| 棉花高质量RNA的提取及MADS-box基因保守区段的克隆 |
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| 引用本文： | 李继刚,郭三堆.棉花高质量RNA的提取及MADS-box基因保守区段的克隆[J].棉花学报,2004,16(1):3-7. |
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| 作者姓名： | 李继刚 郭三堆 |
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| 作者单位： | 中国农业科学院生物技术研究所,北京,100081 |
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| 基金项目： | 国家"863"计划(批准号:BH 863 02 01 01) |
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| 摘 要： | 研究和探讨了一种可用于高质量棉花总RNA提取的热硼酸法,并用于棉花MADS box基因的RT PCR分析,结果表明,该方法制备的RNA质量较高,克隆得到的基因片段序列与已知MADS box基因序列之间具有较高同源性。
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| 关 键 词： | 棉花 热硼酸法 提取 MADS-box基因 |
| 文章编号： | 1002-7807(2004)01-0003-05 |
| 修稿时间： | 2003年9月5日 |
Extraction of Total RNA and Cloning of Conservative Fragments of MADS-box Gene by RT-PCR in Upland Cotton |
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| LI Ji-gang,GUO San-dui.Extraction of Total RNA and Cloning of Conservative Fragments of MADS-box Gene by RT-PCR in Upland Cotton[J].Cotton Science,2004,16(1):3-7. |
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| Authors: | LI Ji-gang GUO San-dui |
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| Abstract: | In this report,a highly effective hot-borate RNA extraction method was used in cotton total RNA extraction from cotton (tissues,)such as cotton fiber(10 dpa ), inflorence and cotton boll shell(10 dpa ). This method is characterized of usage of sodium borate, PVP_(40), NP-40 and deoxycholate. The A_(260/280) and A_(260/230) value of RNA extracted from 3 tissues averaged at over 1.8 and 2.0 respectively, suggesting that the RNA has good quality. In order to study the MADS-box gene in cotton, degenerate primers were designed according the conservative MADS domain. In this experiment, two pairs of primers were used for RT-PCR analysis of inflorence total RNA. Four 131-bp cDNA fragments were obtained. Subsequently, alignment,both at DNA level and amino acid level,was conducted among 3 known Arabidopsis MADS-box gene partial sequence and the 4 new sequences, with the results showing that 52.7% and 72.1% were identical at respective levels and they belong to the same gene family. It has been reported that under weak alkaline conditions, borate may form complex with phenolic compound. Proteinase K, alongside with DTT, can eliminate most protein impurity including RNAse. These may contribute to the effectiveness of the reported method. MADS-box gene family controls flower development in plants. It has found in plants, animals, and fungi. Most of the well-studied plant genes are MIKC-type and have 4 domains MADS; domain(M); intervening (I) domain,keratin-like coiled-coil (K) domain, and C-terminal (C) domain. In addition to controlling plant reproduction, MADS-box gene family has roles in plant vegetative development. The research in MADS-box genes has nearly reached maturity in such crops as tomato. Functional genes related cotton reproductive organ development, especially cotton boll, may be of great significance. Our results showed existence of MADS-box genes in cotton, enabling further full length gene cloning and function analysis. |
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| Keywords: | cotton hot-borate method RNA extraction MADS-box gene |
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