【作者】 刘江；

【导师】 魏国清；

【作者基本信息】 安徽农业大学 ， 特种经济动物饲养， 2021， 硕士

【摘要】 家蚕（Bombyx mori）是一种具有重要经济价值的鳞翅目昆虫。家蚕C₃HC₄型锌指蛋白是一种重要的转录调控因子,能与核酸特异性结合,且与不同的靶基因序列结合会发挥不同的功能,参与多种不同生理过程。本论文对家蚕C₃HC₄型锌指蛋白基因家族进行了鉴定和生物信息分析并对其成员Ring finger protein181（RNF181）进行了c DNA克隆、原核表达、蛋白纯化、抗体制备、表达模式及免疫刺激。获得结果主要如下:1.从家蚕基因组中共鉴定出32个标准的C₃HC₄型锌指蛋白基因。32个C₃HC₄锌指蛋白基因家族可进一步分为4个亚家族。2.对所鉴定的C₃HC₄基因进行了染色体定位。家蚕C₃HC₄基因物理定位在染色体上的分布是广泛且不均匀的,10号染色体上具有明显的聚集现象。除Bm ZFPC₃HC₄6基因包含三个基序外,其余31个基因只含有1个基序。3.构建了家蚕C₃HC₄基因在家蚕各组织中的表达谱,对在脂肪体中特异性表达的6个C₃HC₄基因、丝腺中特异性表达的4个C₃HC₄基因进行了q RT-PCR分析。结果表明Bm ZFPC₃HC₄3、Bm ZFPC₃HC₄11、Bm ZFPC₃HC₄13、Bm ZFPC₃HC₄17、Bm ZFPC₃HC₄26、Bm ZFPC₃HC₄28基因在家蚕免疫刺激后脂肪体中的表达模式不同。在丝腺不同发育时期的不同部位中Bm ZFPC₃HC₄3、Bm ZFPC₃HC₄11、Bm ZFPC₃HC₄13、Bm ZFPC₃HC₄17基因的空间表达模式存在明显的差异。4.从家蚕C₃HC₄型锌指蛋白基因中筛选了RNF181基因,并进行了重组表达质粒构建,成功在大肠杆菌中诱导表达。通过亲和纯化得到较高浓度和纯度的RNF181重组蛋白,成功制备了家蚕RNF181蛋白多克隆抗体。.家蚕各组织中RNF181基因的相对表达水平存在差异。其中脂肪体中的表达量最高,马氏管中表达量最低;RNF181基因在家蚕五龄期表达量最高,其次是卵,其他各龄段均有所表达。在四龄眠期后不同时期丝腺中均有表达,且都呈现先增后降的表达模式。.初步鉴定了RNF181的功能。家蚕RNF181在病原微生物刺激的表达量都表现出一定程度上调,进一步表明RNF181可能参与了家蚕的免疫应答反应。更多还原

【Abstract】 Silkworm,Bombyx mori is a kind of lepidopteran insect with important economic benefits.C₃HC₄zinc finger protein is an important transcription regulator,which can play different biological functions and participate in a variety of physiological processes through specifically binding with nucleic acids and different target gene sequences,In this study,the C₃HC₄zinc finger protein gene family of silkworm was identified and analyzed.The c DNA cloning,prokaryotic expression,protein purification,immune stimulation and antibody preparation of ring finger protein 181（RNF181）gene were carried out.The main results are as follows:1.In this study,32 standard C₃HC₄zinc finger protein genes were identified in Bombyx mori genomic databases.The 32 C₃HC₄zinc finger protein genes can be further divided into four Subfamilies.2.The identified C₃HC₄gene was positioned on chromosome.The physical location of C₃HC₄genes in Bombyx mori were widely and unevenly distributed on chromosomes,and there was obvious aggregation on chromosome 10.31 genes only contain one motif except for Bm ZFPC₃HC₄6 gene which contains three motifs.3.The expression profiles of C₃HC₄gene in different tissues of Bombyx mori were constructed.Six C₃HC₄genes specifically expressed in fat body and four C₃HC₄genes specifically expressed in silk glands were analyzed by q RT-PCR.The expression patterns of Bm ZF_PC₃HC₄3,Bm ZFPC₃HC₄11,Bm ZFPC₃HC₄13,Bm ZFPC₃HC₄17,Bm ZFPC₃HC₄26 and Bm ZFPC₃HC₄28 in the fat body of silkworm were different after immune stimulation.The spatial expression patterns of Bm ZFPC₃HC₄3,Bm ZFPC₃HC₄11,Bm ZFPC₃HC₄13 and Bm ZFPC3HC₄17 genes were significantly different in different parts of silk gland at different developmental stages.4.The gene RNF181 was screened from the C₃HC₄zinc finger protein gene of silkworm,Bombyx mori.The recombinant expression plasmid was successfully constructed and expressed in E.coli（DE3）.The recombinant RNF181 protein with high concentration and purity was obtained by affinity purification,and the polyclonal antibody against ring finger protein 181 was successfully prepared.The expression f RNF181 was different in different tissues of Bombyx mori.The expression level of RNF181 was higher in fat body,followed by silk gland and head,and lowest in Malpighian tubule;RNF181 was expressed in all stages of silkworm development,and higher in 5th instar,followed by egg stage.It was expressed in the silk gland at different stages after the fourth instar dormancy,and the expression pattern of increasing first and then decreasing.The function of RNF181 was preliminarily identified.The expression of RNF181 in silkworm was up-regulated to some extent by pathogenic microorganisms,which further indicated that RNF181 may be involved in the immune response of silkworm.更多还原