Subjects: Biology >> Botany submitted time 2022-10-27 Cooperative journals: 《广西植物》
Abstract： The pollen receptor like kinase (PRK) family, an LRR receptor-like protein kinase, not only played a role in pollen development and fertilization, but also played a role in stress response. Based on the analysis of transcriptome data that generated in our previous study, we found that AhPRK4 was an aluminum-responsive gene. To explore the role of AhPRK4 in response to Al stress, we analyzed the expression of AhPRK4 by qRT-PCR in ‘ZH2’ (Al-sensitive) and ‘99-1507’ (Al-tolerant), clarified the protein structure and genetic relationship of AhPRK4 by sequence analysis, phylogenetic tress construction and other genetic analysis, constructed the recombinant plasmid by homologous recombination, obtained the intracellular domain recombinant protein of AhPRK4 by prokaryotic expression technology and determined the activity of the recombinant protein by incubation with phosphorylated antibodys. The results were as follows: (1) The transcription level of AhPRK4 was up-regulated after different aluminum treatment times and different aluminum concentrations, indicating that AhPRK4 was an aluminum inducible gene; (2) The AhPRK4 protein had 673 amino acids with transmembrane domain, signal peptide and phosphorylation active sites, belonging to the LRR-III protein kinase family; (3) The GSTAhPRK4- CD recombinant protein was induced in vitro and verified by Western Blot. And the recombinant protein had phosphorylated on both serine/threonine and tyrosine residues, but had no significant auto-phosphorylation activity. In conclusion, AhPRK4 was an Al responsive gene, which participated in the regulation of short-term Al stress and was phosphorylated in vitro.
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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