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標題: | 水稻重複性核酸片段的選殖與原生質體基因轉殖之研究 Molecular Cloning of Repetitive DNA and Protoplast Gene Transfer in Rice (Oryza sativa L.) |
作者: | 陳光超 |
出版年 : | 1990 |
學位: | 碩士 |
摘要: | 在植物的基因組(genome)中有著相當數目的重複性DNA(repeated DNA),而適當的重複性DNA對於探討植物基因組成的演化與種間變異言將是一十分有價值的分子標記。在此吾等利用分子選殖之方法來篩選具高套數(high copy number)之核酸片段,希望能作?研究水稻RFLP(restriction fragment length polymorphism)時的良好標記,一方面亦可作?將來從事水稻基因轉殖時之標的位置(target site)。 首先由載體pBR322構築一含300-500 bp之水稻基因庫,經南方氏雜交分析後選擇一雜交形式(hybridization pattern)複雜度高者作進一步之研究。發現此重複DNA片段在經組織培養之水稻癒合組織中有大量擴增(amplify)的現象,且可利用之區分出同種水稻Dular(CY)與(TA)的差異性及相同染色體組(AA genome)與異染色體組(AA, BB, CC, BBCC, CCDD, EE, FF genome)不同稻種間之親緣性。將該段DNA定序後與電腦基因資料庫作比對分析,發現在pURS300中的160 bp與水稻葉綠體DNA rpoB (RNA polymerase β subunit)基因之上游區域序列有相似性,經進一步分析發現此段DNA同時存在於水稻之核與葉綠體中。 另一方面在水稻基因轉殖部分,利用抗藥性基因npt Ⅱ作電導轉殖,並以抗生素G-418篩選具抗性之再生植株,經初步以墨點雜交測試分析(dot blotting analysis)尚無法斷定該抗性的產生確由基因轉殖所造成,此外還須進一步克服水稻組織陪養與原生質體再生植株不穩定性等問題。 There are large amount of repetitive DNA in plant genome. It will be useful to study the plant genome evolution and species variation with proper repetitive DNA as probes. Here, we screened highly repeated rice DNA by molecular cloning method. We wish it will be a useful marker for the study of restriction fragment length polymorphism of rice genomes, on the other hand, it can be as a target site for plant gene transfer. First, we constructed a rice DNA library with the vector pBR322 of which the inserts rice DNA Sau3A fragments are about 300-500bp. Through Southern hybridization analysis, the rice repetitive DNA clone pURS300 which had most complex hybridization pattern had beeen selected for further study. We found it would amplify with in dediffenentiation stage (callus form) . In the same time, it could differentiate the same species rice Dular (CY) from Dular (TA) and could be used to compare the relationship between the same rice genome and different genome rice (AA, BB, CC, BBCC, CCDD, EE and FF). DNA sequences analysis with computer data bank, we found that 160 bp of the pURS 300 sequences are identical to the upstream region of rpoB gene in the rice chloroplast. After further analysis it was found that this sequence was originally cloned from the nucleus genomic DNA , although, part of the sequences were also found in the chloroplast genome. On the other hand, we try to establish the gene transfer system of rice. Antibiotic G-418 resistant gene (npt II) was used as selection marker to select regenerated plant which had resistance to G-418. By dot-blots hybridization analysis of these plants.We can't confirm that the character of antibiotic resistance comes from the npt II gene transfer. Besides of these, the problems of instability of rice tissue culture and protoplast regeneration are still need to be resolved. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75742 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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