巴氏杜氏藻ζ-胡萝卜素脱氢酶基因的克隆与分析

Clone and Analysis ζ- Carotene Desaturase of Dunaliella bardawil

摘要:

ζ-胡萝卜素脱氢酶（ZDS）基因是胡萝卜素生物合成途径中的一个关键酶之一。根据实验室已获得的巴氏杜氏藻ZDS基因的cDNA序列，设计引物，通过分段PCR的方法，获得ZDS基因的编码区序列。然后根据获得编码区序列，利用染色体步移的方法获得其两端侧翼序列：启动子和终止子，并利用生物信息学工具对获得序列进行分析。实验获得的完整ZDS基因全长11896 bp，其中编码区序列（从”ATG”到”TAA”）长度为6435，编码区上游序列4091 bp (包括33 bp 的5’UTR序列)，编码区下游序列1370 bp (包括411 bp的3’UTR序列)。将编码序列与cDNA（开放阅读框）进行比对，发现整个编码区序列含有12个外显子和11个内含子，其中内含子长度达4686 bp，约为外显子长度的2.68倍，内含子均以GT开始，AG收尾，属于最常见的内含子类型。通过生物信息学分，发现ZDS启动子中具有多种转录因子结合位点，包括与光调控有关的GAGA box，ASF1；与植物黄化反应有光的ACGT box等。

Abstract:

ζ-Carotene desaturase (ZDS) is one of the key enzymes in the carotenoid biosynthetic pathway. According to the full-length sequence of the ZDS cDNA in D. bardawil that our laboratory has obtained before, the coding region sequence of ZDS were isolated by the approach of PCR and then flanking sequence of the coding region were amplified by the method of genome walking, including promoter and terminator of ZDS, followed by analysis of these sequences by bioinformatics tools. The whole sequence of ZDS was11896 bp. It contained 6435 bp coding region (from ATG to TAA), 4091 bp upstream of the coding region, and 1370 downstream of the coding region. Blastn the coding region and ORF of ZDS showed that there were 12 exons and 11 introns in coding region. The total length of the introns was 4086 bp, about 2.68 fold of the total length of exons (1749bp). Each intron started with GT and end with AG, belonging to the most common style of exon. By analyzing the promoter of ZDS gene, we found that there are many transcription factor binding sites, including “GAGA box”, ASF1, related with plant light regulation; “ACGT box”, required for etiolation-induced expression of early responsive to dehydration.