应用RNAi技术构建的转基因鲤的检测分析

摘要

The eukaryotic expression vector including myostatin gene with H1 promoter were injected into the nuclear area in a zygote of common carp by microinjection technology and then a group of transgenic carps with RNAi phenotype was obtained.PCR and molecular hybridization detection revealed that the exon-gene was integrated into the receptor fish with integrated level of 32.78％.In the growth experiment,it was found that the transgenic fish had about 99％ more rapid growth,22％ higher body height,and 26％ higher body width than common carp in the control group,in which 31.82％ of individuals in the transgenic population had 60％ higher body width than the ones in the control group.The findings indicated that the hairpin-type dsRNA expressed by the plasmid degraded the transcript products of myo and was useful in inhibition of the expression of its homologue genes in muscle cells and in improvement of the renew power of common carp muscles.This inhibited power was showed that the back muscle of common carp became wider,and body weight was increased,indicating that dsRNA transcripted and expressed by myo has RNAi effect in common carp.This experiment applies a new method to gain transgenic common carp with special functions.%将构建携带H1启动子的肌肉生长抑制基因的真核表达载体,通过显微注射技术导入鲤受精卵核区附近,获得了一批具有RNAi表型的转基因鲤,PCR和分子杂交检测证实外源基因整合到受体鱼的基因组中,阳性率为32.78％；一龄鱼的生长实验表明,转基因鲤比普通鲤平均生长快0.99倍,其体高和体厚分别平均增长0.22和0.26倍,其中有31.82％的群体平均体厚是普通鲤的1.6倍.结果显示,该质粒表达的发夹环型dsRNA可以有效降解其转录产物,对阻抑肌细胞中同源基因的表达、鲤肌肉的再生能力增强起到了重要作用.这种抑制作用表现为鲤背部肌肉增厚、体质量增加,说明该基因经转录产生的双链RNA在鲤体内具有RNAi效应.RNAi技术为获得具有特殊功能的转基因鲤提供了新的手段.