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。
Inhibin α-subunit gene could feedback suppress synthesis and secretion of FSH, regulate animal reproduc tion 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 CI）S sequence of buffalo INH-α gene cloned in our lab, 5 pair of siRNA oligonucleotides were designed, synthsized, annealed and di redtly 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 pSileneer4.1 145 ,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 inhibi- tion ratios were 58.8% ,44.9% ,67.4% and 43.9% ,respectively. Then pSilencer4.1-1013 was transfected and qRT PCR were performed at 2448,72 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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2011年第7期1070-1075,共6页
Chinese Journal of Veterinary Science
基金
农业部重大专项(2008ZX08007-003)
国家自然科学基金资助项目(30960251)
广西区重点资助项目(0991007Z)
