灰绿藜RNA提取方法及在cDNA文库构建中的应用

Methods for High-quality Total RNA Extraction from Chenopodium glaucum and Its Application in cDNA Library Construction

目的:为分离灰绿藜耐盐相关新基因,构建叶片cDNA文库,探索从盐生植物中提取高质量RNA的方法。方法:以灰绿藜叶片为材料,应用RNAPlant Reagent法(Tiangen)、Plant RNA purification Reagent法(Invitrogen)、UNIQ柱式总RNA试剂盒提取法(Sangon)、SDS-LiCl法、TRIZOL试剂法(Invitrogen)等进行总RNA的提取,并构建其全长cDNA文库和抑制消减文库。结果:RNA Plant Reagent法、Plant RNA purification Reagent法和UNIQ柱式总RNA试剂盒提取法获得的RNA完整性均不好;SDS-LiCl法提取的RNA完整性好(28S条带亮度是18S的2倍、条带锐利清晰)、纯度高(A280/A260为1.89±0.03,A260/A230为2.23±0.02),适合用于直接反转录及构建全长cDNA文库,并获得成功;TRIZOL试剂法简单快捷,产率高(2724±82μg/g),完整性较好,可经mRNA纯化后用于抑制消减文库的构建并获得成功。结论:建立了用于构建高质量cDNA文库的灰绿藜RNA的制备方法。

Objective：In present study, the optimal system of total RNA isolation from leaves of Xinjiang halophyte Chenopodiun glaucum was established for cDNA library construction and isolation of new salt tolerance gene. Method： Several commercialized or commonly used methods, such as RNA Plant Reagent （Tiangen）, Plant RNA Purification Reagent （Invitrogen）, UNIQ colamn total RNA isolation kit （Sangon）, SDS - LiCl method and TRIZOL Reagent （Invitrogen）, etc.were used to isolate RNA from leaves of C. glaucum, and then construct the cDNA library and suppression subtracfive library, Result： RNA Plant Reagent, Plant RNA Purification Reagent, UNIQ coliumn total RNA isolation kit could not meet the need for cDNA library construction on the purity or quantity. Wlfile total RNA isolated by SDS- LiCl method showed good integrality （The 28S and 18S bands in agarose gel electrophoresis were clear, and the briglitness ratio of two bands （28S/18S） was more than 2.0） and high purity （The ratio of A280/A260 was 1.89 ±0.03, A260/A230 was 2.23 ± 0.02）, and could be directly trsed in RT- PCR reaction and construction for full - length eDNA library of C. glauoan ; TRIZOL Reagent method had simple and speedy procedttre, total RNA from which displayed very high quantity （2 724 ±82μg/g） and integrality, and could be used in construction of suppression subtractive library of C. glauctan after mRNA being purified. Conclusion：Methods for high-quality total RNA extraction from C. glaucum was established and could be successfully used in the cDNA library construction.