National Natural Science Foundation of China (32060364,32060366), Yunnan Major Science and Technology Project (202102AE090052) and Yunnan Garden plant genetic improvement and efficient Breeding doctoral supervisor team
摘要：通过对新几内亚凤仙(Impatiens hawkeri)指甲花醚合成相关基因CCoAOMT和SAMS的克隆及表达分析，探讨其对新几内亚凤仙指甲花醚合成影响，为解析凤仙花指甲花醚合成途径提供一定的理论依据。本研究基于新几内亚凤仙指甲花醚含量测定，通过转录组测序筛选出目的基因，克隆到1个CCoAOMT和2个SAMS基因，将其命名为IhCCoAOMT、IhSAMS1和IhSAMS2，并对其表达模式进行分析。结果表明：3种处理均能促进指甲花醚积累，且暗培养处理最佳。IhCCoAOMT的cDNA全长为729 bp，编码242 aa，其蛋白具有AdoMet_MTases超家族结构域；IhSAMS1和IhSAMS2的cDNA全长分别为1179 bp和1173 bp，分别编码393 aa和391 aa，其蛋白均具有S-AdoMet_synt超家族结构域。系统进化分析表明，IhCCoAOMT与喜马拉雅凤仙亲缘关系最近；IhSAMS1与IhSAMS2可能为旁系同源。qRT-PCR分析表明，3个基因在新几内亚凤仙的3个处理及4个时期中均有表达，其中IhCCoAOMT在暗培养及前体物 (1,4-dihydroxy-2-naphthoate，DHNA)诱导中表达量均逐渐上调，且暗培养第60 d时表达量最高；IhSAMS1和IhSAMS2在暗培养及DHNA诱导中表达量均逐渐下调，但IhSAMS1在DHNA诱导中表达量较高，IhSAMS2在暗培养中表达量较高且总体表达量较高。综上所述，推测IhCCoAOMT在指甲花醚处理后期起主要作用；而IhSAMS1和IhSAMS2在其早期起主要作用，且IhSAMS2作用更为显著。
Abstract: On the basis of cloning and expression analysis of CCoAOMT and SAMS genes related to 2-methoxy-1,4-naphthoquinone (MNQ) synthesis of Impatiens hawkeri, the molecular mechanism of its MNQ synthesis was discussed, which provided theoretical basis for MNQ synthesis of Impatiens. In this study, based on the determination of MNQ content in I. hawkeri, the target genes were screened by transcriptome sequencing, and 1 CCoAOMT and 2 SAMS genes of I. hawkeri were cloned, named IhCCoAOMT, IhSAMS1 and IhSAMS2 respectively, whose expression patterns were analyzed. The results showed that all three treatments could promote the accumulation of MNQ, and dark culture had the best effect. The cDNA of IhCCoAOMT was 729 bp and encoded 242 aa, its protein has AdoMet_MTases superfamily domain, and the cDNAs of IhSAMS1 and IhSAMS2 were 1179 bp and 1173 bp, encoded 393 aa and 391 aa, respectively, and their proteins have S-AdoMet_synt superfamily domain. Phylogenetic analysis showed that IhCCoAOMT gene of I. hawkeri was most closely related to I. glandulifera. IhSAMS1 and IhSAMS2 of I. hawkeri may be paralogous genes. The qRT-PCR analysis showed that the three genes were expressed in three treatments and four culture periods of I. hawkeri, in which the expression of IhCCoAOMT gradually increased in dark culture and 1,4-dihydroxy-2-naphthoate (DHNA) treatment, and the expression was highest on the 60th day of dark culture. The expression of IhSAMS1 and IhSAMS2 were gradually downregulated in dark culture and DHNA treatment, but the expression level of IhSAMS1 was higher in DHNA treatment, and the expression level of IhSAMS2 was higher in dark culture and the overall expression level was higher. In conclusion, it’s speculated that IhCCoAOMT played a key role in the late stage of MNQ treatment, while IhSAMS1 and IhSAMS2 played an important role in the early stage of MNQ treatment , and IhSAMS2 was more significant.