## 3 Discussion  
JAs play diverse functions across the JA signaling pathway in plants [8]. Notably, JAZ proteins, core negative regulators of JA signaling, are widely reported to play a vital role in JA-mediated biological processes including plant growth, defense against pathogen infection, insect attack, abiotic stress, and plant specialized metabolism [20; 41; 73]. To date, JAZ family proteins have been systematically identified in various plants including _A. thaliana_[33], _T. aestivum_[37], _H. brasiliensis_[39], and _L. aureus_[70]. However, research regarding the _JAZ_ gene family has not yet been reported in the economically and medicinally important plant species _M. canadensis_. Based on the available transcriptome data [67], we performed a comprehensive identification of JAZ proteins in _M. canadensis_ and further investigated their sequence characteristics and protein structure, evolutionary relationships, expression profiles, subcellular locations, and protein-protein interactions. This work provided a basis for subsequent functional analysis of _JAZ_ genes to deepen our understanding of JA signaling in the _Lamiaceae_ family, especially in _M. canadensis_. We isolated seven _McJAZ_ genes from _M. canadensis_, and found that the length of CDs in the _McJAZ_ gene family ranged from 372 to 1041 bp (Figure 1 and Table 1), which was in line with the reported CDs length in the _AtJAZ_ gene family [19]. Moreover, most McJAZ proteins were basic proteins with pI values greater than 7, except for McJAZ with a pI value of 6.12, which was attributed to a higher proportion of acidic amino acids in McJAZ2 such as 21 aspartic acids and 12 glutamic acids than those in the rest of McJAZ proteins, and this phenomenon was ubiquitous in JAZ proteins of other plants like _Camellia sinensis_[74]. In general, JAZ protein at least contains one conserved TIFY and a typical Jas domain at its N- and C-terminal, and some JAZ proteins including _Arabidopsis_ AtJAZ7 and AtJAZ8 have an LxLxL type of EAR motif at the N-terminus [33]. Here, although the amino acid similarity among McJAZ proteins is low (Figure 2 and Supplemental Table S1), each identified McJAZ protein contained one conserved N-terminus TIFY and a C-terminus Jas domain, which further indicated evolutionary conservation of these structures. Furthermore, McJAZ6 and McJAZ7 have an additional EAR motif (LxLxL) in the N-terminus (Figure 2). TIFY has dual functions in regulating homo- and heterodimeric interactions among different JAZ proteins, and recruiting TPL/TPRs by interacting with NINJA protein [22]. The Jas domain is responsible for interacting with COI1 and various TFs including the core regulator MYC2 to complete the JA response [19], and is involved in subcellular localizations of JAZ proteins [75]. The EAR motif in some JAZ proteins is used to directly recruit the TPL-coupled inhibitory complex to attenuate JA response in the absence of NINJA [36]. Given that protein function is tightly associated with conserved domain structure, all of these results suggest that these seven McJAZ proteins may share some common strategy to regulate the perception of JA and JA response in _M. canadensis_, much like JAZ proteins in different plant species. Notably, the Jas domain of the JAZ protein usually contained a conserved sequence (SLx2 Fx2 KKx2 Rx5 PY), but our results showed that McJAZ2 has a truncated Jas motif (DPLIARKNSLARFILEKRDR) without the X\({}_{5}\)PY sequence (Figure 2). Previous studies found that some deviation existed in the Jas domain owing to alternative splicing (AS). For example, AtJAZ10, a splice variant of AtJAZ10 protein in _Arabidopsis_, has a truncated Jas motif (DPLIARRKSLQRFLEKRKRER) lacking LVSTSPY (X\({}_{5}\)PY) sequence in the whole Jas domain of AtJAZ10.1 [76]. Thus, whether McJAZ2 is a splice variant like AtJAZ10.3 merits further investigation. The tissue expression patterns of target genes are closely tied to gene function.