Cloning, Expression and Functional Analysis of MeJA Responsive PgJAZ1 Gene from Panax ginseng
JAs (jasmonates) are important phytohormones involved in regulating the biosynthesis of ginsenosides and other secondary metabolites. JAZ (jasmonate ZIM-domain) protein is a key regulator in jasmonate signaling pathway. In order to explore the relationships among ginsenoside biosynthesis, JAZ gene and jasmonic acid signal regulation in Panax ginseng, the RNA from fresh 4-year roots of P. ginseng was extracted and the synthesized cDNA was used as template. The candidate genes were screened and primers were designed based on the transcriptomic sequencing results of ginseng hairy roots induced by MeJA (methyl jasmonate). The full ORF (open reading frame) of JAZ gene was cloned by RT-PCR (reverse transcription polymerase chain reaction). The promoter sequence of JAZ gene was cloned from genomic library and analyzed by bioinformatics. The expression pattern of JAZ gene in different tissues and hairy roots of P. ginseng were analyzed by qRT-PCR, and the contents of ginsenoside, endogenous JA and MeJA were determined in MeJA-induced hairy roots of P. ginseng. The full ORF of JAZ has 702 bp and encoded 233 amino acids, and it was designated as PgJAZ1. Sequence analysis showed that PgJAZ1 protein contained two conserved domains TIFY and CCT2 (Jas domain). The phylogenetic analysis showed that PgJAZ1 was closely related to AaJAZ1 of Artemisia annua. qRT-PCR results showed that PgJAZ1 was expressed in roots, leaves, flowers, seeds and hairy roots of P. ginseng. The expression level of PgJAZ1 was the highest in flowers and the lowest in adventitious roots. In addition, PgJAZ1 was induced by MeJA and inhibited by SA (salicylic acid), and its expression level reached the highest after treatment by MeJA for 12 h. The PgJAZ1 promoter contained multiple putative cis-acting elements involved in MeJA, SA and hormone response, as well as binding sites of transcription factor such as MYB, MYC and WRKY. Ginsenosides content, endogenous jasmonates (JA and MeJA) induced by MeJA were highly consistent with PgJAZ1 expression. These results suggest that PgJAZ1 is involved in MeJA-mediated ginsenoside biosynthesis.
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