多糖類を多く含む果樹組織からのRNAの抽出とcDNAライブラリーの構築ならびにRT-PCRによる評価
キウイフルーツと温州ミカンの種々の組織を用いて全RNAの抽出法を検討した. 温州ミカンのアルベド組織以外では, Lopez-Gomez•Gomez•Lim (1992) の抽出方法 (多糖類が多く含有される果実に適した抽出方法) を改変した方法により, 十分な全RNAの収量が得られた. アルベド組織からの全RNAの収量は,多量の多糖類の混入のため十分でなかったと思われたそこで, 抽出緩衝液による再抽出や, クロロホルム/イソアミルアルコール抽出を反復し, さらに, 回収した水相に, その2倍量のDEPC処理水を加えた後,終濃度3Mになるように塩化リチウムを添加することにより, アルベド組織から...
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| Published in | Engei Gakkai zasshi Vol. 64; no. 4; pp. 809 - 814 |
|---|---|
| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English Japanese |
| Published |
一般社団法人 園芸学会
1996
|
| Online Access | Get full text |
| ISSN | 0013-7626 1880-358X |
| DOI | 10.2503/jjshs.64.809 |
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| Abstract | キウイフルーツと温州ミカンの種々の組織を用いて全RNAの抽出法を検討した. 温州ミカンのアルベド組織以外では, Lopez-Gomez•Gomez•Lim (1992) の抽出方法 (多糖類が多く含有される果実に適した抽出方法) を改変した方法により, 十分な全RNAの収量が得られた. アルベド組織からの全RNAの収量は,多量の多糖類の混入のため十分でなかったと思われたそこで, 抽出緩衝液による再抽出や, クロロホルム/イソアミルアルコール抽出を反復し, さらに, 回収した水相に, その2倍量のDEPC処理水を加えた後,終濃度3Mになるように塩化リチウムを添加することにより, アルベド組織からの全RNAの収量を改善することができた. これらの方法で抽出した全RNAから, oligo (dT) -celluloseカラムでpoly (A)+RNAに精製した後に, 逆転写-PCR (RT-PCR) やcDNAライブラリーの構築に供することができた. 以上のように, この方法はキウイフルーツの葉, 果実および温州ミカンの砂じょう, アルベド, 葉そして花弁組織からめ全RNAの抽出に有効であることが明らかとなった. |
|---|---|
| AbstractList | キウイフルーツと温州ミカンの種々の組織を用いて全RNAの抽出法を検討した. 温州ミカンのアルベド組織以外では, Lopez-Gomez•Gomez•Lim (1992) の抽出方法 (多糖類が多く含有される果実に適した抽出方法) を改変した方法により, 十分な全RNAの収量が得られた. アルベド組織からの全RNAの収量は,多量の多糖類の混入のため十分でなかったと思われたそこで, 抽出緩衝液による再抽出や, クロロホルム/イソアミルアルコール抽出を反復し, さらに, 回収した水相に, その2倍量のDEPC処理水を加えた後,終濃度3Mになるように塩化リチウムを添加することにより, アルベド組織からの全RNAの収量を改善することができた. これらの方法で抽出した全RNAから, oligo (dT) -celluloseカラムでpoly (A)+RNAに精製した後に, 逆転写-PCR (RT-PCR) やcDNAライブラリーの構築に供することができた. 以上のように, この方法はキウイフルーツの葉, 果実および温州ミカンの砂じょう, アルベド, 葉そして花弁組織からめ全RNAの抽出に有効であることが明らかとなった. |
| Author | 森口, 卓哉 小川, 一紀 生駒, 吉識 徐, 忠伝 矢野, 昌充 大村, 三男 吉岡, 照高 久田, 素 |
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| References | Biggs, M. S., R. W. Harriman and A. K. Handa. 1986. Changes in gene expression during tomato fruit ripening. Plant Physiol. 81 : 395-403. Liang, P. and A. B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257 : 967-971. Fuke, Y. and H. Matsuoka. 1984. Changes in content of pectic substances, ascorbic acid and polyphenols, and activity of pectinesterase in kiwi fruit during growth and ripening after harvest. Studies on constituent in kiwi fruit cultured in Japan. Part II. Nippon Shokuhin Kogyo Gakkaishi 31 : 31-37. MacDiarmid, C. W. B. and R. C. Gardner. 1993. A cDNA sequence from kiwifruit homologous to 1-aminocyclopropane-1-carboxylic acid oxidase. Plant Physiol. 101 : 691-692. Ikoma, Y., M. Yano and K. Ogawa. 1995. Cloning and expression of genes encoding ACC synthase in kiwifruit. Acta Hortic. 398 : 179-186. Callahan, A., P. Morgens and E. Walton. 1989. Isolation and in vitro translation of RNAs from de- veloping peach fruit. HortScience 24 : 356-358. Welsh, J., K. Chada, S. S. Dalai, R. Cheng, D. Ralph and M. McClelland. 1992. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20 : 4965-4970. Christoffersen, R. E., E. Warm and G. G. Laties. 1982. Gene expression during fruit ripening in avocado. Planta 155 : 52-57. Lopez-Gomez, R. and M. A. Gomez-Lim. 1992. A method for extracting intact RNA from fruits rich in polysaccharides using ripe mango mesocarp. HortScience 27 : 440-442. Naohara, J. and M. Manabe. 1987. Quantitative and qualitative changes of pectin during development of satsuma mandarin fruits (Citrus unshiu Marc.). Studies on the properties and function of pectin in satsuma mandarin fruits (Citrus unshiu Marc.). Part II. Nippon Shokuhin Kogyo Gakkaishi 34 : 386-391. Hara, E., T. Kato, 'S. Nakada, S. Sekiya and K. Oda. 1991. Subtractive cDNA cloning using oligo (dT) 30-latex and PCR. Isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells. Nucleic Acids Res. 19 : 7097-7104. Grieson, D., A. Slater, J. Speirs and G. A. Tucker. 