摘要
Hsp70基因的表达量在研究中常作为一个重要的生理指标来反映机体的状况。为了研究象耳豆根结线虫Hsp70基因相关功能,本研究前期成功构建了p EASY-E1-Me Hsp70和p ET30a-Me Hsp70两个原核表达载体,通过热激转化将其分别转入大肠杆菌(Escherichia coli)BL21进行表达。对转入Me Hsp70基因的大场杆菌BL21、转入空载的大肠杆菌BL21和大肠杆菌BL21原始菌种在55℃、65℃进行热稳定性测定,在37℃、200 r/min条件下进行生长曲线测定。热稳定性试验结果表明,转入p ET30a-Me Hsp70基因的大场杆菌生存时间较长,而转入p EASY-E1-Me Hsp70的菌株、转入空载的菌株和原始菌株热稳定性都相对较差。通过荧光定量PCR测定发现Me Hsp70基因均有上调表达,转入p ET30a-Me Hsp70基因的大场杆菌Hsp70基因水平高于其他菌株。生长曲线测定试验结果表明,重组菌株与原始菌株生长曲线基本一致,转入p ET30a-Me Hsp70载体的菌株调整期滞留时间长于其他菌株。该项研究表明Me Hsp70基因能够提升大肠杆菌BL21的抗应激能力和影响大肠杆菌BL21的生长,为进一步开展Me Hsp70基因在象耳豆根结线虫生长过程的功能研究提供了一定的理论基础。
During the research, the expression level of lisp 70 gene, as an important physiological signs, often reflects the condition of the body. The Hsp70 gene will up-regulate to maintain the conformation of the key cellul- ar proteins while the body encountering high temperature, hypoxia, cold or other harsh environment, in order to keep the cellular activities and reduce the damage to the body. In the early, two prokaryotic expression vectors （pEASY-E1-MeHsp 70 and pET3Oa-MeHsp 70） that were transformed into Escherichia coli BL21, respectively, by the heat shock transformation way have been constructed successfully for the research. Keeping the original Escherichia coli BL21 and the transferred Escherichia coli BL21 into which the pEASY-E1-MeHsp70 recombina- nt plasmid, the pET3Oa-MeHsp 70 recombinant plasmid and the no-load pEASY-E1 were transferred respectively in 55~C or 65~C for thermal stability of determination. Growth curves were determined at 37~C, 200 r/min and 30~C and 200 r/min. The results showed that the transferred Escherichia coli BL21 of pET3Oa-MeHsp70 gene survive longer and have a better thermal stability than others. By real-time fluorescence quantitative PCR test, MeHsp70 gene had a certain amount of up-regulated expression, which indicated that the increasing heat stability of Escherichia coli BL21 directly related to the up-regulation of MeHsp 70 gene.The growth curve test showed that the recombinant strain almost corrsponded with the original strain＇s, and the strain in the pET3Oa-MeHsp 70 vector was longer than the others. The research showed that the MeHsp70 gene could increase the thermal stability of Escherichia coli BL21 and affect the growth of Escherichia coli BL21. It suggested that Hsp70 gene play an important role for growth of Meloidog＇yne enterolobii.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2016年第10期2653-2661,共9页
Genomics and Applied Biology
基金
国家自然科学基金项目(31360432,31360424,31160024)
公益性行业(农业)科研专项(201103018)共同资助
关键词
象耳豆根结线虫
热休克蛋白70基因
大肠杆菌
抗逆性
生长曲线
Meloidogyne enterolobii, Heat shock protein 70 gene （Hsp 70）, Thermal stability, Escherichia coli BL-2 I, Growth curve
