池蝶蚌IκBα和c-Rel的结构特征及在LPS刺激下的表达分析  

STRUCTURAL CHARACTERISTICS OF IΚBΑAND C-REL AND EXPRESSION ANALYSIS UNDER LPS STIMULATION IN HYRIOPSIS SCHLEGELII

作　　者：王小敏 周叶 马慧妹 ZAHID Anwar 胡蓓娟[1,2] 彭扣 洪一江[1,2] WANG Xiao-Min;ZHOU Ye;MA Hui-Mei;ZAHID Anwar;HU Bei-Juan;PENG Kou;HONG Yi-Jiang(School of Life Sciences,Nanchang University,Nanchang 330031,China;Key Laboratory of Aquatic Animal Resources and Utilization of Jiangxi,Nanchang University,Jiangxi 330031,China)

机构地区：[1]南昌大学生命科学学院,南昌330031 [2]江西省水产动物资源与利用重点实验室,南昌330031

出　　处：《水生生物学报》2023年第5期786-795,共10页Acta Hydrobiologica Sinica

基　　金：国家重点研发计划(2018YFD0901400);国家自然科学基金(31660337和31860737);现代农工技术研究体系(CARS-49)资助。

摘　　要：为研究淡水贝类NF-κB/Rel信号通路中核因子κB(Nuclear factor-κappaB,NF-κB)和NF-κB抑制因子(Inhibitors of NF-κB,IκB)的功能,对池蝶蚌(Hyriopsis schlegelii)IκBα和c-Rel(以下简称HsIκBα和Hsc-Rel)的序列结构、表达特征及Hs IκBα和Hsc-Rel之间的相互关系进行了分析。结果表明,Hs IκBα的c DNA全长为1783 bp,其开放阅读框(Open Reading Frame,ORF)为1083 bp,编码360个氨基酸。Hsc-Rel的cDNA为2414 bp,ORF为2298 bp,编码765个氨基酸。通过构建HsIκBα-ORF-GST和Hsc-Rel-RHD-HIS重组质粒、原核诱导表达和纯化,进行GST-pull down,通过SDS-PAGE和Western Blot检测研究,发现HsIκBα和Hsc-Rel-RHD存在直接的相互作用。通过对池蝶蚌进行脂多糖(Lipopolysaccharide,LPS)刺激后,分别在7个时间点(0、6h、12h、24h、36h、48h和72h)对10个组织中HsIκBα和Hsc-Rel的mRNA表达差异性进行分析,结果显示:HsIκBα和Hsc-Rel的mRNA在所有组织都有表达,且在肝胰腺组织表达最高,在血细胞表达最低。在6h时,HsIκBα在8个组织中和Hsc-Rel在7个组织中的表达水平都出现下调;在12h时,HsIκBα在10个组织中的表达量都上调,并且都达到了峰值。Hsc-Rel只有血细胞、心脏、肝胰腺和肠出现上调;在24h时,HsIκBα在10个组织中的表达值呈现下调,Hsc-Rel却都出现上调(肾组织除外),其中血细胞、斧足、肠的表达值达到峰值;在36—72h,HsIκBα和HscRel的部分组织的表达值逐渐恢复到基础水平,以上结果表明,HsIκBα和Hsc-Rel的表达量随着LPS的诱导而产生明显变化,说明其参与免疫应答反应,且可能存在应答LPS的NF-κB通路。The functions of nuclear factor-κappaB(NF-κB)and inhibitors of NF-κB(IκB)in freshwater shellfish are still unclear.In this study,we analyzed the sequence structure and expression characteristics of IκBαand c-Rel(HsIκBαand Hsc-Rel)and the correlation between HsIκBαand Hsc-Rel in Hyriopsis schlegelii.The results showed that the fulllength cDNA of HsIκBαwas 1783 bp with an 1083 bp open reading frame that encoded a total of 360 amino acids.The cDNA of Hsc-Rel gene was 2414 bp with the ORF of 2298 bp that encoded a total of 765 amino acids.The GST-pull down experiment was completed by constructing HsIκBα-ORF-GST and Hsc-Rel-RHD-HIS recombinant plasmids,prokaryotic expression and purification of two fusion proteins.The direct interaction between HsIκBαand Hsc-Rel-RHD was found.HsIκBαand Hsc-Rel mRNA level was the highest in hepatopancreas tissues and the lowest in hemocytes.At 6h after LPS stimulation,the expression of HsIκBαin 8 tissues and Hsc-Rel in 7 tissues were down-regulated;At 12h after LPS stimulation,HsIκBαwas upregulated in 10 tissues,and all reached the peak.Hsc-Rel was only upregulated in hemocytes,heart,hepatopancreas and intestine.At 24h after LPS stimulation,HsIκBαwas down-regulated in 10 tissues,but Hsc-Rel was up-regulated in all tissues except kidney,where the expression of hemocytes,foot,and intestine reached its peak.Between 36h—72h after LPS stimulation,the expression of HsIκBαand Hsc-Rel in some tissues gradually returned to basic levels,the results showed that the expression of HsIκBαand Hsc-Rel significantly changed with LPS stimulation,indicating that they were involved in the immune response,and there may be a NF-κB pathway in response to LPS.

关 键 词：HsIκBα Hsc-Rel 克隆 蛋白互作 LPS刺激 池蝶蚌 

分 类 号：Q344.1[生物学—遗传学]