1985. The appearance of polygalacturonase mRNA in tomatoes. One of a series of changes in gene expression during development and ripening. Planta 163 : 263-271. Podivinsky, E., E. F. Walton and P. L. McLeay. 1994. Extraction of RNA from kiwifruit tissues. Bio-Techniques 16 : 396-398. Yoshida, K. T., S. Naito and G. Takeda. 1994. cDNA cloning of regeneration-specific genes in rice by differential screening of randomly amplified cDNAs using RAPD primers. Plant Cell Physiol. 35 : 1003-1009. Hubank, M. and D. G. Schatz. 1994. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 22 : 5640-5648. Birnboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7 : 1513-1523. |
| References_xml | – reference: Grieson, D., A. Slater, J. Speirs and G. A. Tucker. 1985. The appearance of polygalacturonase mRNA in tomatoes. One of a series of changes in gene expression during development and ripening. Planta 163 : 263-271. – reference: Hubank, M. and D. G. Schatz. 1994. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 22 : 5640-5648. – reference: Callahan, A., P. Morgens and E. Walton. 1989. Isolation and in vitro translation of RNAs from de- veloping peach fruit. HortScience 24 : 356-358. – reference: Christoffersen, R. E., E. Warm and G. G. Laties. 1982. Gene expression during fruit ripening in avocado. Planta 155 : 52-57. – reference: Birnboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7 : 1513-1523. – reference: Hara, E., T. Kato, 'S. Nakada, S. Sekiya and K. Oda. 1991. Subtractive cDNA cloning using oligo (dT) 30-latex and PCR. Isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells. Nucleic Acids Res. 19 : 7097-7104. – reference: Ikoma, Y., M. Yano and K. Ogawa. 1995. Cloning and expression of genes encoding ACC synthase in kiwifruit. Acta Hortic. 398 : 179-186. – reference: Yoshida, K. T., S. Naito and G. Takeda. 1994. cDNA cloning of regeneration-specific genes in rice by differential screening of randomly amplified cDNAs using RAPD primers. Plant Cell Physiol. 35 : 1003-1009. – reference: Welsh, J., K. Chada, S. S. Dalai, R. Cheng, D. Ralph and M. McClelland. 1992. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20 : 4965-4970. – reference: Lopez-Gomez, R. and M. A. Gomez-Lim. 1992. A method for extracting intact RNA from fruits rich in polysaccharides using ripe mango mesocarp. HortScience 27 : 440-442. – reference: Fuke, Y. and H. Matsuoka. 1984. Changes in content of pectic substances, ascorbic acid and polyphenols, and activity of pectinesterase in kiwi fruit during growth and ripening after harvest. Studies on constituent in kiwi fruit cultured in Japan. Part II. Nippon Shokuhin Kogyo Gakkaishi 31 : 31-37. – reference: MacDiarmid, C. W. B. and R. C. Gardner. 1993. A cDNA sequence from kiwifruit homologous to 1-aminocyclopropane-1-carboxylic acid oxidase. Plant Physiol. 101 : 691-692. – reference: Podivinsky, E., E. F. Walton and P. L. McLeay. 1994. Extraction of RNA from kiwifruit tissues. Bio-Techniques 16 : 396-398. – reference: Biggs, M. S., R. W. Harriman and A. K. Handa. 1986. Changes in gene expression during tomato fruit ripening. Plant Physiol. 81 : 395-403. – reference: Naohara, J. and M. Manabe. 1987. Quantitative and qualitative changes of pectin during development of satsuma mandarin fruits (Citrus unshiu Marc.). Studies on the properties and function of pectin in satsuma mandarin fruits (Citrus unshiu Marc.). Part II. Nippon Shokuhin Kogyo Gakkaishi 34 : 386-391. – reference: Liang, P. and A. B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257 : 967-971. |
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| Title | 多糖類を多く含む果樹組織からのRNAの抽出とcDNAライブラリーの構築ならびにRT-PCRによる評価 |
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