| 适用于棉花荧光原位杂交的DNA纤维高效制备技术 |
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| 引用本文： | 彭仁海,宋国立,刘方,黎绍惠,王春英,张香娣,王玉红,王坤波.适用于棉花荧光原位杂交的DNA纤维高效制备技术[J].作物学报,2009,35(3):412-417. |
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| 作者姓名： | 彭仁海 宋国立 刘方 黎绍惠 王春英 张香娣 王玉红 王坤波 |
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| 作者单位： | 1中国农业科学院棉花研究所/农业部棉花遗传改良重点实验室,河南安阳455000;2安阳工学院生物与食品工程学院,河南安阳455000 |
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| 基金项目： | 国家自然科学基金，国家高技术研究发展计划(863计划)，国家转基因生物新品种培育重大专项基金 |
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| 摘 要： | 棉花富含酚类和多糖等高分子次生化合物，细胞质浓厚，且染色体形态小、数目多、制片困难，至今还未见棉花DNA纤维制备方面的报道。本研究用“刀切引流法”，在含有Triton X-100和PVP40的冰冷细胞核提取缓冲液中，用锋利的刀片切割发育一周的棉花黄化子叶以释放棉花细胞核，所得细胞核干净完整杂质少，不需要研磨和巯基乙醇等处理，方便快捷无毒害，成功率达到100%。细胞核在室温下经温和碱裂解去除染色质上的蛋白质后，以前端导引裂解液铺展载玻片，即“引流法”拉伸制备DNA纤维，避免了液体表面张力的影响，消除了因载玻片推抹用力不均而导致的DNA纤维堆积和断裂，所制备的DNA纤维平直完整、伸展程度均匀、背景清晰。用基因组和45S rDNA 分别标记探针进行杂交，结果表明所制备的棉花DNA纤维适用于荧光原位杂交。本研究探索出一套简单、高效、快捷、无毒害的适宜于棉花荧光原位杂交的DNA纤维制备技术，必将为棉花基因组研究和全基因组序列的最终完成提供强有力的技术支持。
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| 关 键 词： | 棉花 荧光原位杂交 DNA纤维 高效制备 |
| 收稿时间： | 2008-10-13 |
An Efficient Method of Cotton DNA Fibers Preparation for FISH |
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| PENG Ren-Hai,SONG Guo-Li,LIU Fang,LI Shao-Hui,WANG Chun-Ying,ZHANG Xiang-Di,WANG Yu-Hong,WANG Kun-Bo.An Efficient Method of Cotton DNA Fibers Preparation for FISH[J].Acta Agronomica Sinica,2009,35(3):412-417. |
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| Authors: | PENG Ren-Hai SONG Guo-Li LIU Fang LI Shao-Hui WANG Chun-Ying ZHANG Xiang-Di WANG Yu-Hong WANG Kun-Bo |
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| Institution: | 1.Key Laboratory of Cotton Genetic Improvement, Ministry of Agriculture/Cotton Research Institute, Chinese Academy of Agricultural Sciences,Anyang 455000,China;2.College of Biology and Food Technology, Anyang Institute of Technology, Anyang 455000,China |
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| Abstract: | Fluorescence in situ hybridization (FISH) has become the most important technique applied in molecular cytogenetics, especially in developing physical maps in plants. As a key technique, FISH on cotton DNA fibers stretched has not been reported yet, possibly owning to the difficulty in their DNA fiber preparation as well as the existence of thick cytoplasm and hard cell walls. Here we present a method of highly efficient preparation of stretched DNA fibers in cotton. Cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish that contained ice-cold nucleus isolation buffer(MgSO4 10 mmol L-1, KCl 5 mmol L-1, HEPES 0.5 mmol L-1, DTT 1 mg mL-1, Triton X-100 0.25%, PVP40 2%) followed by sequential filtration through 100, 50, and 30 µm nylon meshes. Nuclei were obtained by centrifuging the filtrates at 16 000 g for 1 min. Mixture of nucleus lysis buffer(0.5% SDS, 5 mmol L-1 EDTA, 100 mmol L-1 Tris, pH7.0) and nuclei was incubated on the slide for 9 min, DNA fibers obtained by dragging and stretching with a clean slide edge from the end to another end on the liquid surface. After incubated at 60℃ overnight, the slides were pretreated with DNase-free RNase and then rinsedin 2×SSC. The probes and DNA fibers were denatured separately, and hybridization mixture was incubated on the slide overnight, followed by post-hybridization rinses in 2×SSC, 1×4T. The slides were blocked with 5% BSA and covered with antibody for 1h. After rinsed with 1×TNT the slides were counterstained with 4'', 6-diamidino-2-2phenylin-dole (DAPI) and followed by rinsing in 1×PBS. After mounting the slides with Vectashield mounting medium the hybridization signals were observed under a fluorescence microscope. Images were captured by a charge-coupled device (CCD) system and brought together to make the plate using Adobe Photoshop 7.0 software. The results indicated that it was easier to release nuclei from cells in nucleus isolation buffer by chopping cotyledon, and slowly and smoothly dragging the nuclei solution with a slide edge from the end to another end on slide treated with poly-L-lysine. Highly stretched and intact DNA fibers were obtained. This method is very simple and rapid, which takes only 30 min to finish the entire process, and it is also safe because poisonous mercaptoethanol is replaced by dithiothreitol. The linear or near-linear stretches of beads on-a-string signals with cotton genomic DNA and 45S rDNA as probes showed that the DNA fibers were suitable for FISH. |
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| Keywords: | Cotton FISH DNA fibers Efficient preparation |
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