榛子胚珠发育相关基因ChARF1的原核表达及多克隆抗体制备  

Procaryotic expression and polyclonal antibody preparation of ovule development related gene ChARF1 in hazel

作　　者：黄立龙 魏珩 王秋缘 刘春明[2] 刘剑锋[1] 程云清[1] HUANG Li-long;WEI Heng;WANG Qiu-yuan;LIU Chun-ming;LIU Jian-feng;CHENG Yun-qing(Key Laboratory of Plant Resources Science and Green Production,Jilin Normal University,Siping 136000,China;School of Life Sciences,Jilin Normal University,Siping 136000,China)

机构地区：[1]吉林师范大学吉林省植物资源科学与绿色生产重点实验室,吉林四平136000 [2]吉林师范大学生命科学学院,吉林四平136000

出　　处：《东北师大学报（自然科学版）》2022年第2期106-112,共7页Journal of Northeast Normal University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(32171840);吉林省科技厅中青年科技创新创业卓越人才项目(20210509033RQ)。

摘　　要：为解析ChARF1基因在榛子胚珠发育中的调控作用,以平欧杂交榛(Corylus heterophylla×C.avellana)“达维”成熟期胚珠为实验材料,克隆了在榛子胚珠发育不同时期显著差异表达的ARF同源基因,构建了其原核表达载体,并制备了该蛋白的多隆抗体.结果表明:ChARF1与胡桃(Juglans regia)ARF(XM_035695460.1)参考序列亲缘关系最近,与拟南芥AtARF1/2和番茄SlARF2聚类于同一大进化枝.以ChARF1基因N端前380个氨基酸(N-380)特异序列构建pET-28a-SUMO-ChARF1(N-380)原核表达载体,诱导表达后获得了包涵体形式存在的相对分子质量为66 000的抗原蛋白,纯化后免疫2只实验级雄性日本大耳白兔,最终获得能够特异性检测抗原蛋白的ChARF1(N-380)多克隆抗体E7200和E7201,二者效价均达到1∶512 000.Auxin response factor(ARF) is an important transcription factor in auxin transduction pathway, playing a key role in flower and fruit development.In this study, a highly differentially expressed ARF homologous gene was screened in different stages of ovule development.Then the gene was cloned from mature ovules of Corylus heterophylla × C.avellana, "Dawei" and named as ChARF1.The phylogenetic analysis showed that ChARF1 had a closest relationship with Juglans regia ARF(XM_035695460.1) and clustered in the same branch with Arabidopsis thaliana AtARF1/2 and Solanum lycopersicum SlARF2.The specific N-terminal sequence of ChARF1 was selected to construct the prokaryotic expression vector pET-28 a-SUMO-ChARF(N-380).The antigen protein in the form of inclusion body was successfully expressed under the induction of IPTG.Two experimental flap-eared Japanese rabbits were immunized with the purified antigen protein.The ChARF1(N-380) polyclonal antibodies E7200 and E7201 with titer of 1∶512 000 were obtained.

关 键 词：榛子 ARF 原核表达 多克隆抗体 Western Blot 

分 类 号：Q785[生物学—分子生物学] S664.4[农业科学—果树学]