基因的四個(gè)拷貝。由于脲孢子是雙核的,這表明在豆銹基因組中可能存在四個(gè)等位基因,兩個(gè)等位基因位于兩個(gè)基因座的每個(gè)基因座上。進(jìn)一步的證據(jù)表明,INF56基因家族相對(duì)同源,具有一些限制性位點(diǎn)差異。所有副本均包含內(nèi)含子。在pATH表達(dá)載體中將INF56 cDNA與TrpE啟動(dòng)子融合。兩種衍生自INF56的ORF1和ORF2的融合蛋白在大腸桿菌中表達(dá),分別進(jìn)行分離和純化以誘導(dǎo)特異性抗體。該抗體將使用Western印跡進(jìn)行表征,并將在將來(lái)用于細(xì)胞學(xué)研究。

著錄項(xiàng)

  • 作者

    Xuei, Xiaoling.;

  • 作者單位

    Cornell University.;

  • 授予單位 Cornell University.;
  • 學(xué)科 Agriculture Plant Pathology.; Biology Molecular.; Biology Genetics.
  • 學(xué)位 Ph.D.
  • 年度 1991
  • 頁(yè)碼 153 p.
  • 總頁(yè)數(shù) 153
  • 原文格式 PDF
  • 正文語(yǔ)種 eng
  • 中圖分類(lèi) 植物病理學(xué);分子遺傳學(xué);遺傳學(xué);
  • 關(guān)鍵詞

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