Guangxi Zhiwu (Feb 2023)
Cloning, prokaryotic expression and subcellular localization of PtCHI gene from Pueraria montana var. thomsonii
Abstract
In order to explore the differences in the molecular mechanism of the Pueraria cultivars between isoflavone metabolic enzyme gene PtCHI, and to preliminarily reveal the difference content causes of the isoflavones. The materials of the study were Pueraria montana var. lobata and P. montana var. thomsonii. Puerarin and total flavonoids of P. montana var. lobata and P. montana var. thomsonii were extracted by ethanol, and their contents were measured by high-performance liquid chromatography. Based on the reported CHI gene of Pueraria montana var. lobata, the PtCHI gene from P. montana var. thomsonii was isolated by homologous cloning method, and the protein was expressed in vitro. At the same time, the location of the PtCHI gene was studied in Arabidopsis thaliana protoplasts. The results were as follows: (1)The content of puerarin in P. montana var. lobata was significantly higher than the P. montana var. thomsonii, and the content of total flavonoids was also higher but not significant.（2) The gene PtCHI was successfully isolated from P. montana var. thomsonii. The gene was 742 bp in length, containing a complete ORF frame of 672 bp, encoding 223 amino acids, and had up to 99% homology with P. montana var. lobata. (3)This study found that the expression of CHI gene in P. montana var. thomsonii was stem>root>leaf, P. montana var. lobata was root>stem>leaf. The expression of CHI gene from P. montana var. lobata was significantly higher than P. montana var. thomsonii besides in leaves. (4) Through the online tool prediction analysis, PtCHI was found to be stable hydrophilic protein and the size was 27.8 kD. The secondary and tertiary structures were based on α-helix, with 25 phosphorylation sites, closely relating P. montana var. lobata, Glycine max and Glycyrrhiza uralensis, and were more likely to interact with F3H2, F3H, 4CL4, DFR2 and CHS. (5)At the same time, the protein of PtCHI was successfully induced and isolated in vitro, with a single protein of 27.8 kD. (6) Through the Arabidopsis thaliana protoplasts revealed that PtCHI was mainly located in the chloroplasts. This study further analyzed the difference in flavonoids in P. montana var. lobata and P. montana var. thomsonii, as well as provides the reference for the functional verification of P. montana var. thomsonii PtCHI and the research on the mechanism of isoflavone metabolism.
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