摘要
为获得纯度高、质量好的酿酒葡萄植株叶片RNA以用于荧光实时定量分析,为后续以酿酒葡萄叶片RNA为材料研究相关转录水平的调控提供技术保障,以经典酿酒葡萄赤霞珠叶片为试验材料,比较分析了重蒸酚法、Trizol法、改良的十六烷基三甲基溴化铵(CTAB)法和LiCl法(基于改良的SDS法)4种方法对酿酒葡萄叶片RNA提取的影响。结果表明,重蒸酚法提取RNA浓度较低,完整性较差,存在部分降解;Trizol法提取的RNA则有部分DNA污染;改良CTAB法提取浓度较高,但蛋白和DNA污染严重;LiCl法提取结果显示,28S的条带亮度是18S的2倍,条带无拖尾,完整性较好,存在DNA污染和少量的蛋白污染。进一步在LiCl法基础上利用醋酸钠、DNAase消化、苯酚抽提法进一步优化,并采用荧光实时定量验证,结果表明,醋酸钠的加入RNA质量无明显改善;然而,经氯仿和水饱和酚(体积比1∶1)抽提2次,氯仿抽提1次,氯仿和水饱和酚(体积比1∶1)抽提1次,并经DNAase进行消化,最终提取出质量浓度为173.611μg/mL、A_(260/280)为1.803纯度高、完整性较好且无蛋白和DNA污染的葡萄叶片总RNA;经实时荧光定量PCR进一步验证,该方法提取的RNA经反转录后获得较好的扩增曲线以及融解曲线,并能够对相关基因进行定量分析。
In order to obtain RNA from wine grape leaves with high purity and good quality for fluorescence real-time quantitative analysis,and to provide technical support for subsequent studies on the regulation of transcription levels using wine grape leaf RNA as materials and have certain application value,in this study,classic wine grape Cabernet Sauvignon leaves were used as the experimental materials.Effects of four methods,including resteaming phenol,Trizol,improved cetyltrimethyl ammonium bromide(CTAB),and LiCl(based on improved SDS),on RNA extraction from wine grape leaves were compared and analyzed.The results showed that the concentration of RNA extracted by resteaming phenol was low,the integrity was poor,and there was partial degradation.In addition,the RNA extracted by Trizol method had some DNA contamination.In the modified CTAB method,the extraction concentration was high,but the protein and DNA contamination was serious.In the RNA swim lane extracted by LiCl method,the brightness of the 28S band was twice of that of the 18S band.The band has no dragging phenomenon,and the extracted RNA integrity was good,but there was still DNA contaminationn and a small amount of protein contamination.Further,on the basis of LiCl method,sodium acetate,digestion by DNAase,and phenol extraction method were used for optimization.The results showed that the addition of sodium acetate did not improve the quality of RNA.However,total RNA from grape leaves with high purity,good integrity,and no protein and DNA contamination(173.611µg/mL,A_(260/280)=1.803)was extracted by extracting chloroform and water-saturated phenol(1∶1)twice,chloroform once,chloroform and water-saturated phenol(1∶1)once,and digestion by DNAase.The results of real-time fluorescence quantitative PCR further verified that the RNA extracted by this method obtained good amplification curve and melting curve after reverse transcription,and could be used to quantitatively analyze related genes.
作者
成丹丹
范丽娜
王美娇
于放
王燕燕
CHENG Dandan;FAN Lina;WANG Meijiao;YU Fang;WANG Yanyan(School of Biological Engineering,Dalian Polytechnic University,Dalian 116033,China)
出处
《山西农业科学》
2024年第2期50-57,共8页
Journal of Shanxi Agricultural Sciences
基金
国家自然科学基金面上项目(31970324)
辽宁省教育厅基础研究项目(J2020042)。
关键词
酿酒葡萄
赤霞珠
RNA提取
LiCl法优化
QRT-PCR
wine grape
Cabernet Sauvignon
RNA extraction
LiCl method optimization
Real-time fluorescence quantita-tive PCR(qRT-PCR)
