Cloning and functional analysis of the PLkF3H2 promoter in Larix kaempferi

The flavanone 3-hydroxy-lase2 ( F3H2 ) gene is one of the key genes in flavonoid biosynthesis pathways in Larix kaempferi (Lam.) Carrière (Japanese larch). However, the transcriptional regulation mechanism of this gene in flavonoid metabolism has not been studied. In this study, we cloned a 2398 bp upstream promoter sequence and tested its transcriptional activity in larch protoplasts using transient transformation. We also constructed the 5’ shortened fragments of this sequence into the upstream of the β-glucuronidase ( GUS ) gene in the pBI121 expression vector. In transient and stable transgenic tobacco, GUS expression could be initiated by both full-length and shortened promoters, but its activity gradually decreased as the 5’ end was continuously removed. The GUS histochemical staining revealed that the key promoter region was located from − 526 bp to − 429 bp. Moreover, in transgenic tobacco, the PLkF3H2 promoter responded to environmental signals and hormones, such as methyl jasmonate (MeJA), abscisic acid (ABA), NaCl, and low temperature. The NaCl and low-temperature responsive elements were located at − 2325 bp ~ − 1570 bp and − 526 bp ~ − 429 bp, respectively. But in larch, LkF3H2 gene expression was positively regulated by MeJA, ABA, and NaCl via PLkF3H2 promoter elements, while it was negatively regulated by low temperature. Our study provides a theoretical reference for the use of the PLkF3H2 in biological adversity resistance as well as the functional verification of the LkF3H2 gene and regulation of flavonoid synthesis in Japanese larch.