从江香猪PHKG2基因超表达载体和RNA干扰载体的构建及其表达

Construction and Expression of PHKG2 Gene Overexpression Vector and RNA Interference Vector in Congjiang Pig

旨在克隆从江香猪磷酸化酶激酶γ2(phosphorylase kinase gamma 2,PHKG2)基因编码区,并对PHKG2基因功能进行初步探究。本研究利用RT-PCR法扩增从江香猪PHKG2基因完整CDS区。通过构建pEGFP-N3-PHKG2超表达载体,设计并合成4对针对从江香猪PHKG2基因的RNAi表达载体,瞬时转染至C2C12细胞株和从江香猪肾细胞中,以正常生长的细胞为空白对照,24h后检测各个重组载体的绿色荧光蛋白的表达情况。48h后提取细胞总RNA,利用qRT-PCR方法检测目的基因PHKG2和糖原代谢相关基因糖原磷酸化酶(glycogen phosphorylase,muscle form,PYGM)、肌肉糖原合成酶(glycogen synthase 1(muscle),GYS1)、磷酸甘油酸变位酶(Phosphoglyce rate mutase,PGAM2)基因mRNA的表达情况,并且测定各组细胞中的糖原含量。结果表明,经过双酶切、测序检测以及脂质体瞬时转染至C2C12细胞中,验证超表达载体pEGFP-N3-PHKG2和4对RNAi表达载体均构建成功。转染至从江香猪肾细胞后,相对于空白对照(Blank)和阴性对照(pEGFP-N3),超表达载体pEGFP-N3-PHKG2能极显著提高PHKG2基因的表达量(P<0.01),同时显著上调PGAM2、PYGM基因的表达量(P<0.05),并且显著降低细胞中糖原的含量(P<0.05)。各RNAi载体中,相对于空白对照(Blank)和阴性对照(NC),shRNA-1的干扰效率较高,极显著下调PHKG2基因的表达量(P<0.01),显著下调PGAM2、PYGM、GYS1基因的表达量(P<0.05),并且显著升高细胞中糖原含量(P<0.05)。shRNA-2极显著下调PHKG2基因的表达量(P<0.01);shRNA-3对PHKG2基因的表达也能起到显著的干扰作用(P<0.05);shRNA-4对于所检测的4个基因干扰效果不明显;shRNA-2、shRNA-3、shRNA-4转染细胞后,细胞中糖原含量升高水平不明显。PHKG2基因超表达和RNAi后可分别明显上调和抑制PHKG2、PYGM、PGAM2、GYS1基因的表达,并且分别降低和提高糖原含量,可能是影响从江香猪猪肉品质的重要候选基因,为进一步研究PHKG2基因在细胞糖原代谢通路中的作用机制奠定基础。

The aim of this study was to clone the coding region of phosphorylase kinase gamma 2(PHKG 2)gene in Congjiang pig and to explore the function of PHKG2 gene.In this study,the complete CDS region of PHKG 2 gene in Congjiang pig was amplified by RT-PCR.By constructing the overexpression vector pEGFP-N3-PHKG 2,four pairs of RNAi expression vectors targeting the Congjiang pig PHKG 2 gene were designed and synthesized,and transiently transfected into C2C12 cell line and Congjiang pig kidney cells with normal growing cells as a blank control,the expression of green fluorescent protein of each recombinant vector was detected after 24 h.RNA was extracted from cells after 48 h.The expression levels of PHKG 2 and glycogen metabolism related genes(glycogen phosphorylase(PYGM),glycogen synthase 1(muscle)(GYS 1),phosphoglyce rate mutase(PGAM2))were detected by qRT-PCR,and the glycogen content of each group in cells was determined.The results showed that,after double enzyme digestion,sequencing detection and transient transfection of liposomes into C2C12 cells,it was verified that the overexpression vectors pEGFP-N3-PHKG 2 and 4 RNAi expression vectors were successfully constructed.After transfecting into Congjiang pig kidney cells,compared with the blank and negative control(pEGFP-N3),the overexpression vector pEGFP-N3-PHKG 2 transfected into cells extremely significantly increased the expression level of PHKG 2 gene(P<0.01),and PGAM 2 and PYGM genes expression were significantly up-regulated(P<0.0 5),and the glycogen content in cells was significantly reduced(P<0.05).In each RNAi vector,the interference efficiency of shRNA-1 was higher than that of the blank and negative control(NC).The expression levels of PHKG2 gene was extremely significantly down-regulated(P<0.01),and the expression levels of PGAM 2,PYGM and GYS 1 genes were significantly down-regulated(P<0.05),and significantly increased the glycogen content in the cells(P<0.05).shRNA-2 extremely significantly down-regulated the expression of PHKG 2 gene(P<0.01);shRNA-3 also significantly interfered with the expression of PHKG 2(P<0.0 5);shRNA-4 did not interfere with the expression of the 4 genes detected obviously.The glycogen contents in cells didn’t significantly increase after shRNA-2,shRNA-3,shRNA-4 transfected into the cells.In this study,the overexpression vectors and RNAi vectors of PHKG 2 gene of Congjiang pig were successfully constructed.The expression of PHKG2,PYGM,PGAM2 and GYS1 genes and glycogen content were significantly affected.PHKG2 gene may be an important candidate gene affecting meat quality of Congjiang pig and to lay the foundation for further studying the role of PHKG2 gene in glycogen metabolism pathway.