转座子介导的插入诱变(insertional mutagenesis)策略在养殖鱼类功能基因发掘和解释方面有着重要的研究意义.本研究以高效Tgf2转座子为研究基础,构建了pTgf2-β-actin-eGFP、pTgf2-EF1α-eGFP、pTgf2-MyoD-eGFP和pTgf2-Krt8-eGFP这4种带有不同启动子的供体质粒,以及pCS2-gfTP这种gfTP转座酶mRNA的体外表达的辅助质粒.将供体质粒和gfTP转座酶mRNA一起注射到草鱼(Ctenopharyngodon idellus)、团头鲂(Megalobrama amblycephala)和鲫(Carassius auratus)的1～2细胞期受精卵中,经对子代增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)整合率的检测和筛选,初步构建了1个以转座子为工具介导的插入诱变库.实验表明,在早期胚胎中,鲫和团头鲂的荧光率达到90％,在草鱼中则高达95％,随着供体质粒注射的改变,表现出肌肉、表皮及全身不同的绿色荧光发光特征.eGFP基因平均整合率在3种鱼类子代平游期的为50％～53％,在1龄阶段为33.0％～36.1％.在3种鱼类中得到插入诱变突变体,获得草鱼阳性个体162尾,其中有45尾表型明显差异;鲫阳性个体60尾,其中有14尾表型明显差异;团头鲂阳性个体51尾,其中有10尾表型明显差异.结果表明,通过插入Tgf2转座子,可在草鱼、团头鲂和鲫等鲤科鱼类中实现基因组的插入诱变,本研究结果可为目标性状功能基因的挖掘和功能机制研究积累一定的基础.%Transposon mediated insertional mutagenesis has great value in the discovery and interpretation of functional genes in aquaculture fish.In this study,a preliminary study of insertion mutagenesis was carried out in 3 Cyprinidae fish mediated by Tgf2 transposon.On the basis of high efficient Tgf2 transposon,4 donor plasmids pTgf2-β-actin-eGFP,pTgf2-EF1α-eGFP,pTgf2-MyoD-eGFP,pTgf2-Krt8-eGFP and the in vitro expression of mRNA helper plasmid gfTP in pCS2-gfTP were constructed.The donor plasmids and in vitro expressed gfTP transposase mRNA were co-injected into 1～2 cell stage fertilized eggs of grass carp (Ctenopharyngodon idellus),crucian carp (Carassius auratus) and blunt snout bream (Megalobrama amblycephala).By detection and screening of the eGFP gene integration rate,the insertional mutagenesis library mediated by Tgf2 transposon was initially established.In the early stage of embryos,the fluorescence ratio was very high.The fluorescence ratio in both blunt snout bream and crucian carp reached 90％,and even 95％ in grass carp.According to the different donor plasmid,there were 3 types of fluorescence,such as in the muscle,skin and whole body.The integration ratio in larva fish was 50％～53％,and the integration ratio in the one year old fish was 33.0％～36.1％.In total,in grass carp,162 positive mutants,and 45 individuals were got in them had obvious phenotypes.There were 60 positive individuals in crucian carp,including 14 obvious phenotypes.In blunt snout bream,there were 51 positive individuals,including 10 obvious phenotypes.These results suggest that insertional mutagenesis could be realized in grass carp,blunt snout bream and wild crucian carp by Tgf2 transposon inserted into fish genome.Our studies can accumulate some basis for the study of mining and functional mechanism of target character gene function.
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