Title:Cloning, Expression, and Functional Analysis of the Full-Length cDNA of
Acetyl-CoA C-acetyltransferase (AACT) Genes Related to Terpenoid
Synthesis in Platycodon grandiflorus
Volume: 29
Issue: 12
Author(s): Mengli Liu, Hanwen Yu, Jing Li, Nan Dong, Bowen Chen, Rui Xu, Junxian Wu, Xiangwei Chang, Jutao Wang, Huasheng Peng, Liangping Zha*Shuangying Gui*
Affiliation:
- School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
- Anhui Province Key Laboratory of Research
& Development of Chinese Medicine, Hefei 230012, China
- School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
- Institute of Pharmaceutics, Anhui Academy of Chinese
Medicine, Hefei 230012, China
Keywords:
Platycodon grandiflorus, acetyl-CoA C-acetyltransferase, prokaryotic expression, western blot, MeJA, full-length cDNAs.
Abstract:
Platycodon grandiflorus is a well-known and widely distributed traditional herbal
medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots.
Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and
plays an important role in the biosynthesis of terpenoids.
Objective: The objective of this study was to clone and identify the PgAACT function in P.
grandiflorus.
Methods: The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus
by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a
vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was
observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR)
was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in
AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect
the changes in P. grandiflorus saponins.
Results: In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2
(PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2
proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed,
and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl
sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT
genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63
kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the
cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that
PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression
level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly
increased, suggesting that PgAACT genes play an important role in regulating plant defense systems.
Conclusion: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful
in understanding the role of these two genes in terpene biosynthesis.
