红色荧光蛋白基因在转基因唐鱼中遗传和表达的稳定性分析

Inheritance and expression stability of red fluorescent protein gene in transgenic (Tanichthys albonubes)

为了评估转红色荧光蛋白(red fluorescent protein,RFP)基因唐鱼新品系的稳定性,研究了RFP基因在不同世代转基因唐鱼中的遗传和表达情况。荧光显微镜下观察显示,RFP基因在F6和F10代所检测的组织器官中均有表达,并且两个世代间相同组织部位的表达水平相似。F6和F10代个体分别配对繁殖实验表明,RFP基因在转基因唐鱼后代中的遗传仍然符合孟德尔分离规律,而且培育出的转基因个体表型特征无显著差异。利用PCR技术在F2、F6和F10代转基因唐鱼基因组中扩增外源性肌球蛋白轻链2启动子、RFP基因编码区和整合位点上下游侧翼区域(片段总长度为4 883 bp),测序结果显示,3个世代间的外源基因序列完全相同,没有发生碱基缺失或突变等现象。研究表明,红色荧光蛋白基因在转基因唐鱼传代培育过程中保持了稳定的遗传和表达。

The stability of the red fluorescent protein （RFP） transgenes is important for application of the transgenic Tanichthys albonubes line.This paper investigated comparatively the inheritance and expression of RFP gene in different transgenic T. albonubes generations. Under the observation of fluorescence microscope, RFP gene was expressed in almost all body tissues, and both F6 and F10 generations had a similar expression pattern. Results of self-cross and test-cross breeding experiments of transgenic F6 and F10 individuals indicated that segregation of RFP gene still followed Mendelian single-gene inheritance, and their transgenic progenies had a uniform phenotype. The regions including myosin light polypeptide 2 promoter, RFP gene coding sequence and transgene flanking junctions were amplified by PCR from genomic DNA of F2, F6 and F10 generations. Sequence alignment analysis showed that these important regions were well preserved in transgenic fish, and any kind of alteration at nucleotide level was not found. These results suggested that, RFP gene in transgenic T. albonubes maintained stable inheritance and expression through successive generations.