摘要
针对巴西橡胶树胶乳RNA的提取方法存在操作复杂、步骤多、时间长、保存条件要求苛刻等问题以及橡胶树胶乳远距离、长时间采样的诸多不便等问题,介绍一种具有操作简单、实验时间短、步骤少等优点的简易巴西橡胶树胶乳RNA提取方法和一种适于长时间保存胶乳的方法。该方法仅仅以DEPC水为提取液,提取的RNA浓度可以达到为0.722μg/mL,纯度OD260/OD280在2.0~2.2之间,总RNA提取量为36.1μg/mL,可以满足后续分子生物学实验RT-PCR和RACE的要求;对在冰上保存1~10 d的胶乳提取RNA,浓度在0.502~0.722μg/mL之间,OD260/OD280在1.8~2.4之间,总RNA的提取量在20.8~35.44μg/mL之间。
Purpose：The existing methods for RNA extraction from latex of Hevea brasiliensis are somewhat complicated,high labor intensive and time consuming.In order to solve these problems,we introduced a simple method of RNA extraction from rubber latex of H.brasiliensis in which DEPC water was used as a new extract buffer.At the same time,to solve the problem of long-term transportation and long-time latex RNA preservation,we devised a new protocol to preserve the fresh latex for future RNA extraction.This newly developed method provided an efficient technical support for RNA extraction and latex sample storage.Methods and Results：The pure DEPC-treated water was used as an extracting solution.The obtained RNA concentration was 0.722 μg/mL,and the RNA purity was about 2.0～2.2 with the OD~/OD^0 ratio.The yield of total RNA extracted from fresh latex was 36.1 μg/mL.The extracted RNA was compatible with the further molecular analysis such as RT-PCR and RACE.After keeping the fresh latex in mixtures of DEPC-treated water and phenol chloroform on ice for 1～10 days,the latex could still be used to produce quantitative and qualified RNA for further research.The concentration of RNA extacting from the long-time preserved latex was between 0.502 and 0.722 μg/mL,with OD260/OD280 ratio between 1.8～2.4.The finale yields of total RNA obtained from the latex were ranged from 20.8 to 35.44 μg/mL.
出处
《热带作物学报》
CSCD
北大核心
2013年第10期1907-1913,共7页
Chinese Journal of Tropical Crops
基金
国家自然科学基金项目(No.31270712
No.31000291
No.31370681)
海南省重大科技项目子课题(No.ZDZX2013023-1)
中国热带农业科学院青年拔尖人才基金项目(No.ITBB130102)
