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构建包含RAc1基因cDNA片段的质粒,作为水稻肌动蛋白基因RAc1之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参。从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAc1基因中设计的目的片段,将纯化的目的片段与pMD19-TSimple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。建立了RAc1基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102～107拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系(r=1.000),扩增效率高(E=98.2%)。建立了基因RAc1实时定量PCR的质粒标准品。
The plasmid containing the cDNA fragment of RAc1 gene was constructed and used as a standard for the quantitative detection of the mRNA of RAc1 in rice actin gene. The detection method was established for the quantitative determination of other genes in rice. Total RNA was reverse transcribed from total RNA of rice leaves. PCR was used to amplify the target fragment designed in RAc1 gene. The purified target fragment was ligated with pMD19-TSimple vector and transformed into host strain JM-109. The recombinant plasmid DNA was extracted and PCR Identification and sequencing analysis. The plasmid was purified and the absorbance at 260 nm was detected to determine the copy concentration of the recombinant plasmid stock solution. Then, a real-time fluorescence quantitative PCR test was performed to prepare a fluorescent quantitative PCR gradient concentration standard. The real-time fluorescent quantitative PCR method was established for the detection of RAc1 gene mRNA. The detection sensitivity was 102 copies with a linear range of 102-107 copies. The threshold cycle number (Ct) and the logarithm of the initial template amount in the PCR system There was a good linear relationship (r = 1.000) and high amplification efficiency (E = 98.2%). Established a plasmid RAc1 real-time quantitative PCR standards.