摘要
利用RT-PCR扩增获得拟南芥At RALF1基因的全长cDNA序列,将其构建入携带His标签的原核表达载体p ET28b中,获得重组表达载体p ET28b-At RALF1,并将其转入到大肠杆菌BL21(DE3)中。随后在不同的IPTG浓度、温度和诱导时间条件下,进行At RALF1蛋白的表达研究,建立了At RALF1融合蛋白的高效表达体系。结果表明,在30℃、1 mmol/L IPTG的条件下诱导4 h,At RALF1融合蛋白的表达量最大。进一步用获得的At RALF1融合蛋白处理苗龄5 d的野生型拟南芥(Col-0)植株,发现其根的生长受到了抑制,表明我们获得了具有活性的At RALF1小肽,为进一步研究该小肽奠定了基础。
The full-length cDNA sequence of AtRALF1 was amplified from Arabidopsis thaliana by RT-＇ PCR. The verified sequence of AtRALF1 was inserted into the corresponding region of pET28b vector to construct the recombinant plasmid pET28b-AtRALF1. The plasmid pET28b-AtRALF1 was then introduced into the bacterial host E.coli BL21 （DE3） to analyze the expression of AtRALF1. Through the analysis of dif- ferent concentration of IPTG, temperature and time course, the expression conditions were optimized, and 30 ℃ for 4 h with I mmol/L IPTG induction was found to be the optimal condition for prokaryotie expres- sion. The AtRALF1 protein was then purified for further research. When the purified AtRALF1 protein was added to 5-day-old seedlings （Col-0）, the growth of the seedling root was obviously inhibited in Arabidop- sis thaliana. It is suggested that the recombinant AtRALF1 protein has the inhibition activity of signaling peptides, which can be used for further study.
出处
《生命科学研究》
CAS
CSCD
2016年第4期320-324,共5页
Life Science Research
基金
湖南大学中央高校专项基金(531107040814)
关键词
拟南芥
信号肽
快速碱化因子(RALF)
原核表达
蛋白质纯化
活性检测
A rabidopsis
signaling peptides
rapid alkalinization factor （RALF）
prokaryotic expression
protein purification
activity identification
