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目的:对刺五加鲨烯环氧酶基因的cDNA进行克隆及序列分析。方法:采用改良的异硫氰酸胍法提取刺五加总RNA,逆转录为cDNA,根据已报道的人参SE基因cDNA序列设计引物,利用RT-PCR法克隆刺五加SE基因的cDNA序列。结果:克隆了2个序列不同的cDNA(SE1和SE2),开放阅读框分别长1 665,1 629 bp,分别编码554,542个氨基酸。SE1,SE2间的核苷酸和氨基酸一致性分别为91.49%,92.55%,两者与三七SE1的氨基酸序列相似性最高,分别为93.45%,94.87%。SE1,SE2均含有1个FAD结合区域,SE1,SE2推测的氨基酸分别存在2个和4个跨膜螺旋。结论:首次分离并报道了刺五加的2个SE基因cDNA序列,为刺五加的次生代谢工程研究奠定了基础。
Objective: To clone and sequence the cDNA of squillose epoxidase from Acanthopanax senticosus. Methods: The total RNA of Acanthopanax senticosus was extracted by modified guanidium isothiocyanate method and reverse transcribed into cDNA. According to the published sequence of SE gene, primers were designed and the cDNA sequence of Acanthopanax senticosus SE was cloned by RT-PCR. Results: Two cDNAs (SE1 and SE2) with different sequences were cloned. The open reading frames were 1 665 and 1 629 bp, encoding 554 and 542 amino acids, respectively. The nucleotide and amino acid identities between SE1 and SE2 were 91.49% and 92.55%, respectively. The two strains shared the highest similarity with the amino acid sequence of SE1 (93.45% and 94.87%, respectively). SE1, SE2 contain a FAD binding region, SE1, SE2 presumed amino acids exist in two and four transmembrane spiral. Conclusion: The cDNA sequence of two SE genes of Acanthopanax senticosus was isolated and reported for the first time, which laid the foundation for the secondary metabolic engineering of Acanthopanax.