赤桉木质素合成途径OMT基因家族的原核表达与纯化研究

Prokaryotic expression and purifi cation of OMT family genes involved in lignin niosynthesis of Eucalyptus camaldulensis

双子叶植物的木质素主要由愈创木基单体和紫丁香基木质素单体聚合形成,这两种单体的比例对造纸过程中木质素去除难易程度具有重要的影响。植物OMT基因家族的CCoAOMT和COMT基因是木质素合成途径的关键酶基因,可调控植物体内木质素单体的比例与含量。本文以所克隆的赤桉COMT、CCoAOMT1、CCoAOMT2三个基因的cDNA为模板,分别构建pET-32a的原核表达质粒,并优化在大肠杆菌BL21(λDE3)中的表达体系。结果显示,在加入0.4 mmol IPTG诱导表达后,COMT、CCoAOMT1、CCoAOMT2的原核表达质粒分别在4 h、2 h和3 h后达到可溶性重组蛋白表达峰值。通过渗透休克纯化方法,可在原核表达菌株的周质空间提纯CCoAOMT1和CCoAOMT2重组蛋白,在原核表达菌株的细胞质中可提纯COMT重组蛋白。

In dicotyledons,the lignin polymer is constructed by the guaiacyl and syringyl units,and the ratio of this two units decide the wood characteristics and the pulping performance. The CCoAOMT and COMT gene of OMT gene family plays a key role in the lignin biosynthesis,with a function to determine the contents of lignin and the ratio of monolignol. In this study,three target cDNAs（COMT,CCoAOMT1,CCoAOMT2） of Eucalyptus camaldulensis were inserted into the prokaryotic expression vector pET-32a,and constructed plasmids were transformed into E. coli BL21（λDE3）. The expression condition was optimized. The analysis result showed that the optimal induction time after 0.4 mmol ITPG been added for COMT,CCoAOMT1 and CCoAOMT2 expression is 4 hours,2 hours and 3 hours,respectively. The CCoAOMT1 and CCoAOMT2 recombinant protein can be purifi ed by the osmotic shock-mediated method,and soluble COMT recombinant protein can be purifi ed from the cytoplasm of E. coli..