鲤mstn基因RNA干涉载体构建与检测分析

Construction and Detection of RNA Interference Vectors for mstn Gene in Common Carp

肌肉生长抑制素(Myostation,MSTN)是影响肌肉生长的负调控因子,其缺失或突变会导致肌肉量的增加。本研究参照鲤(Cyprinus carpio)高通量转录组测序数据的拼接结果,筛选出针对mstn基因的si RNA序列(19bp保守同源序列),合成si RNA转录模板的发夹结构,构建包含斑马鱼(Danio rerio)U6启动子、干涉片段si MSTN和鲤mstn的供体质粒pzu6p-si MSTN。通过显微注射方法,获得一批转鲤mstn基因的转基因鲤。经PCR检测,转基因鲤mstn外源基因的平均整合率为36.2%,对其中5尾转基因阳性鲤的扩增产物进行回收、克隆和测序验证。结果表明:转基因阳性鲤中均含有转基因目的片段,说明鲤mstn基因RNA干涉载体可在鲤中实现基因整合,为进一步获得具有特殊功能的转基因鱼类提供新的方法。

Myostatin(Myostation, MSTN)is a negative regulator of muscle growth, in which the deletion and mutation can produce the double muscle phenotype. Si RNA sequences of mstn genes, a 19 bp conservative homologous sequence, were screened by referring to the splicing results of high-throughput transcriptome sequencing data of common carp(Cyprinus carpio), and the hairpin structure of the si RNA transcription template was synthesized. The donor plasmid of pzu6p-siMSTN was constructed, which contained the zebrafish(Danio rerio)U6 promoter, the interference fragment of si MSTN and the mstn of common carp. Transgenic common carp were obtained using co-microinjection of the donor with the donor plasmid of pzu6p-siMSTN. PCR analysis revealed that the average integration rate of the exogenous gene in transgenic common carp was 36.2%. The amplified products were recovered, cloned and sequenced in five positive transgenic common carp to confirm that all of them contained the target fragment. The findings indicate that RNA interference vector of mstn gene can achieve gene integration in common carp, which provides a new method for further development of transgenic fish with special functions.