长期继代培养的转基因苹果外源基因遗传及表达稳定性分析

Genetic and expression stability analysis of exogenous gene in long-term subcultured transgenic apples(Malus×domestica Borkh.)

为研究长期继代培养的转基因苹果组培苗中外源基因的遗传及表达稳定性,以继代培养9年的7个转GFP(绿色荧光蛋白)基因苹果株系组培苗为试材,分析转基因株系对Kan(卡那霉素)的抗性、DNA水平、转录水平和转录后水平GFP基因的遗传及表达稳定性。结果表明,在7个苹果转基因株系组培苗中均可检测出GFP特异基因片段,并且均可在含有50mg/L卡那霉素的培养基上正常生长;绝对定量qRT-PCR检测发现,各转化株系GFP基因拷贝数并不相同;利用相对定量qRT-PCR法检测发现7个株系中GFP基因mRNA表达量有明显差异,同时荧光显微镜下观察各转化株系叶片,绿色荧光强度有明显差异,利用SPSS软件对7个转基因株系中GFP基因拷贝数与GFP mRNA表达量相关性分析结果呈无显著相关性。以上结果表明,外源基因可以在长期继代培养的转基因苹果组培苗中保持其遗传稳定性,但不同转化株系中外源基因的表达量有显著差异,其表达量与拷贝数无显著相关,可能与外源基因在植物基因组中的插入位点有关。

In order to assess the inheritance and expression stability of exogenous genes in the long-term subcultuted transgenic apple lines,the GFPtransgenic apple tissue culture plantlets of 7 lines cultured for 9 years were used as test materials.The genetic and expression stability of GFP gene were analyzed at the levels of resistance,DNA,transcription and post transcription.The results showed that GFPgene could be detected in the tissue culture seedlings of 7 transgenic apple lines which could grow normally on the culture medium containing 50 mg/L kanamycin;Absolute quantitative qRT-PCR analysis showed that the copy numbers of GFPgene were different in each transformed lines,and the expression levels of GFP mRNA in 7 transgenic lines were significantly different by relative quantitative qRT-PCR detection.At the same time,the green fluorescence intensity of each transformed line was significantly different under the fluorescence microscope.There was no significant correlation between GFP gene copy number and GFP mRNA expression in 7 transgenic lines by SPSS software.In conclusion,the exogenous gene could remain stability in long-term cultured transgenic apple plantlets.However,there were significant differences of the expression of foreign genes in different transformation strains,and their expressions were not significantly related to the copy number,which might be related to the insertion site of exogenous genes in the plant genome.