SATB1与肌动蛋白基因的异源表达与体外相互作用  

Heterologous Expression and Interaction in Vitro between SATB1 and Actin Gene

作　　者：齐文靖 鄂明菊 卜庆盼 韩德复 Qi Wenjing;E Mingju;Bu Qingpan;Han Defu(College of Life Science,Changchun Normal University,Changchun,130032)

机构地区：[1]长春师范大学生命科学学院,长春130032

出　　处：《分子植物育种》2023年第17期5671-5676,共6页Molecular Plant Breeding

基　　金：吉林省教育厅项目(JJKH20200825KJ,JJKH20220833KJ);吉林省科技厅项目(YDZJ202201ZYTS661);长春师范大学自然科学项目(长师大自科合字[2019]第017号)共同资助。

摘　　要：SATB1通过调节染色质高级结构变化,影响细胞内基因的表达。前人研究发现,细胞核肌动蛋白(Actin)与SATB1共同调控细胞凋亡相关基因的表达。然而,二者之间的相互作用机制并不清楚。本研究以人T淋巴细胞瘤细胞Jurkat c DNA为模板,通过PCR扩增获得β-actin和SATB1基因编码序列,分别插入p ET32a和p GEX4T-2载体中构建原核表达质粒pET32a-his-β-actin和pGEX4T-2-GST-SATB1,并对重组质粒进行酶切和DNA测序验证。然后,将pET32a-his-β-actin和pGEX4T-2-GST-SATB1质粒转化BL21(DE3)菌中进行诱导表达和纯化。通过体外蛋白pulldown实验检测肌动蛋白与SATB1二者体外结合情况。结果表明,重组质粒pET32a-his-β-actin和pGEX4T-2-GST-SATB1构建成功。体外蛋白pulldown实验发现肌动蛋白与SATB1在体外能够结合,二者具有相互作用。本研究为深入研究肌动蛋白和SATB1在基因表达中的相互作用机制提供了科学依据。SATB1 regulates gene expression by modulating the chromatin structure.Previous study found that nuclear actin and SATB1 jointly regulate the expression of apoptosis related genes.However,the mechanism between nuclear actin and SATB1 is not clear.In this study,we used PCR to amplify the coding sequences of actin and SATB1 genes from Jurkat cell c DNA,and separately inserted into pET32a and pGEX4T-2 vectors to construct pET32a-his-β-actin and p GEX4T-2-GST-SATB1 recombinant plasmids.The recombinant plasmids were verified by enzyme digestion and DNA sequencing.Then,pET32a-his-β-actin and pGEX4T-2-GST-SATB1 plasmids were transformed into BL21(DE3) for protein induction and purification.We utilized in vitro protein pulldown assay to detect the binding of actin and SATB1.The results showed that the recombinant plasmids pET32a-his-β-actin and p GEX4T-2-GST-SATB1 were successfully constructed.In vitro protein pulldown assay showed that actin could bind to SATB1 in vitro,indicating that they could interact with each other.This study provides a theoretical basis for further studying the interaction mechanism of actin and SATB1 in gene expression.

关 键 词：SATB1 肌动蛋白 异源表达 蛋白相互作用 

分 类 号：Q78[生物学—分子生物学]