【摘要】 转基因动物的外源基因通过使用体细胞克隆与胚胎移植的方法将表达载体转入细胞后获得,大多以随机整合的形式存在于受体动物的基因组中,而转基因动物中外源基因的整合拷贝数是影响外源基因表达水平及遗传稳定性的重要因素之一,作为转基因动物遗传稳定性的鉴定指标,获得存活的转基因动物后便有必要对其外源基因拷贝数进行检测。为了研究转基因阿尔巴斯白绒山羊(Capra hircus)基因组内外源海葵红色荧光蛋白(red fluorescence protein from Discosoma sp.,Ds Red)基因的拷贝数与其表达量之间是否存在一定的联系,本研究采用较鉴定外源基因拷贝数的传统方法DNA印迹杂交(Southern blot)更为高效率、高灵敏度、高准确性的绝对定量q RT-PCR法检测了6只转基因白绒山羊外源Ds Red基因表达框中3个不同片段的拷贝数。结果表明,外源基因3个片段的拷贝数分别为巨细胞病毒(Cytomegalovirus,CMV)启动子:2.80±0.38~13.68±0.16;Ds Red基因:1.34±0.04~11.84±0.02;CMV启动子与Ds Red连接处:1.58±0.04~19.22±0.38。通过q RT-PCR中相对定量的方法检测了6只转基因白绒山羊外源Ds Red基因m RNA表达量,将其与外源基因中3个不同片段的拷贝数数据分别使用SPSS软件进行相关性分析后,相关性系数P值的结果为:CMV启动子为P=0.763;Ds Red基因为P=0.665;CMV启动子与Ds Red连接处为P=0.803,3组P值均大于0.05,无显著性相关。上述结果表明,在所检测的6只转基因阿尔巴斯白绒山羊中外源CMV启动子启动的Ds Red基因的表达量与其在转基因白绒山羊基因组内的拷贝数在0.05的水平没有显著相关性。另外验证了绝对定量q RT-PCR法在大型转基因家畜的外源基因拷贝数的研究中的可靠性:其重复性良好,误差可控,可以成为检测转基因动物外源基因拷贝数的首选方法。更多还原

【Abstract】 Exogenous genes mostly random integrated in the genome of transgenic animals which generatedby somatic cell cloning and embryo transplantation.The copy number of exogenous gene is one of the important factors which affect the expression level of exogenous gene and genetic stability in transgenic animals.As the identification index of genetic stability of transgenic animals,it is necessary to detect the copy number of the exogenous gene after the survival of transgenic animals.In order to test whether there is relationship between the copies of exogenous red fluorescence protein from Discosoma sp.(Ds Red) gene,and its expression level in the transgenic Arbas cashmere goat(Capra hircus) genomic,Absolute q RT-PCR method which was more efficient highly sensitive and accurate than the traditional method of Southern blot was used to identify the copy number of exogenous gene to test the copy number of 3 different fragments in exogenous Ds Red gene in 6 transgenic cashmere goats.The results showed that the copy number of Cytomegalovirus(CMV) promoter ranged from 2.80±0.38 to 13.68±0.16,Ds Red gene from 1.34±0.04 to 11.84±0.02,joint of CMV promoter and Ds Red gene from 1.58 ± 0.04 to 19.22 ± 0.38.The expression of exogenous Ds Red gene m RNA in 6 transgenic cashmere goats was detected by relative q RT-PCR method.The exogenous gene in 3different fragments of copy number data respectively using SPSS software for correlation analysis,the correlation coefficient P value was,CMV promoter,P=0.763;Ds Red gene,P=0.665;joint of CMV promoter and Ds Red gene:P=0.803.The P values of the 3 groups were all greater than 0.05.The results showed that in our detection of 6 transgenic Arbas cashmere goats in exogenous Ds Red gene expression and the copy number of the exogenous gene had no significant correlation at 0.05 level.In addition,the reliability of the q RT-PCR method in the study of the copy number of exogenous genes in large transgenic livestock was verified:With good reproducibility and controllable error,it can be the preferred method to detect the copy number of exogenous gene in transgenic animals.更多还原