您好,欢迎访问上海市农业科学院 机构知识库!

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene

文献类型: 外文期刊

作者: Weng, H 1 ; Pan, AH 2 ; Yang, LT 2 ; Zhang, CM 2 ; Liu, ZL 3 ; Zhang, DB;

作者机构: 1.Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai 200240, Peoples R China

2.Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai 200240, Peoples R China; Nanjing Univ, Dept Biol Sci & Technol, Nanjing 210093, Peoples R China; Shanghai Acad Agr Sci, Agrobiotech Res Ctr, Shaghai Key Lab Agr Genet & Breeding, Shanghai 201106, Peoples R China

3.Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai 200240, Peoples R China; Nanjing Univ, Dept Biol Sci &

关键词: Brassica napus;HMG I/Y;real-time PCR;transgene copy number;QUANTITATIVE PCR;COPY NUMBER;PLANTS;QUANTIFICATION;EXPRESSION

期刊名称:PLANT MOLECULAR BIOLOGY REPORTER ( 影响因子:1.595; 五年影响因子:2.042 )

ISSN:

年卷期:

页码:

收录情况: SCI

摘要: In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Trans-gene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous beta-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. The GUS and nptII copy numbers in primary transformants (T-0) were calculated by comparing threshold cycle (C-T) values of the GUS and nptII genes with those of the internal standard. HMG I/Y This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing its C-T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.

  • 相关文献

[1]Isolation and characterization of an oilseed rape MAP kinase BnMPK3 involved in diverse environmental stresses. Yu, SW,Zhang, LD,Zuo, KJ,Tang, DQ,Tang, KX.

[2]Rice straw decomposition affects diversity and dynamics of soil fungal community, but not bacteria. Li, Peng,Pan, Aihu,Tang, Xueming,Li, Peng,Ming, Feng,Li, Yongchun,Zheng, Xianqing,Lv, Weiguang,Ding, Lina. 2018

[3]Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. Ding, JY,Jia, JW,Yang, LT,Wen, HB,Zhang, CM,Liu, WX,Zhang, DB. 2004

[4]Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Yang, LT,Ding, JY,Zhang, CM,Jia, JW,Weng, HB,Liu, WX,Zhang, DB. 2005

[5]Cloning, expression, purification, and characterization of an organophosphate-degrading enzyme in Escherichia coli. Jinbin Wang,Dachao Chen,Dongxiao Hu,Xuefeng Su,Xueming Tang. 2013

[6]CHEMICAL SYNTHESIS OF TOXIN DOMAIN OF CRYIA (c) GENE AND EXPRESSION IN ESCHERICHIA COLI. Rihe Peng,Aisheng Xiong,Xian Li,Huiqin Fan,Quanhong Yao. 2001

[7]Fine mapping and candidate gene analysis of hwh1 and hwh2, a set of complementary genes controlling hybrid breakdown in rice. Jiang, Wenzhu,Chu, Sang-Ho,Piao, Rihua,Lee, Joohyun,Qiao, Yongli,Koh, Hee-Jong,Jiang, Wenzhu,Chu, Sang-Ho,Piao, Rihua,Lee, Joohyun,Qiao, Yongli,Koh, Hee-Jong,Chin, Joong-Hyoun,Jin, Yong-Mei,Jin, Yong-Mei,Han, Longzhi,Piao, Zongze.

[8]High efficiency and throughput system in directed evolution in vitro of reporter gene. Xiong, Ai-Sheng,Peng, Ri-He,Liu, Jin-Ge,Zhuang, Jing,Qiao, Yu-Shan,Xu, Fang,Cai, Bing,Zhang, Zhen,Chen, Jian-Min,Yao, Quan-Hong.

[9]Molecular cloning, characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum. Shi, Liang,Qin, Lei,Ren, Ang,Fang, Xing,Mu, Dashuai,Zhao, Mingwen,Xu, Yingjie,Tan, Qi.

[10]Identification and Quantification of Three Genetically Modified Insect Resistant Cotton Lines Using Conventional and TaqMan Real-Time Polymerase Chain Reaction Methods. Yang, LT,Pan, AH,Zhang, KW,Guo, JC,Yin, CS,Chen, JX,Huang, C,Zhang, DB.

[11]Expression profile analysis of 9 heat shock protein genes throughout the life cycle and under abiotic stress in rice. Ye ShuiFeng,Yu ShunWu,Shu LieBo,Wu JinHong,Luo LiJun,Ye ShuiFeng,Wu AiZhong,Wu AiZhong. 2012

[12]Evaluation of Four Genes in Rice for Their Suitability As Endogenous Reference Standards in Quantitative PCR. Wang, Chong,Rao, Jun,Liu, Yinan,Yang, Litao,Zhang, Dabing,Jiang, Lingxi,Zhang, Dabing.

[13]Event-Specific Quantitative Detection of Nine Genetically Modified Maizes Using One Novel Standard Reference Molecule. Yang, Litao,Guo, Jinchao,Pan, Aihu,Zhang, Haibo,Zhang, Kewei,Wang, Zhengming,Zhang, Dabing.

[14]Molecular characteristics and expression profiles of glycerol-3-phosphate dehydrogenase 1 (GPD1) gene in pig. Gao, Y. Z.,Jiang, Y.,Bai, C. Y.,Pan, Y. C.,Wu, X.,Sun, Y. Z..

[15]Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola. Peng, RH,Yao, QH,Xiong, AS,Cheng, ZM,Li, Y. 2006

[16]Expression analysis of a novel pyridoxal kinase messenger RNA splice variant, PKL, in oil rape suffering abiotic stress and phytohormones. Yu, Shunwu,Luo, Lijun. 2008

[17]Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis. Xiong, Ai-Sheng,Wang, Feng,Jiang, Hai-Hua,Peng, Ri-He,Jin, Xiao-Fen,Zhu, Bo,Yao, Quan-Hong,Zhuang, Jing,Zhang, Jian.

[18]Isolation and characterization of BnMKK1 responsive to multiple stresses and affecting plant architecture in tobacco. Yu, Shunwu,Chen, Chen,Li, Jiajia,Ye, Shuifeng,Liu, Guolan,Mei, Xiaohan,Luo, Lijun,Zhang, Lida,Tang, Kexuan.

[19]Isolated and characterization of a cDNA encoding ethylene-responsive element binding protein (EREBP)/AP2-type protein, RCBF2, in Oryza sativa L.. Liu, Jin-Ge,Zhang, Zhen,Qin, Qiu-Lin,Peng, Ri-He,Xiong, Ai-Sheng,Chen, Jian-Min,Xu, Fang,Zhu, Hong,Yao, Quan-Hong.

[20]High-level Expression of a Manganese Superoxide Dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris. Yin, Chaomin,Zhao, Wenxia,Ma, Aimin,Zheng, Liesheng,Chen, Liguo,Tan, Qi,Shang, Xiaodong.

作者其他论文 更多>>