利用实时荧光定量PCR方法分析转基因水稻外源基因拷贝数

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

为分析转基因水稻外源基因拷贝数 ,利用新型、灵敏、高通量的实时荧光定量PCR方法进行转基因水稻外源基因拷贝数的分析。转基因外源基因的拷贝数通过转基因水稻外源基因 (GUS和HPT基因 )和水稻内标准SPS基因含量的比较计算获得。定量分析了 14株T0 代的转基因水稻植株 ,得到了外源基因插入分别为 1、2、3和 4的转基因植株 ,同时利用SouthernBlot方法进行验证分析。随机选择 18个已经过定量PCR检测分析的转基因水稻植株 ,用SouthernBlot的方法分析转基因水稻植株中的HPT或GUS基因的拷贝数 ,SouthernBlot分析结果显示有 15个转基因水稻植株的分析结果与定量PCR分析的结果是一致的 ,3个植株定量PCR分析的转基因拷贝数稍高于SouthernBlot的分析结果 ,主要原因是SouthernBlot方法在同一个插入位点有多拷贝的T -DNA片段插入时 ,转基因植株的基因组在完全酶切时会产生相似的DNA片段 ,电泳分析时很难分辨清楚。定量PCR方法则完全避免了这种情况的发生 ,除非目的基因DNA片段在PCR引物处发生断裂。两种方法分析结果的比较显示定量PCR方法分析转基因拷贝数更加有效、适用。

In transgenic plants, the transgene copy number can greatly influence the expression level and genetic stability of the target gene. Estimation of transgene copy number is an important part of genetically modified (GM) crop research. Currently, transgene copy numbers are estimated by Southern analysis, which is laborious, time consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. Here, report the development of a sensitive, high throughput real time PCR technique for estimating transgene copy number in GM rice was reported. This system uses TaqMan quantitative real time PCR and comparison to a novel rice endogenous reference gene, sucrose phosphate synthase (SPS), to determine the copy numbers of exogenous β glucuronidase ( GUS ) and hygromycin phosphortransferase ( HPT ) genes in transgenic rice. The GUS and HPT copy numbers 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 1, 2, 3 and 4 transgene copies in the T 0 transformants. Furthermore, the copy number estimations for 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.