枇杷果实总RNA的提取及八氢番茄红素合成酶基因(PSY)片段的克隆

Extraction of total RNA from loquat fruit and cloning of phytoene synthase gene fragment

【目的】为从枇杷果实中提取高质量的RNA,【方法】以枇杷果实为材料,对6种总RNA提取方法进行分析和比较。【结果】结果表明,在裂解前先用70%丙酮和80%乙醇分别对枇杷果皮和果肉进行预处理,再结合CTAB-LiCl法,得到的总RNA质量较好,OD260/OD280在1.8～2.2,OD260/OD230超过2.0,得率分别为幼果果皮56.78μg.g-1,幼果果肉40.62μg.g-1,成熟果果皮40.86μg.g-1,成熟果果肉9.24μg.g-1;并根据其他植物的八氢番茄红素合成酶基因(PSY)保守区设计引物,用以上方法提取的RNA为模板进行RT-PCR,得到453 bp的基因片段,其推导的氨基酸序列与其他植物的同源性高达95%,推测该片段为PSY基因。【结论】先用70%丙酮和80%乙醇分别对枇杷果皮和果肉进行预处理,再结合CTAB-LiCl法,得到的RNA质量好,可用于后续的分子生物学研究。

[Objective] The objective of the study is to isolate high quality of RNA from loquat fruit. [Method] Six RNA extraction methods were analyzed and compared.[Result] The results showed that the total RNA with desirable yield and quality can be extracted with CTAB-LiC1 protocol with preprocessing peel and pulp of fruit by 70% acetone and 80% alcohol before lysis respectively. The OD260/OD280 were among 1.8 and 2.2, OD260/OD230 were higher than 2.0, and the yields of peel of young fruit, pulp of young fruit, peel of ripe fruit, pulp of ripe fruit were 56.78 μg·g^（-1）, 40.62 μg·g^（-1）, 40.86 μg·g^（-1） and 9.24 μg.g-1 respectively. In addition, according to conserving sequence of phytoene synthase （PSY） gene from several other plants, the primers were designed for the reverse transcription-polymerase chain reaction （RT- PCR）, and the 453 bp fragment of loquat was obtained. [Conclusion] The studies suggested that the total RNA with desirable yield and quality can be extracted with CTAB-LiC1 protocol with preprocessing peel and pulp of fruit by 70% acetone and 80% alcohol respectively, and the extracted RNA could be used in further molecular research.