不同抗性番茄叶片接种晚疫病后总RNA提取方法及实时荧光定量PCR比较

为筛选出适合番茄晚疫病接种材料总RNA的提取方法,本研究选取番茄感病5#自交系和抗病CLN2037E自交系正常生长和晚疫病接种的叶片为材料,比较Takara公司(货号)9109、9752Q、9769S和北京华越洋公司ZH120等4种RNA提取试剂盒。结果表明:方法9109和ZH120均能获得较清晰的RNA电泳条带。ZH120提取RNA的OD260 nm/OD280 nm在1.92~2.08之间,得率在316.38~359.71g/g之间,其他3种方法提取RNA的完整性和纯度均不高。四种方法提取的RNA均能通过反转录PCR克隆出番茄Actin和RPL2基因片段。以ZH120方法提取的RNA为模板时,基因RPL2实时荧光定量PCR的溶解曲线峰值单一,扩增曲线和Ct值重复性好,变异系数低。结果表明:ZH120试剂盒可作为番茄正常及晚疫病接种叶片RNA提取的适宜方法,并为后续基因克隆及表达谱分析等分子生物学研究提供高质量的RNA。

Comparative of Methods and RT-qPCR for RNA Extraction from Leaves Inoculated by Late Blight of Tomato with Different Resistance

In order to screen one optimal RNA extract method for tomato inoculated by late blight, the leaves of disease-susceptible 5# and disease-resistant CLN2037E inbred line under normal and late blight infection conditions were selected as materials to compare four RNA extraction kits: No.9109, 9752Q, 9769S from Takara Biotechnology Company and No.ZH120 from Huayueyang Biotechnology Company. The results were showed as follows. Clear RNA electrophoresis bandings were obtained using method 9109 and ZH120. The average values of OD260 nm/OD280 nm and productivity ranged from 1.92 to 2.08 and 316.38 to 359.71g/g, respectively. However, the purity and integrity of RNA extracted by other three methods was lower than ZH120 method. Tomato genes Actin and RPL2 were cloned through RT-PCR using the RNA as template extracted by the four kits. Real-time PCR was also performed to identify the RNA quality employing the tomato reference gene RPL2. The RNA from ZH120 possessed one single specific melting curve, good repeatable amplification curve and Ct value, low variable coefficient, comparing with other three methods. The results indicated that method ZH120 was suitable for the tomato leaves' RNA extraction under normal and late blight infection conditions. Furthermore, ZH120 provided high quality RNA for subsequent molecular biology studies such as gene clone and expression profile analysis.