一种高效提取棉花细胞核的技术

An Efficient Method of Nuclei Extraction in Cotton

[目的]研究一种高效提取棉花细胞核的技术,为棉花大片段基因组文库的构建和全基因组测序提供技术支持。[方法]采用"刀切法",在含有Triton X-100和PVP40的冰冷细胞核提取缓冲液中,用锋利刀片切割发育1周的棉花黄化子叶以释放细胞核,并经过100、50、和30μm的膜过滤后离心收集细胞核,同时选取棉花幼嫩叶片和黄化处理时间不同的子叶分别用研磨法和刀切法作对照。[结果]刀切法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100%。[结论]建立了一种简单、高效、快捷、无毒害的棉花细胞核提取技术。

[Objective] The experiment aimed to study an ~ffieient method of nuclei extraction of cotton and provided technical support for constructing large-insert genomic library and sequencing complete genome. [Method] The cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish which contained ice-cold nucleus isolation buffer （10 mmoL/L MgSO4, 5mmoL/L KCl, 0.5 mmol/L HEPES, 1 mg/ml DTT, 0.25% Triton X-100 and 2% PVP40） , then the nuclei were collected after selected through 100, 50 and 30μm nylon meshes and eentrifugation. Meanwhile, the tender leaves and cotyledons with different germination time in dark were treated by grinding method and Jackknife method.[Result] The chopping with a sharp scalpel method was very.simple and rapid, which did not need grind and the success rate was 100%.[Conclusion] An efficient method of nuclei extraction of cotton with simple, high efficiency, rapid reaction and poison free were established .