龙眼DlPPO1基因的克隆及其表达调控分析

Cloning and Expression Analyses of DlPPO1 from Dimocarpus longan Lour.

该研究根据同源克隆技术,利用RT-PCR和RACE技术,以‘四季蜜’龙眼叶片cDNA为模板,获得龙眼多酚氧化酶基因(polyphenol oxidase,PPO)的3个转录本DlPPO1-a、DlPPO1-b和DlPPO1-c的cDNA全长序列(KM387405、KM516087和KM516088)和1条DNA序列DlPPO1(KU837229)。DlPPO1-a、DlPPO1-b和DlPPO1-c的全长分别为1 969、1 960和1 920bp,包含相同的完整开放阅读框1 800bp并编码599个氨基酸;该基因与荔枝、橄榄和枣等物种的PPO基因同源性较高。生物信息学分析表明,DlPPO1保守结构域具有多酚氧化酶的典型结构域特征。利用实时荧光定量PCR技术检测DlPPO1表达结果表明,在龙眼体胚发生过程中,DlPPO1从心形胚时期开始上调表达至子叶胚时期达到最高,推测其在龙眼体胚发生中后期可能发挥重要作用;DlPPO1在龙眼叶片中表达量最高,其次是花芽,而在其他组织部位表达量较低。激素和非生物胁迫处理下的表达分析表明,水杨酸(SA)、低浓度茉莉酸甲酯(MeJA)、NaCl、甘露醇及PEG可诱导DlPPO1基因上调表达,这些表达模式暗示其可能参与多种非生物胁迫应答过程。

Based on homology cloning techniques,we isolated the polyphenol oxidase gene（3transcripts：DlPPO1-a,DlPPO1-b,and DlPPO1-c;Genbank：KM387405,KM516087,and KM516088）from leaves of longan（‘Sijimi＇cultivar）by RT-PCR and RACE.The full-length cDNA sequence of DlPPO1-a,DlPPO1-b,and DlPPO1-c were 1 969 bp,1 960 bp and 1 920 bp,respectively,containing a 1 800 bp open reading frame（ORF）which encoded 599 amino acids;DlPPO1shared high homology with PPOgene of Litchi chinensis,Canarium album,and Populus euphratica,etc.Bioinformatic analysis revealed that the deduced DlPPO1 protein with conserved domains shared the typical characteristics of the PPO family.QPCR analysis indicated that during somatic embryogenesis（SE）in longan,the expression level of DlPPO1 rose from the stage of heart embryo and then reached the highest at the stage of the cotyledon embryo,which suggested that DlPPO1 might play important roles during the middle and late stages of longan SE.It was detected that DlPPO1 abundantly accumulated in longan leaves,followed by flower buds;there was lower expression in other longan tissues.After exposure to phytohormones and abiotic stress,the expression of DlPPO1 was induced by salicylic acid（SA）,methyl jasmonte（MeJA）,NaCl,mannitol,and PEG treatments.Consequently,it suggested that DlPPO1 might participate in abiotic stress responsiveness.