| 转植酸酶基因(phyA2)玉米定性、定量PCR检测 |
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| 引用本文： | 于彩虹,田少亭,路兴波,李凡,杨淑珂,孙红炜.转植酸酶基因(phyA2)玉米定性、定量PCR检测[J].农业生物技术学报,2012,20(4):356-361. |
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| 作者姓名： | 于彩虹 田少亭 路兴波 李凡 杨淑珂 孙红炜 |
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| 作者单位： | 1. 中国矿业大学(北京)化学与环境工程学院,北京,100083
2. 山东省农业科学院植物保护研究所,济南,250100 |
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| 基金项目： | 国家转基因生物新品种培育科技重大专项 |
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| 摘 要： | 转植酸酶基因玉米检测是对转植酸酶基因玉米释放进行有效监管的重要前提和技术支撑。本研究根据转植酸酶基因玉米(Zeamays)外源植酸酶基因phyA2序列设计基因特异性引物,进行了定性、定量PCR检测。定性结果显示,该引物可有效检测植酸酶玉米中phyA2基因,而在其他不含植酸酶基因的材料中则检测不到相应基因,表明该引物具有很高的特异性。以玉米内标准基因玉米淀粉合酶异构体zSTSII-2基因(zSSIIb)和植酸酶基因phyA2为对象,进行SYBRGreenⅠ染料法荧光定量PCR反应,绘制2种基因扩增循环数-拷贝数标准曲线图,根据混合样品中(zSSIIb)和植酸酶基因phyA2的拷贝数计算样品中的转基因含量,并做熔解曲线分析。结果表明,zSSIIb和phyA2标准曲线相关系数分别为0.998和0.995,相关性较好,两个基因的熔解曲线均呈单峰型,表明引物特异性较强,混合玉米样品(植酸酶玉米含量分别为1%、0.5%和0.1%)中植酸酶玉米含量分别为1.1%、0.54%和0.17%,实测值与理论值接近。本研究建立了转植酸酶基因玉米定性、定量PCR检测体系,可有效地对转植酸酶基因玉米进行检测。
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| 关 键 词： | 转植酸酶基因玉米 定性PCR 定量PCR zSSIIb 植酸酶基因(phyA2) |
Qualitative and Quantitative PCR Detection of the Phytase (phyA2)Transgenic Corn |
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| YU Cai-Hong
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TLAN Shao-Ting
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LU Xing-Bo
,
LI Fan
,
YANG Shu-Ke
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SUN Hong-Wei.Qualitative and Quantitative PCR Detection of the Phytase (phyA2)Transgenic Corn[J].Journal of Agricultural Biotechnology,2012,20(4):356-361. |
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| Authors: | YU Cai-Hong TLAN Shao-Ting LU Xing-Bo LI Fan YANG Shu-Ke SUN Hong-Wei |
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| Institution: | 1 School of Chemical & Environmental Engineering, China University of Mining & Technology(Beijing), Beijing 100083, China; 2 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan 250100, China |
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| Abstract: | The detection of phytase transgenic corn is important to supervise its release and provides technical support. According to phytase gene (phyA2) sequences in transgenic phyA2 corn(Zea mays), the primers for qualitative and quantitative were designed. The qualitative PCR results showed that the primers were only effective to detect phyA2 transgenic corn, which indicated that the primers were highly specific. The SYBR Green I fluorescence quantitative PCR of endogenous reference zSSIIb zea mays starch synthase isoform zSTSII-2 and phyA2 genes were studied in this paper. Transgenic contens were calculated according to the standard Ct-copies linear graphs of the two genes. The results showed that the standard curves of zSSIIb and phyA2 genes had higher R2 value as 0.998 and 0.995, respectively, and the melting curves had single peak which mean the primers were specific. The mixed corn samples were detected to be 1.1%, 0.54% and 0.17%, respectively, which were close to the true values. The qualitative and quantitative PCR methods were developed in this study which can be effective at detecting phyA2 transgenic corn. |
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| Keywords: | Transgenic phytase corn Qualitative PCR Quantitative PCR zSSIIb Phatase gene(phyA2) |
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