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当前位置: 首页 > 文章 > 水牛抑制素α亚基基因RNAi载体构建与检测 中国兽医学报 2011,31 (7) 1070-1075
Position: Home > Articles > Construction and analysis of buffalo inhibin α-subunit gene RNAi vectors Chinese Journal of Veterinary Science 2011,31 (7) 1070-1075

水牛抑制素α亚基基因RNAi载体构建与检测

作  者：
谢琴;蒋建荣;王丹;石德顺;谢体三;陈施蓓;刘庆友
单  位：
广西大学动物繁殖研究所
关键词：
水牛;抑制素α亚基;RNAi
摘  要：
抑制素通过反馈抑制促卵泡素的合成与分泌影响动物的生殖功能,将动物的繁殖力控制在种属特有的水平。为探讨通过抑制素基因沉默提高水牛繁殖力的可行性,本文构建并筛选了水牛抑制素α亚基基因的RNAi载体。根据实验室克隆的水牛INH-α基因序列,设计合成了5对特异性单链siRNA序列,经退火形成双链,连入pSilenc-er4.1-CMV neo载体。重组质粒经酶切及测序正确后转染水牛卵泡颗粒细胞。48h后,采用实时荧光定量PCR(qRT-PCR)技术检测不同组转染细胞中INH-α基因的相对表达水平,再选择沉默效率高的载体分别检测转染后24、48、72和96h抑制效率的变化。结果显示,经酶切和测序验证成功构建pSilencer4.1-145、pSilencer4.1-308、pSilencer4.1-769、pSilencer4.1-1013和pSilencer4.1-1053共5个重组RNAi表达载体,其中pSilencer4.1-308、pSi-lencer4.1-769、pSilencer4.1-1013和pSilencer4.1-1053具有抑制效果,抑制效率分别为58.8%、44.9%、67.4%和43.9%。选择pSilencer4.1-1013载体转染颗粒细胞24、48、72和96h后的抑制效率分别为27.1%、61.0%、57.0%、41.2%,转染48h后抑制效率最高。本研究成功构建了有效抑制的水牛INH-α基因表达的RNAi载体,为研制INH-α沉默的转基因水牛新品系奠定了基础。
译  名：
Construction and analysis of buffalo inhibin α-subunit gene RNAi vectors
作  者：
XIE Qin,JIANG Jian-rong,WANG Dan,SHI De-shun,XIE Ti-san,CHEN Shi-bei,LIU Qing-you(Animal Reproduction Institute,Guangxi University,Nanning 530005,China)
关键词：
buffalo;inhibin-α gene;RNAi
摘  要：
Inhibin α-subunit gene could feedback suppress synthesis and secretion of FSH,regulate animal reproduction trait at its species level.To investigate feasibility of increasing buffalo reproduction trait by silencing inhibin gene,buffalo inhibin-α gene RNAi vectors were constructed and screened in this study.According to CDS sequence of buffalo INH-α gene cloned in our lab,5 pair of siRNA oligonucleotides were designed,synthsized,annealed and directly cloned into pSilencer4.1-CMV neo expression vector.The constructed vectors were transfected to in vitro culture buffalo granulose cells,relative transcription level of INH-α gene mRNA were determined by Real-time PCR 48 h late.Then the high efficiency knock down vector was chosen to detect the variation of inhibition ratios at 24,48,72 and 96 h.The results showed that:5 buffalo inhibin a-subunit RNAi vectors were constructed including pSilencer4.1-145、pSilencer4.1-308、pSilencer4.1-769、pSilencer4.1-1013 and pSilencer4.1-1053,which were conformed correctly by restriction and sequencing analysis.The relative transcription levels of inhibin-α mRNA in buffalo granulose cells transfected with pSilencer4.1-308、pSilencer4.1-769、pSilencer4.1-1013 and pSilencer4.1-1053 decreased.The inhibition ratios were 58.8%、44.9%、67.4% and 43.9%,respectively.Then pSilencer4.1-1013 was transfected and qRT-PCR were performed at 24、48、72 and 96 h,inhibition ratios were 27.1%、61.0%、57.0% and 41.2%,respectively.In conclusion,effective RNAi vectors for buffalo inhibin-α gene have been successfully constructed,which is of great important in producing INH-α silencing transgenic buffalo.

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