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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (10): 72-80    DOI: 10.13523/j.cb.20171010
技术与方法     
Taqman定量PCR技术检测基因编辑番茄中外源基因拷贝数体系的建立
任爽, 朱鸿亮
中国农业大学食品科学与营养工程学院 北京 100083
Establishment of Taqman Quantitative PCR System to Estimate Copy Numbers of Exogenous Transgene in Genome Edited Tomato
REN Shuang, ZHU Hong-liang
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
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摘要： 近些年CRISPR/Cas9基因编辑技术已广泛应用于作物遗传育种中，经过拷贝数筛选得到的单拷贝基因编辑株系，可以进行遗传分离获得具有改良性状但不携带外源基因成分的非转基因植株，消除转基因安全性的风险。目前拷贝数的检测主要采用Southern杂交，但是该方法存在操作复杂，需要相对大量的植物材料等缺点，不能进行高通量的筛选。为建立简便高效的基因编辑番茄植株中外源基因拷贝数的检测体系，以内源性基因APX（抗坏血酸过氧化物酶基因）作为内参基因，外源性基因HPT（潮霉素磷酸转移酶基因）作为目的基因，利用Taqman法的实时荧光定量PCR技术，检测有12株PDS基因（八氢番茄红素脱氢酶基因）编辑番茄中外源抗性基因HPT的拷贝数为1，初步建立了一种检测基因编辑植株中外源基因整合拷贝数的方法，为快速可靠的筛选单拷贝改良株系奠定了一定的技术基础。
关键词： 实时荧光定量PCR;  CRISPR/Cas9;  基因编辑番茄;  拷贝数;  Taqman探针    
Abstract: Recently, CRISPR/Cas9 genome editing technology has been broadly utilized to crop breeding. Genome-edited but transgene-free plants can be segregated away from T0 transgenic plants by genetic separation, which will eliminate the risk of transgenic safety. Copy numbers of transgene which has been integrated into the transformed plant genome is a critical factor affecting the genetic separation of the offspring. Copy numbers are currently obtained by Southern blot analysis, but this method is complicated and requires relatively large amounts of plant materials. As to avoid these shortcomings, Real-time Fluorescent Quantitative Polymerase Chain Reaction provides a new solution. The endogenous ascorbate peroxidase (APX) is selected as reference gene. The exogenous hygromycin phosphortransferase (HPT) is selected as target gene. With the Taqman RT-PCR conditions, one copy was significantly estimated in the 12 genome edited tomatos of slyPDS (Phytoene desaturase). Initially, a sensitive and efficient detection system is developed for estimating copy number in genome edited tomato, which laid a foundation for the rapid and reliable screening of improved crops.
Key words: Real-time Fluorescent Quantitative PCR    Taqman probe    CRISPR/Cas9    Copy number    Genome edited tomato
收稿日期: 2017-04-13 出版日期: 2017-10-25
ZTFLH:  Q786  
通讯作者: 朱鸿亮,hlzhu@cau.edu.cn     E-mail: hlzhu@cau.edu.cn
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引用本文:

任爽, 朱鸿亮. Taqman定量PCR技术检测基因编辑番茄中外源基因拷贝数体系的建立[J]. 中国生物工程杂志, 2017, 37(10): 72-80.

REN Shuang, ZHU Hong-liang. Establishment of Taqman Quantitative PCR System to Estimate Copy Numbers of Exogenous Transgene in Genome Edited Tomato. China Biotechnology, 2017, 37(10): 72-80.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171010    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I10/72

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