CAJ | 학술논문

该实验拟构建甘蔗14-3-3基因的真核表达载体，为体外研究该蛋白功能和高级构象特征奠定基础。用RT-PCR技术从甘蔗茎中扩增出编码14-3-3蛋白的全长基因，并连入pGEM-T载体中，双酶切和测序用于序列正确性的鉴定。结果表明，该基因的最大开放阅读框为771bp，编码256个氨基酸。后将所得cDNA片段亚克隆至真核表达载体pPIC9K，经电转后在酵母细胞中表达，表达产物经Western blot鉴定分子量约为28kD。表明已成功构建了甘蔗14-3-3蛋白真核表达载体，并获得了表达产物。
해실험의구건감자14-3-3기인적진핵표체재체，위체외연구해단백공능화고급구상특정전정기출。용RT-PCR기술종감자경중확증출편마14-3-3단백적전장기인，병련입pGEM-T재체중，쌍매절화측서용우서렬정학성적감정。결과표명，해기인적최대개방열독광위771bp，편마256개안기산。후장소득cDNA편단아극륭지진핵표체재체pPIC9K，경전전후재효모세포중표체，표체산물경Western blot감정분자량약위28kD。표명이성공구건료감자14-3-3단백진핵표체재체，병획득료표체산물。
To lay the foundation for in vitro researching the function and advanced conformation of sugarcane 14-3-3 protein,the eukaryotic expression vector was constructed. RT-PCR was performed for cloning the full-length 14-3-3 gene from sugarcane stalk. The gene was inserted into the pGEM-T vector. The double enzyme diges-tion and sequencing were used to identify the sequence correct. The results showed that the 14-3-3 gene con-tains an open reading frame of 771 bp and encodes 256 amino acids. And then,the cDNA fragment was cloned into eukaryotic expression vector pPIC9K. A specific fragment was showed after double enzyme digestion. The mo-lecular weight is about 28 kD by analysis of protein purification and Western blot. It indicated that the eukaryot-ic expression vector of 14-3-3 protein was successfully constructed and the expression product was obtained by this system.