【摘要】 从毛白杨(Populus tomentosa)木质化茎中提取总RNA,利用RT-PCR技术进行cDNA扩增,获得1条1 621 bp的片段。测序结果表明:该片段编码536个氨基酸组成的多肽,序列中包含所有已知4-CL蛋白的2个特征序列boxⅠ(SSGTTGLPKGV)和boxⅡ(GEICIRG)。将cDNA克隆到原核表达载体pET-32a(+)中,并转化大肠杆菌(Escherichia coli)感受态细胞BL21(DE3)。IPTG诱导后,经SDS-PAGE和Western-blot分析表明,4-CL基因在大肠杆菌BL21中获得表达,所表达融合蛋白的分子量约为70 ku。酶活性分析表明:该重组蛋白对底物PA、FA、CA均表现出催化活性,其偏爱性顺序为PA>FA>CA,对SA则没有活性。更多还原

【Abstract】 Total RNA was extracted from the ligninified stem tissue of Populus tomentosa and a（1 621） bp fragment of cDNA was amplified by RT-PCR.The sequencing results showed that the peptide coded by the cDNA contained 536 amino acids,including two characteristic sequences boxⅠ（SSGTTGLPKGV）and boxⅡ（GEICIRG）of all known 4-CL proteins.The cDNA of 4-CL gene was cloned into protokaryotic expression vector pET-32a（+）and transformed into competent BL₂₁(DE₃) cells.The recombinant protein was analyzed with SDS-PAGE and Western blot after IPTG induction.The result showed that 4-CL gene was expressed in E.coli BL₂₁ and the molecular weight of the recombinant protein was estimated to be 70 ku.The enzyme activity analyses showed that the recombinant protein could catalyze substrates PA（4-coumaramic acid）,FA（ferulic acid） and CA（caffeic acid）,with the preference order being PA> FA> CA,but showed no reactivity on SA（sinapic acid）.The study provides evidence affords proof for the regulation of 4-CL activities and biosynthesis of the downstream products in plants via gene engineering.更多还原