請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75743
標題: | 竹嵌紋病毒部分基因之研究 STUDIES ON THE PARTIAL GENOME OF BAMBOO MOSAIC VIRUS |
作者: | 劉秀鸞 |
出版年 : | 1990 |
學位: | 碩士 |
摘要: | 竹嵌紋病毒?Potexvirus之一員。SDS-PAGE分析結果顯示其具有一28KD的主要鞘蛋白,及一個26KD的次要蛋白;以Western電泳轉印法,發現二者皆與病毒鞘蛋白抗血清有免疫反應。竹嵌紋病毒基因組?單一正向的單股RNA,經電泳分析,估計其大小約?6.5Kb。試管內轉譯RNA可得大分子的產物,而在28KD處也有一明顯的蛋白帶,推測即?鞘蛋白。核?酸序列分析,係利用載體的特定引線(primer)及合成的寡核?酸(oligonucleotide, 17mer)當作引線,首先完成一長約2.3Kb之互補選殖株(cDNA clone)的序列分析;又直接以病毒RNA?範本定序,確定此cDNA係由病毒RNA所合成之互補選殖株。將此段cDNA進行試管內轉錄及轉譯作用,得到的主要蛋白分子量應可與整段cDNA所可譯讀產物大小相若,故此段cDNA可能不是單獨成?一完整的開放讀碼(open reading frame; ORF),而?一長ORF之一部分。以BV279,BV281及BV280?引線,作延伸反應,定出此段cDNA位置約位在距RNA5' 1Kb, 3' 3.2Kb處;同時,當以BV279及BV281?引線時,二者分別可合成約0.5kb及2.6Kb之較小DNA片段,顯示在RNA5'末端約0.5Kb處,似乎存在有一不尋常之二次結構。因此,利用BV279?引線,合成cDNA時,選殖到最大段的cDNA選殖株(clone),多?0.5Kb片段,選取二株選殖方向相反的cDNA clone,完成核酸序列分析,其3'端與前述2.3Kb cDNA之5'端約有60個核?酸序列重疊,即共定出2787個核?酸序列,但仍?一長ORF之一部分。 Bamboo mosaic virus (BoMV), a member of potexvirus group, is a plant virus with a single-stranded, positive polarity genome of 6.5 kb. The molecular weight of the major virion protein (coat protein; cp) has been determined to be 28 kd on SDS-PAGE, however, a minor band of 26 kd could be detected. The major in vitro translation products directed by BoMV genome RNA were high molecular weight protein bands as shown on SDS-PAGE, however, a distinct band at 28 kd was found to be similar to cp molecular wight. The nucleotide sequences of a 2.3 kb cDNA clone of BoMV were analyzed and compared with the direct DNA sequence on the RNA template to identify its fidelity. A read through reading frame without a termination codon could be found on the entire 2.3 kb cDNA fragment. To confirm this read through coding capacity, the 2.3 kb cDNA clone and a 1.1 kb fragment have been subcloned onto downstream of phage SP6 promotor in the plasmid pGEM-1 and pGEM-2 respectively. RNA transcripts obtained from these two clones by in vitro transcription were translated in a reticulocyte in vitro translation system. The largest molecular weight of the translated products could be encoded by the entire transcripts. So the 2.3 kb cDNA clone could be a part of a large open reading frame. Primer extension experiments were used to map the location of the clone on the RNA genome of BoMV. The 2.3 kb clone were found to locate on the genome at a distance of 1.0 kb from the 5'end and 3.2 kb from 3' end of the RNA molecule. In the meantime, smaller DNA fragments about 0.5 kb and 2.6 kb were also detected when primers (BV279 & BV281 respectively) were used to map the 5' end position. The results indicate that an unusual secondary structure could be found at this region. Also, we have obtained a cDNA clone about 0.5 kb, which has a 60 nucleotide overlapping with the previous 2.3 kb cDNA clone. The total sequences of 2787 bp analyzed from these cDNA clones still have a read through reading frame and could be a part of a large open reading frame of the polymerase gene. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75743 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
文件中的檔案:
沒有與此文件相關的檔案。
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。