家蚕bHLH转录因子Bmdimm与Bmchip的相互作用  

Interaction of b HLH Transcription Factor Bmdimm and Bmchip in Bombyx mori

作　　者：赵朋[1] 王叶菁[1,2] 位曙光 刘莉娜 李珍珍[1] 赵萍[1] 何华伟[1,2,3] 

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400715 [2]西南大学生物技术学院,重庆400715 [3]重庆市蚕丝纤维新材料工程技术研究中心,重庆400715

出　　处：《中国农业科学》2016年第10期2027-2038,共12页Scientia Agricultura Sinica

基　　金：国家重点基础研究发展计划("973"计划)(2012CB114602);国家自然科学基金(31402139;31572465);重庆市基础与前沿研究计划(cstc2015jcyj A00040);中央高校基本科研业务费(XDJK2013C049;XDJK2013A019;SWU112086);西南大学博士基金(SWU112111)

摘　　要：【目的】bHLH转录因子Bmdimm是家蚕(Bombyx mori)5龄后期调控蚕丝蛋白丝素重链基因表达的重要转录因子。分析发现Bmdimm蛋白中包含有可能发生泛素化修饰的赖氨酸残基K43,因此推测Bmdimm可能发生泛素化修饰而被降解。Bmchip属于家蚕泛素连接酶E3家族中的U-box亚家族,在家蚕5龄期后部丝腺中有表达。论文旨在明确Bmdimm与Bmchip之间的相互作用,以便于更好地理解Bmdimm在家蚕体内的泛素化修饰及其对丝素重链基因转录调控的生物学意义。【方法】利用家蚕各组织基因芯片表达谱分析Bmdimm和Bmchip在后部丝腺中的表达情况;分别通过PCR和基因合成获得Bmdimm和Bmchip的CDS序列,将其构建到不同的原核表达载体并转化大肠杆菌表达目的蛋白,筛选最优的表达条件;利用镍柱亲和层析纯化融合蛋白,加入蛋白酶酶切,并再次通过镍柱亲和层析去除融合标签,纯化Bmdimm和Bmchip蛋白;利用凝胶过滤层析分析Bmdimm和Bmchip在溶液中的聚集状态;利用圆二色光谱研究Bmdimm和Bmchip蛋白的二级结构;利用Far-western blotting和Pull-down研究Bmdimm和Bmchip在体外的相互作用;利用微量热泳动研究Bmdimm和Bmchip结合的亲和力。【结果】基因芯片表达谱揭示Bmdimm和Bmchip在家蚕5龄期后部丝腺中都有表达。构建了Bmdimm和Bmchip多种表达载体,发现ppSUMO-Bmdimm和pET-32M·3C-Bmchip表达载体在16℃,0.3 mmol·L^(-1) IPTG诱导下表达最好,并以此为基础表达纯化了Bmdimm和Bmchip蛋白;凝胶过滤分析表明Bmdimm和Bmchip蛋白在溶液中主要以二聚体的形式存在;圆二色光谱显示Bmdimm和Bmchip蛋白都含有α-螺旋结构;Far-western blotting和Pull-down结果表明Bmdimm和Bmchip可以在体外结合;利用微量热泳动实验计算出Bmdimm和Bmchip结合的平衡解离常数为(750±28.6)μmol·L^(-1)。【结论】家蚕bHLH转录因子Bmdimm和泛素连接酶Bmchip在家蚕5龄期后部丝腺中均有表达,二者可以在体�【Objective】 Bmdimm is a member of the bHLH transcription factors family, which is one of key transcription factors that regulate the expression of fibroin heavy chain gene in the late stage of the fifth instar of Bombyx mori. Bmdimm was predicted to have a potential ubiquitination site of lysine residue K43, and therefore it is presumed that Bmdimm may be modified by ubiquitin and thus degraded. Bmchip belongs to the U-box subfamily of the B. mori ubiquitin ligase E3 family, which is expressed in the posterior silk gland during the fifth instar of B. mori. Studying on the interaction of Bmdimm and Bmchip will promote us to better understand the ubiquitylation of Bmdimm in vivo and its biological significance on the transcriptional regulation of fibroin heavy chain gene. 【Method】 The expression profiles of Bmdimm and Bmchip in the posterior silk gland of B. mori were analyzed based on the B. mori genome microarray database. The CDS sequences of Bmdimm and Bmchip were obtained by PCR cloning and gene synthesis, respectively. Multiple expression vectors were constructed and then transformed into E. coli to express the target proteins to screen the best expression conditions. The fused Bmdimm and Bmchip were purified using Ni^（2＋） affinity chromatography, and then digested with protease. The fused tag and target proteins were separated via Ni^（2＋） affinity chromatography again. The oligo states of Bmdimm and Bmchip in solution were assessed by gel filtration chromatography. The secondary structures of Bmdimm and Bmchip were studied by circular dichroism spectroscopy. The interaction of Bmdimm and Bmchip in vitro were investigated using Far-western blotting and Pull-down. The affinity of Bmdimm and Bmchip were determined by microscale thermophoresis.【Result】The B. mori genome microarray expression profiles indicate both Bmdimm and Bmchip were expressed in the posterior silk gland during the fifth instar of B. mori. Multiple expression vectors of Bmdimm and Bmchip were constructed, and it was fo

关 键 词：家蚕 Bmdimm Bmchip 相互作用 泛素化 

分 类 号：S881.2[农业科学—特种经济动物饲养]