三角褐指藻岩藻黄素合成关键酶PtHMGR基因的克隆与诱导表达调控研究  

Cloning and Induced Expression of the Key Enzyme HMGR Gene Involved in Fucoxanthin Synthesis in Phaeodactylum tricornutum

作　　者：王博 龚一富[1] 叶奔 李荣辉 舒明雨 贾泽茗 王何瑜[2] WANG Bo;GONG Yi-fu;YE Ben;LI Rong-hui;SHU Ming-yu;JIA Ze-ming;WANG He-yu(Key Laboratory of Marine Biotechnology of Zhejiang Province,School of Marine Sciences,Ningbo University,Ningbo 315832,China;College of Food and Pharmaceutical Sciences,Ningbo University,Ningbo 315832,China)

机构地区：[1]宁波大学海洋学院,浙江省海洋生物工程重点实验室,浙江宁波315832 [2]宁波大学食品与药学学院,浙江宁波315832

出　　处：《中国药学杂志》2023年第3期222-230,共9页Chinese Pharmaceutical Journal

基　　金：宁波市社发重大项目资助(2017C510002)。

摘　　要：目的研究茉莉酸甲酯(methyl jasmonate,MeJA)、硫酸铈铵(ammonium cerous sulfate,ACS)、花生四烯酸(arachidonic acid,AA)、光和促进剂(photosynthetic induction factor,PIF)、光合抑制剂[3-(3,4-dichlorophenyl)-1,1-dimethylurea,DCMU]和不同光质对三角褐指藻3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutarate monoacyl-CoA reductase,HMGR)基因PtHMGR表达的影响,研究不同外源因素处理下PtHMGR基因表达与岩藻黄素含量之间的关系。方法通过转录组测序获得PtHMGR基因cDNA序列,并对其进行生物信息学分析,有机溶剂提取法测定MeJA、ACS、AA、PIF、DCMU和不同光质处理下三角褐指藻岩藻黄素含量,荧光定量多聚酶链式反应(polymerase chain reaction,PCR)检测PtHMGR基因表达。结果PtHMGR基因全长2161 bp,其ORF长1914 bp,编码637个氨基酸。PtHMGR蛋白分别含有2个烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADP)和3-羟基-3-甲基戊二酸单酰辅酶A(3-hydroxy-3-methyl glutaryl coenzyme A reductase,HMG-CoA)结合基序,催化结构域包含L-domain、S-domain和N-domain,N端存在3个跨膜螺旋区。系统进化树分析结果表明,动物和植物HMGR明显分为2支,三角褐指藻与假微型海莲藻(Thalassiosira pseudonana)HMGR蛋白的亲缘关系最近,同处于水生植物一支。荧光定量PCR分析结果表明,12.5 mg·L^(-1)AA、0.4 mg·L^(-1)ACS、0.05μg·L^(-1)PIF、0.4 mg·L^(-1)DCMU和紫光处理下PtHMGR基因表达水平最高。相关性分析结果表明,ACS和不同光质处理下岩藻黄素含量与PtHMGR基因表达呈正相关。结论PtHMGR基因是参与ACS和不同光质调控三角褐指藻岩藻黄素合成的关键基因之一,PtHMGR与三角褐指藻岩藻黄素的合成与积累具有密切关系。OBJECTIVE To study the effects of methyl jasmonate(MeJA),arachidonic acid(AA),ammonium cerous sulfate(ACS),photosynthetic induction factor(PIF),photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU)and different light quality on expression of 3-hydroxy-3-methylglutarate monoacyl-coA reductase(HMGR)gene in Phaeodactylum tricornutum and study the relationship between the expression of PtHMGR gene and fucoxanthin content in P.tricornutum treated by different exogenous factors.METHODS The cDNA of PtHMGR gene in P.tricornutum was obtained by transcriptome sequencing,and bioinformatics analysis was conducted.An organic solvent extraction method was used to determine fucoxanthin content in P.tricornutum and PtHMGR gene expression was detected by real-time fluorescence quantitative PCR.RESULTS A 2161 bp PtHMGR gene was cloned,containing a complete open reading frame of 1914 bp,encoding 637 amino acids.P.tricornutum PtHMGR protein contains two NADP and HMG-COA binding motifs respectively.It has a typical structure with a catalytic regionincluding L,N and S domains,and there are three transmembrane helical regions at the N-terminal.The phylogenetic tree analysis showed that PtHMGR proteins are divided into two branches,animal HMGRs group and plant HMGRs group.The P.tricornutum and Thalassiosira pseudonana HMGR are closely related,and both belong to the branch of aquatic plants.The analysis of PtHMGR gene transcription results showed that 12.5 mg·L^(-1)AA,0.4 mg·L^(-1)ACS,0.05μg·L^(-1)PIF,0.4 mg·L^(-1)DCMU,and purple light promoted the expression of P.tricornutum PtHMGR gene.Correlation analysis results showed that fucoxanthin content was positively correlated with PtHMGR gene expression under ACS and different light quality treatments.CONCLUSION PtHMGR gene is one of the key genes involved in the regulation of fucoxanthin synthesis in P.tricornutum under ACS and different light quality treatment,and HMGR is closely related to the synthesis and accumulation of fucoxanthin.

关 键 词：三角褐指藻 岩藻黄素 3-羟基-3-甲基戊二酸单酰辅酶A还原酶 克隆 诱导表达 

分 类 号：Q943[生物学—植物学]