蓝莓组织RNA提取方法的研究

Methods for RNA isolation from blueberry tissues

为建立蓝莓组织RNA提取方法,以蓝莓幼果为材料,比较了5种RNA提取方法,建立了改良的十六烷基三甲基溴化铵-乙酸钾(CTAB-KAc)提取方法,并比较了CTAB-KAc法对蓝莓根、成熟叶、幼茎、花、幼果和成熟果实组织RNA的提取效果.结果表明:CTAB-KAc法提取RNA的过程只需3 h,蓝莓幼果RNA的A260/A280和A260/A230值分别达到2.02和2.46,且无基因组DNA污染,RNA产量达到143.37μg·g-1;用CTAB-KAc法成功提取了蓝莓成熟叶、幼茎、花、幼果和成熟果实组织的RNA,但是根的RNA提取方法还需优化.把上述RNA反转录成c DNA后进行PCR,扩增出预期大小的目的片段,可满足后续分子生物学实验的要求.

Tissues of blueberry were generally rich in polysaccharides, proteins, and polyphenols, resulting in the difficulties of RNA isolation with both high quantity and quality.To optimize the technology of RNA isola-tion from blueberry tissues, five methods for RNA extraction from immature fruit of blueberry were evaluated. The results showed that the most efficient technique for RNA isolation from blueberry fruits was the optimized CTAB-KAc-based method.A subsequent experiment showed that the optimized CTAB-KAc-based technique was also efficient in successful RNA isolation from full-expanded leaf, green stem, flower, immature fruit and mature fruit of blueberry, although protocol for roots still needed to be optimized.The method yielded 143.37μg· g-1 high-quality RNA without DNA contamination within 3 h.The ratios of A260/A280 and A260/A230 of the total RNA from immature fruit of blueberry were 2.02 and 2.46, respectively.Reverse transcription-PCR con-firmed that the isolated RNA was of appropriate quality and integrity for subsequent molecular biology studies.