厚荚相思RNA提取方法的建立与优化

The Establishment and Optimization of RNA Isolation in Acacia crassicarpa

针对厚荚相思叶片中单宁、酚类、色素、纤维素多糖物质含量高的特点,对Trizol法、改良的热硼酸盐法、CTAB(hexadecyl trimethy ammonium bromide)法和超声波破碎结合RNA提取试剂盒法从厚荚相思体外培养的叶片中提取总RNA进行比较研究,结果表明:在RNA提取试剂盒的基础之上,引入20 MHz的超声波,破碎2 min(puls on 5 s,puls off 5 s)为假叶RNA提取的优化方法.运用此方案获得的RNA片段较完整,其A260/A280值均在1.82～1.98之间,并成功进行了基因片段的RT-PCR(reverse transcription PCR).

Extraction of high-quality RNA from the phyllode of Acacia crassicarpa is very difficult due to high levels of tannin,phenols,pigment,cellulose polysaccharide that bind or coprecipitate with RNA.There-fore,the preferred RNA isolation protocols were optimized from the phyllode of Acacia crassicarpa,compar-ing the Trizol method,improved hot-borate methods,CTAB and RNA isolation kit combined with ultrasonic method.It was found that the RNA isolation kit combined with 20 MHz ultrasonic processor（puls on 5 s,puls off 5 s） was optimal for the RNA isolation from Acacia crassicarpa.The extracted total RNA with this method had high integrity,the ratio of A260/A280 was in the range of 1.82～1.98,and had been successfully used for RT-PCR experiment.