【摘要】 采用PCR和RT-PCR技术,以‘E1’甘蓝基因组DNA和柱头cDNA为模板对THL1基因进行扩增克隆,得到的片段长度分别为732bp和455bp。序列分析表明,克隆的DNA和cDNA序列与甘蓝‘西园四号’THL1的DNA和cDNA同源性分别为97.9%和98.3%,两条序列内含子的大小不同;同时,前者第2内含子不符合典型的GT-AG规则:即第2个内含子3′端碱基为AT。将THL1基因cDNA序列定向克隆到原核表达载体pET-43.1a(+),构建融合表达质粒pET43.1a(+)-THL1,在大肠杆菌BL21中表达出分子量为74kD的融合蛋白,经胰岛素检测,THL1有氧化还原活性,表明THL1在大肠杆菌中得到了正确表达。更多还原

【Abstract】 The DNA and cDNA fragments of the THL1 were amplified from genomic DNA and stigma cDNA of Brassica oleracea’E1’ by PCR and RT-PCR methods. Their lengths were 732 bp and 455 bp respectively. Sequence analysis indicated that the identities of DNA and cDNA with those cloned from Brassica oleracea ’Xiyuan 4’ previously were 97.9% and 98.3% respectively, the introns of the two sequences were different in size. Moreover, the second intron of THL1 from Brassica oleracea’E1’ was not comply with the typical GT-AG rule: AT existed in the 3′ end of the second intron. The cDNA of THL1 was cloned into vector pET-43.1a(+) to be pET43.1a(+)-THL1, transferred into E.coli BL21 and expressed as a 74 kD fusion protein when induced with IPTG. Thioredoxin activity was measured by ability of the THL1 to reduce insulin. The result showed the THL1 gene had been correctly expressed.更多还原