柑橘果实汁囊总 RNA提取方法的比较研究

摘要

To isolate high quality total RNA from Citrus juice sacs is difficult because the tissues contain sugars,water.In this study,three methods for extracting high quality total RNA of juice sacs from “wuzisha-tangju” collected on different sampling dates by using Column Plant RNA OUT kit ( Beijing TianDZ ) , Quick RNA isolation kit ( Beijing Hua Yue Yang ) and improved extraction kit respectively .The results showed that two bands of 28S and 18S rRNA were distincted by agarose gel electrophoresis with improved Column Plant RNAOUT(Beijing TianDZ),whose values of A260/A280 and A260/A230 were 1.86-1.93 and 2.1-2.3 respectively, and the yield was over 28.9 μg/g,the CrAI was cloned successfully using the cDNA as templates which were synthesized total RNA extraction ,and the expression level of CrAI was detected on different development period using Real-Time PCR.%针对柑橘果实水分多、含糖量高而难以提取高质量的总RNA，本研究以无籽沙糖橘不同发育时期的果实汁囊组织为试材，比较了3种RNA提取方法。结果表明天恩泽改良法提取效果最好，可快速得到28S和18S条带清晰、完整性好，OD260/280介于1．86～1．95，OD260/230介于2．1～2．3，平均浓度都在28．9μg/g以上的总RNA。以反转录后cDNA为模板，成功克隆到柑橘酸性转移酶基因（ CrAI），经Real-Time PCR 实验，检测到不同发育时期CrAI的表达水平。