Subjects: Biology >> Botany >> Applied botany submitted time 2022-04-29 Cooperative journals: 《广西植物》
Abstract： In order to explore the difference in the molecular mechanism of the variety Puerarial between isoflavones metabolic enzyme genes PtCHI, to preliminarily reveal the difference content of the isoflavones. The material of this study is Pueraria montana and Pueraria thomsonii. Puerarin and total flavonoids of Pueraria montana and Pueraria thomsonii were extracted by ethanol, and their content was measured by high-performance liquid phase. Based on the CDS sequence of the CHI gene of Pueraria montana, the PtCHI gene from Pueraria thomsonii was isolated by homologous cloning, and the protein was expressed in vitro. At the same time, the location of the PtCHI gene was studied in Arabidopsis protoplasts. The results were as follows: (1)The content of puerarin in Pueraria montana is significantly higher than the Pueraria thomsonii, and the content of total flavonoid was also, but not significant. (2)Successfully isolated the gene PtCHI from Pueraria thomsonii. The gene was 742 bp in length, contains a complete ORF frame of 672 bp, encodes 223 amino acids, and has up to 99% homology with Pueraria montana; it was predicted to be a stable hydrophobic protein with a size of 27.8 kD. (3)This study found that the expression of CHI gene in Pueraria thomsonii was stem> root> leaves, Pueraria thomsonii was root> stem> leaves. The expression of CHI genes from Pueraria thomsonii was significantly higher than Pueraria 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-based, with 25 phosphorylation sites, closely related Pueraria montana, 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, respectively. (6) Through the Arabidopsis protoplasts revealed that PtCHI was only located in the chloroplasts. This study is in further analyzing the difference in flavonoids in Pueraria montana and Pueraria thomsonii, as well as laying the foundation for the functional verification of Pueraria thomsonii PtCHI and the research on the mechanism of isoflavone metabolism.
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