Gene Cloning and Interaction Proteins Screening of DcPMK in Dioscorea composite

doi:10.7525/j.issn.1673-5102.2022.05.017

Abstract

Abstract:

To explore the synthesis path of terpenoid in Dioscorea composite and to provide theoretical reference for studying， D. composite was used as material， DcPMK gene was cloned by RT-PCR and the bioinformatic analysis， tissue-specific expression analysis and expression analysis after salicylic acid （SA） treatment were carried out respectively， and the bait vector of DcPMK was constructed to screen interacting proteins from Arabidopsis yeast library. The results showed that the open reading frame （ORF） of DcPMK was 1 536 bp （GenBank accession No. MZ171241）， which encoded 511 amino acids. By multiple sequence alignment， the results showed that similarity between DcPMK and PMK amino acid sequence of other plants was as high as 88.85%， DcPMK obtained 1 ATP binding site Gly-X-Gly-XX-Ala. The result of phylogenetic tree showed that DcPMK was closely related to monocotyledon such as Phonenix dactylifera．HPLC analysis showed that the content of diosgenin was the highest in rhizome， qRT-PCR analysis indicated that DcPMK was constitutively expressed among flower， leaf， stem， rhizome and root， with the highest levels in old stem and the lowest in rhizome. After SA treatment， the changes of diosgenin content were in parallel with the expression levels of DcPMK， the diosgenin content was improved as DcPMK up-regulated expression in leaf. Yeast two-hybrid results showed that DcPMK was no autoactivation and cell toxicity， then 27 significant interactional genes were screened， which were growth and development-related genes AtKCR1（AT1G67730）， AtRPS9M（AT3G49080）， AtASY4（AT2G33793）， abiotic and biotic stress-related genes AtVDAC2（AT5G67500）， AtVDAC3（AT5G15090）， AtRH8（AT4G00660）， anthocyanin accumulation-related gene AtPHR2（AT2G47590）. The results illustrated that DcPMK could participate in the synthesis of terpenoids in D. composite， and was widely involved ingrowth and stress response pathways through protein-protein interaction.

Key words: Dioscorea composite, PMK, bioinformation, expression pattern, interaction proteins

CLC Number:

S567.9

Hongpeng WANG, Yidan LI, Yao WANG, Xiaoyu TAN, Chengbin CHEN, Lipeng ZHANG. Gene Cloning and Interaction Proteins Screening of DcPMK in Dioscorea composite[J]. Bulletin of Botanical Research, 2022, 42(5): 855-865.

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