Guangxi Zhiwu (Jul 2023)
Cloning and functional analysis of promoter of TcALDH and TcGLIP genes in Tanacetum cinerariifolium
Abstract
Natural pyrethrin is a green botanical insecticide that extracted from the aboveground tissues of pyrethrum (Tanacetum cinerariifolium). Aldehyde dehydrogenase (TcALDH) and GDSL lipase (TcGLIP) are key rate-limiting enzymes involved in pyrethrin biosynthesis pathway in pyrethrum. The promoters of TcALDH and TcGLIP genes were cloned from the genomic DNA of pyrethrum clone ‘W99’ in order to investigate the regulatory mechanism of these genes. The regulatory elements, activity, hormone specificity and tissue inducibility of the two promoters were analyzed through bioinformatics analysis, histochemical staining (GUS staining), luciferase reporting, and exogenous hormone treatment. The results were as follows: (1) Using pyrethrum genomic DNA as a template, specific primers were used to clone the pTcALDH and pTcGLIP fragments. The sequence lengths of pTcALDH and pTcGLIP were 2 848 and 1 343 bp, respectively, and the promoter analysis software the PlantCARE predicted that they both contained multiple cis-elements related to stress response and hormone signals. (2) The plant expression vectors fused by pTcALDH and pTcGLIP and luciferase report gene were constructed, and were transformed into tobacco (Nicotiana benthamiana) to analyse hormone inducibility by observing the fluorescence imaging in tobacco leaves. The results demonstrated that the pTcALDH displayed typical hormone inducibility of methyl jasmonate (MeJA) and abscisic acid (ABA), whereas the pTcGLIP showed no response. (3) The tissue culture seedlings of pyrethrum ‘W99’ were treated with MeJA and ABA, the expression of TcALDH was up-regulated by ABA within 12 h, and first increased and then decreased under MeJA treatment; the expression of TcGLIP was down-regulated by ABA and MeJA. (4) We constructed the expression vectors of pTcALDH and pTcGLIP fused with GUS reporters and transformed them into tobacco, then the transient transformation of tobacco drived the expression of GUS gene and showed initiating activity. It was found that the pTcALDH expressed in the glands, glandular hair heads and mesophyll of the leaves, while the pTcGLIP was only expressed in the parenchyma cell. These results indicated that the pTcALDH and pTcGLIP were tissue-specific promoters, and the pTcALDH appeared MeJA-inducible and ABA-inducible characteristics. This study provides a new insight into the regulatory mechanism of TcALDH and TcGLIP genes involved in pyrethrin synthesis.
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