鸡BRD2基因及其剪接体的克隆测序与亚细胞定位分析

Cloning,sequence analysis and subcellular localization of chicken BRD2 gene and its splices

【目的】克隆鸡含溴结构域蛋白2(BRD2)基因及其剪接体,并对其进行序列分析和亚细胞定位,为深入研究鸡BRD2基因及其剪接体在细胞转录调控中的作用机理打下基础。【方法】通过RT-PCR扩增鸡BRD2基因及其剪接体的编码区(CDS)序列,采用BioEdit、ProtParam、SOPMA、SWISS-MODEL、CDART等在线软件进行生物信息学分析;鸡BRD2基因及其剪接体经Xho I和Bam H I双酶切后,分别亚克隆至真核表达载体pEGFP-C1多克隆位点上构建重组真核表达载体,通过转染HEK-293T细胞进行亚细胞定位分析。【结果】鸡BRD2基因CDS序列全长2340 bp,编码779个氨基酸残基,分子量为85.66 k D,理论等电点(pI)为8.74,其编码蛋白二级结构主要由无规则卷曲(60.59%)和α-螺旋(28.50%)组成,含有3个明显的功能结构域(2个BD结构域和1个ET结构域)。与鸡BRD2基因相比,其剪接体BRD2-X1、BRD2-X2和BRD2-X3的CDS序列长分别为2301、2283和1983 bp,编码766、760和660个氨基酸残基。以鸡BRD2氨基酸序列为参照,BRD2-X1表现为第526~537位氨基酸缺失,BRD2-X2表现为第1~19位氨基酸缺失,BRD2-X3表现为第1~119位氨基酸缺失;与鸡BRD2蛋白相比,鸡BRD2基因剪接体BRD2-X1和BRD2-X2并未影响鸡BRD2蛋白的功能结构域,而剪接体BRD2-X3导致鸡BRD2蛋白第一个BD结构域部分缺失。鸡BRD2基因融合蛋白EGFP-BRD2及其剪接体融合蛋白EGFP-BRD2-X1、EGFP-BRD2-X2和EGFP-BRD2-X3均定位在细胞核,EGFP-BRD2和EGFPBRD2-X1在细胞核中呈均匀分布,而EGFP-BRD2-X2和EGFP-BRD2-X3在细胞核中呈不均匀的点状分布。【结论】鸡BRD2蛋白及其剪接体均定位在细胞核,但BD结构域缺失可引起BRD2剪接体BRD2-X3细胞核定位模式的变化。

【Objective】This study aimed to clone chicken bromodomain-containing protein 2(BRD2)gene and its splices,and analyze their sequences and subcellular localization,which laid a foundation for further study of mechanism of chicken BRD2 gene and its splice in cellular transcriptional regulation.【Method】The coding region(CDS)region sequence of chicken BRD2 gene and its splice were amplified by RT-PCR and then analyzed by online softwares including BioEdit,ProtParam,SOPMA,SWISS-MODEL,CDART,etc.The PCR products of chicken BRD2 gene and its splices were digested by Xho I and BamH I,and subcloned into eukaryotic expression vector pEGFP-C1 multiple cloning site to construct recombinant eukaryotic expression vectors.The subcellular localization of chicken BRD2 gene and its splices were analyzed by transfecting the constructed plasmids into HEK-293T cells.【Result】The results revealed that the fulllength CDS region sequence of chicken BRD2 gene was 2340 bp and encoded 779 amino acids with a molecular weight of 85.66 kDa,and the theoretical pI was 8.74,The secondary structure of chicken BRD2 protein was mainly composed of random coil(60.59%)andα-helix(28.50%),and contained three obvious functional domains(2 BD domains and 1 ET domain).Compared with chicken BRD2 gene,the splices BRD2-X1,BRD2-X2 and BRD2-X3 were 2301,2283 and 1983 bp in length,and encoded 766,760 and 660 amino acid residues,respectively.Taking the amino acid sequence of chicken BRD2 as reference,BRD2-X1,BRD2-X2 and BRD2-X3 showed amino acid deletion at positions 526-537,1-19 and 1-119,respectively.In addition,BRD2-X1 and BRD2-X2 did not affect the functional domain of chicken BRD2 protein,while the splice BRD2-X3 caused partial deletion of the first BD domain of chicken BRD2 protein.Moreover,the fusion protein EGFP-BRD2,and its splices fusion proteins EGFP-BRD2-X1,EGFP-BRD2-X2 and EGFP-BRD2-X3 were mainly located in the nucleus.However,the fusion proteins EGFP-BRD2 and EGFP-BRD2-X1 showed even distribution in the nucleus,but EGFP-BRD2-X2 and EGFP-BRD2-X3 exhibited unevenly spotty pattern distribution in the nucleus.【Conclusion】The chicken BRD2 protein and its different splices locate in the nucleus,but the deletion of BD domain lead to the nuclear localization pattern changes of BRD2 splice BRD2-X3.