龙眼褪黑素合成途径SNAT、ASMT和COMT家族基因鉴定及表达分析

Whole-genome Identification and Expression Analysis of SNAT,ASMT and COMT Families of Melatonin Synthesis Pathway in Dimocarpus longan

基于第3代龙眼(Dimocarpus longan)基因组及转录组数据库,对褪黑素合成路径上可能的限速基因SNAT(serotonin N-acetyltransferase)、ASMT(N-acetylserotonin methyltransferase)和COMT(caffeic acid O-methyltransferase)进行基因家族成员鉴定,利用实时荧光定量PCR技术研究其在龙眼体胚早期的表达谱,并研究外源IAA、GA3、MeJA、SA和ABA对龙眼胚性愈伤组织中褪黑素含量的影响,以及褪黑素合成途径相关基因的表达模式。结果显示:龙眼SNAT(DlSNAT)、ASMT(DlASMT)和COMT(DlCOMT)基因家族分别含有2、18和7个成员,蛋白质结构域保守,相同家族的成员之间保守基序相差极小,DlASMT和DlCOMT家族成员高度相似。基于氨基酸序列进行系统进化树分析,龙眼、拟南芥、水稻、小麦、番茄、辣椒和卷柏的SNAT家族可分为3个亚组;ASMT和COMT家族具有高度同源性,共同建树并分为3个亚组。DlSNAT、DlASMT和DlCOMT家族成员的启动子中含有大量响应生长素、赤霉素、茉莉酸甲酯、水杨酸和脱落酸的顺式作用元件。表达谱分析显示,DlASMT和DlCOMT家族成员主要在龙眼不完全胚性紧实结构和球形胚阶段高表达,DlSNAT1和DlSNAT2在龙眼体胚发生早期始终处于高水平表达。用0.1 mmol·L^(-1)IAA处理龙眼胚性愈伤组织,内源褪黑素含量在24 h内显著降低,GA;、MeJA、SA和ABA处理则显著提高了褪黑素含量;同时分析DlSNAT、DlASMT、DlCOMT成员表达情况,内源褪黑素含量变化趋势和DlSNAT表达基本一致。综上,DlSNAT、DlASMT和DlCOMT可能通过影响内源褪黑素的合成参与调控龙眼早期体胚发生。

In this study,on the basis of the third-generation longan(Dimocarpus longan Lour.)genome and transcriptome database,the possible rate-limiting genes of melatonin synthesis:SNAT(serotonin N-acetyltransferase),ASMT(N-acetylserotonin methyltransferase)and COMT(caffeic acid O-methyltransferase)family genes were identified and analyzed. The effect of different exogenous phytohormones on the melatonin content was detected,and,the expression patterns of SNAT,ASMT,and COMT genes in respond to exogenous phytohormone treaments and during early somatic embryogenesis(SE)in longan were determined by quantitative real time polymerase chain reaction(qRT-PCR). The results showed that the longan SNAT(DlSNAT),ASMT(DlASMT),and COMT(DlCOMT)family genes have two,18,and seven members,respectively;the protein domains and motifs in each same gene family are conserved,and DlASMTs and DlCOMTs are highly similar;phylogenetic analysis showed that the SNAT family of Dimocarpus longan,Arabidopsis thaliana,Oryza sativa,Triticum aestivum,Solanum lycopersicum,Capsicum annuum,and Selaginella tamariscina could be divided into three subgroups,while the ASMT and COMT families were highly homology and were divided into three subgroups;the cis-acting elements analysis revealed that a large number of auxin-,gibberellin-,methyl jasmonate-,salicylic acid-,and abscisic acid-related elements existed in the promoters of DlSNAT,DlASMT and DlCOMT family members;qRT-PCR analysis showed that most of DlASMTs and DlCOMTs were highly expressed in the incomplete embryonic compact structure and globular embryo stages of somatic embryo(SE),while DlSNAT1 and DlSNAT2 were highly expressed during early SE in longan;the content of endogenous melatonin was significantly reduced under exogenous 0.1 mmol·L^(-1)of indole-3-acetic acid(IAA)treatment,whereas significantly increased under exogenous gibberellin A3(GA;),methyl jasmonate(MeJA),salicylicacid(SA)and abscisic acid(ABA)treatments;at the same time,the expression changes of DlSNATs consist with the change trends of endogenous melatonin content. The results indicated that DlSNATs,DlASMTs and DlCOMTs may involved in the synthesis of endogenous melatonin thereby regulate the process of early SE in longan.