巴氏杜氏藻八氢番茄红素脱氢酶基因的克隆与分析  

Cloning of Phytoene Desaturase Gene from Dunaliella bardawil and Promoter Activity Analysis

作　　者：姜建国[1] 陈善立[2] 劳永民[1] 

机构地区：[1]华南理工大学轻工与食品学院,广东广州510006 [2]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《现代食品科技》2013年第8期1756-1760,共5页Modern Food Science and Technology

基　　金：国家自然科学基金项目(31171631)

摘　　要：八氢番茄红素脱氢酶(PDS)是巴氏杜氏藻(Dunaliella bardawil)类胡萝卜素生物合成途径中的上游关键酶。本研究根据NCBI上已发布的巴氏杜氏藻mRNA序列(GenBank:Y14807),设计特异性引物,通过基因组步移法及半巢式PCR的方法,获得PDS编码区序列。在编码区序列获取过程中,发现5’端编码区有325 bp序列与NCBI上公布的cDNA序列有所不同,故采用了5’RACE技术进行验证,经过比对发现结果与本实验基因组步移所获得序列相匹配,推测NCBI公布mRNA序列的PDS与本实验获取的PDS可能为同工酶。然后根据获得的序列,再利用基因组步移PCR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其进行分析。实验获得的完整PDS基因全长12678 bp,其中编码区序列长度为8113 bp,编码区上游序列3010 bp,编码区下游序列1555 bp,编码区序列含有12个外显子和11个内含子。通过生物信息学分,发现PDS启动子中具有多种转录因子结合位点。Phytoene desaturase （PDS） is the upstream key enzyme of carotenoid metabolic process ofDunaliella bardawil. According to the mRNA that NCBI published before （GenBatlk： Y14807）, the PDS coding region was obtained through genome walking and nesting PCR. Due to that 325 bp in 5＇ coding region were not matched with the mRNA published before., 5＇ RACE was used to validate the 5＇ sequences and the final result of 5＇ RACE was matched with the 5＇ sequences isolated. The sequence of promotor and terminator were also obtained by genome walking. The full length ofPDS gene isolated included 8113 bp coding region （From ATG to TAA）, 3010 bp upstream sequence of coding region and 1555 downstream sequence. 12 Exons and 11 introns were found in the eDNA ORF and coding region. Several kinds of transcription factor binding sites were predicted in the promotor ofPDSby bioinformatic software.

关 键 词：八氢番茄红素脱氢酶 巴氏杜氏藻 基因组步移 巢式PCR 启动子 

分 类 号：TS201.25[轻工技术与工程—食品科学]