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Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR

文献类型： 外文期刊

作者： Yang, LT 1 ; Ding, JY 2 ; Zhang, CM 2 ; Jia, JW 3 ; Weng, HB; Liu, WX; 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 Univ, Sch Life Sci, Shanghai 200436, Peoples R China; Shanghai Acad Agr Sci, Agrobiotech Res Ctr, 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 & Technol, Nanjing 210093, Peoples R China; Shanghai Univ, Sch Life Sci, Shanghai 200436, Peoples R China; Shanghai Acad Agr Sci, Agrobiotech Res Ctr, Key Lab Agr Gen

关键词： transgenic rice;TaqMan real-time PCR;GUS;HPT;copy number

期刊名称：PLANT CELL REPORTS （ 影响因子：4.57； 五年影响因子：4.463 ）

ISSN： 0721-7714

年卷期： 2005 年 23 卷 10-11 期

页码：

收录情况： SCI

摘要： In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous beta-glucuronidase (GUS) and hygromycin phosphortransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T-0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T-0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.

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