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標題: | 野生型及抗鏈黴素突變體縐葉菸草葉綠體16S rRNA基因的核?酸序列 Nucleotide Sequence Analysis of Chloroplast 16S rRNA Gene in Wild-Type and Streptomycin-Resistant Nicotiana plumbaginifolia |
作者: | 葉國楨 |
出版年 : | 1991 |
學位: | 碩士 |
摘要: | 本研究可分為二個部分。第一,為了建立縐葉菸草(Nicotiana plumbaginifolia)葉綠體16S rRNA基因更完整的資料,我們以質體Bluescrip為載體,選殖出一個包含完整16S rRNA基因的BamH I片段,並且分析其核?酸序列。發現有1490bp,只比栽培菸草(Nicotiana tabacum)葉綠體16S rRNA在第356個位置多一個C,其他序列完全相同。第二部分,為快速分析12個呈母系遺傳的抗鏈黴素(streptomycin)突變植株在葉綠體16S rRNA基因上相當於E.coli 530環及912附近二個區域的突變情形,我們先以PCR的方法大量複製一段橫跨530環至912附近(共517bp)的DNA片段,直接將產物就530,912二個區域定序,發現12株中有8株在912的位置發生由C→T的突變,這個結果與在其他生物中的研究一致。所以,可確定突變植株是因為此處發生單點突變而使其對鏈黴素具抗性。另外4株在此二區域皆未發生突變,推測很可能在葉綠體基因的其他部位(如核醣蛋白質S12的基因)發生突變。其中一株(SR1021)發生G834→A834(相當於E.coli之G885→A885),至目前為止並沒有此處發生突變而導致抗鏈黴素的報導,所以可能為靜突變(silent mutation),但是由細菌16S rRNA的二級結構看來,它和530環及912也相當靠近(圖1),而且經DNA序列的比對,發現此處也具有相當高的保守性,所以我們不排除因此處突變而導致抗性的可能。 There were two parts in this study. First, using plasmid Bluescript as a vecter, we successfully cloned a full length 16S rRNA gene from the chloroplast genome of wild-type Nicotiana plumbaginifolia. Sequence analysis of this gene shows that it is 1490bp in length, and that except for the presence of an additional C at position 356, it is identical to the chloroplast 16S rRNA gene of N. tabacum. Second, we analyzed the nucleotide sequence in two regions in the chloroplast 16S rRNA gene of 12 maternally inherited streptomycin-resistant mutants of N. plumbaginifolia. These regions correspond to the 530 loop and 900 stem/loop of Escherichia coli 16S rRNA. To do this, we amplified, using the technique of polymerase chain reaction (PCR), a 517-bp fragment covering these two regions from the chloroplast genome of the mutants, and analyzed the PCR products directly. Our results show that: (1) eight mutants possess a C to T transition at positiion 861 (equivalent to nucleotide 912 of the E. coli 16S rRNA); (2) one mutant has a G to A transition at position 834 (equivalent to nucleotide 885 of the E. coli 16S rRNA; and (3) three mutants do not contain any changes in the two regions analyzed. The G to A transition at position 834 is a novel mutation, but its relation with streptomycin resistance remains to be explored. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75848 |
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顯示於系所單位: | 植物科學研究所 |
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