摘要
为了获得一定纯度的Nt LBM1蛋白,本研究通过RT-PCR从野生型烟草叶片中扩增得到NtLBM1cDNA序列。生物信息学分析表明,NtLBM1全长1 459 bp,开放阅读框为846 bp,编码281个氨基酸,含1个SANT保守结构域;构建pET30a-NtLBM1原核表达载体,并转化到E. coli BL21 (DE3)中,NtLBM1蛋白以包涵体形式存在,包涵体蛋白经8 mol/L尿素溶解后,采用Q Sepharose FF离子交换层析和Ni-IDA亲和层析洗脱纯化,4℃透析后置于2 mol/L尿素缓冲液中复性,最终获得浓度为0.390 mg/mL、纯度90%以上的Nt LBM1蛋白。目的蛋白的成功表达为进一步研究NtLBM1蛋白的互作及其对植物生长发育的调控机制提供了理论参考和数据支持。
In order to obtain a purified NtLBM1 protein, the cDNA sequence of NtLBM1 was amplified by RTPCR from wild-type tobacco leaves. According to the bioinformatics analysis, the length of NtLBM1 was 1 459 bp,and the open reading frame was 846 bp, encoding 281 amino acids with a SANT conserved domain. Then, the pET30 a-NtLBM1 prokaryotic expression vector was constructed and transformed into E. coli BL21(DE3). NtLBM1 protein existed in the form of inclusion bodies was dissolved in 8 mol/L urea and purified by Q Sepharose FF ion exchange chromatography and Ni-IDA affinity chromatography, ultimately refolding in 2 mol/L urea buffer after dialysis at 4 ℃. Nt LBM1 protein with a concentration of 0.390 mg/mL and a purity of more than 90% was obtained. The successful expression of the target protein provided theoretical reference and data support for further research on the interaction of NtLBM1 protein and its regulation mechanism on plant growth and development.
作者
黎忠杰
陶怡
黄璐
杨洁
吴拥军
Li Zhongjie;Tao Yi Huang;Lu Yang Jie;Wu Yongjun(Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Collaborative Innovation Center for Mountain Ecology&Agro-Bioengineering(CICMEAB),College of Life Sciences/Institute of Agro-bioengineering,Guizhou University,Guiyang,550025)
出处
《分子植物育种》
CAS
北大核心
2022年第2期414-421,共8页
Molecular Plant Breeding
基金
国家自然科学基金项目(310717551/C1408)资助。
关键词
NtLBM1
原核表达
蛋白纯化
NtLBM1
Prokaryotic expression
Protein purification
