【作者】 王玉军；

【导师】 徐世清； 司马杨虎；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2006， 硕士

【摘要】 家蚕滞育生物钟是依赖卵内酯酶A4（EA4）蛋白的倒计时型生物钟。EA4是最早被确认的具时间间隔测定功能的酶。根据家蚕EA4测定的氨基酸序列,以3d龄雌蛹脂肪体和卵巢总RNA为模板,利用简并引物RT-PCR方法,得到308bp的部分ea4序列,其1-144nt和145-308nt分别与家蚕2个WGS（AADK01019126.1）5595-5738nt和WGS（AADK01014467.1）的8636-8473nt有95%和98%的一致性,根据预测的ea4的cDNA序列,设计特异引物,以雌蛹脂肪体和卵巢RNA为模板,RT-PCR得到的519bp的cDNA序列与预测序列完全一致,并且其推测的氨基酸序列与已知的EA4部分一致性98%,证实获得了ea4全长cDNA。根据预测的ea4 DNA序列设计引物,以家蚕全基因组的DNA为模板作PCR,得到WGS（AADK01019126.1）和WGS（AADK01014467.1）之间的拼接序列,最终查清了ea4基因组结构。ea4DNA全长5281bp,有3个外显子和2个内含子,位于家蚕同一条染色体上。将ea4克隆到具His6序列标签的表达载体pET28a（+）,构建大肠杆菌融合表达质粒pET28a（+）-ea4;将重组质粒转化大肠杆菌BL₂₁,IPTG诱导,成功表达了家蚕ea4基因的融合蛋白,为In vitro研究EA4功能奠定了基础。更多还原

【Abstract】 The diapause bioclock protein of Bombyx silkworm is count down clock which depends on esterase A4 （EA4）, EA4 is the first confirmed time interval measuring enzyme.A 308bp of ea4 sequence was got by degenerate primer RT-PCR, the primers were designed according to the determined amino acids and the total RNA of fat body and ovary from 3 days old female pupa was used for the template. The consistence of 1-144nt and 145-308nt of the sequence with 5595-5738nt of WGS （AADK01019126.1） and 8636-8473nt of WGS （AADK01014467.1） are 95% and 98% respectively. A pair of special primers was designed according to the cDNA sequence of ea4, and used the total RNA of fat body and ovary of female pupa as the template for RT-PCR, then got a 519bp sequence which is consist with the predicted sequence completely and the consistence of the predicted amino acids with the determined is 98%, which proved to get the overall cDNA of ea4. Designed another pair of special primers according to the DNA sequence of ea4, and used the DNA of Bombyx silkworm as the template for PCR, the link sequence between WGS （AADK01019126.1） and WGS （AADK01014467.1） were gained, naturally, the genome of ea4 was clear, the full length DNA of ea4 is 5281bp including 3 extrons and 2 introns, they are at the same chromosome.Clone the ea4 gene to the express vector pET28a（+） which hold the sequence express tag of His₆ for construct the fusion express vector pET28a（+）-ea4. Transform the fusion vector to E.coli BL₂₁ and induced by IPTG, the fusion protein was expressed, which set the foundation of the protein function for In vitro research.更多还原