关键词: 普通菜豆, PvEG261, 镰孢菌枯萎病, 干旱胁迫, 响应机制

Abstract:

【Objective】By analyzing the sequence and expression pattern characteristics of PvEG261 from common beans, and studying its resistance to Fusarium wilt and drought, the foundation was laid for the signal regulation network analysis of Fusarium wilt and drought-resistance and molecular breeding in common beans. 【Method】 Bioinformatics analysis was performed on the open reading frame (ORF) of PvEG261 to predict the physical and chemical properties, secondary structure, signal peptide sequence of the protein encoded by the PvEG261, and search for highly homologous protein sequence in NCBI database through Blastp tool online for sequence alignment and phylogenetic tree construction; the tissue expression specificity of PvEG261 and the expression pattern in response to Fusarium wilt pathogen and drought stress were analyzed by qRT-PCR; PvEG261 overexpression vector was constructed and transformed into Agrobacterium rhizogenes K599 to induce the generation of hairy transgenic roots in common beans. Meanwhile, the PvEG261 silencing vector was constructed, and the transcription product in vitro was inoculated on the seedlings of common bean to interfere with PvEG261 expression. Through inoculation with the pathogen and drought treatment, the phenotypes of control, PvEG261-overexpressed and silenced plants were observed, disease and drought-resistance were both identified, and hydrogen peroxide (H₂O₂) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and peroxidase (POD) activity as physiological and biochemical indicators were all assayed. 【Result】 The cDNA sequence of PvEG261 was 471 bp, which encodes a protein composed of 156 amino acids. The structure prediction indicated that it contained 10 strand structures, the predicted molecular mass of the encoding product was 38.89 kD, and the theoretical pI was 5.21. PvEG261 belonged to the members of dirigent gene superfamily, it contained a signal peptide sequence of 10 amino acids, and belonged to a secreted protein. The relationship between PvEG261 and cowpea DIR22 protein is the closest, which reached 91.61%. The results of qRT-PCR showed that the expression in the root tissues increased significantly after inoculation with Fusarium wilt pathogen and drought treatment, and the gene has obvious tissue expression specificity, with the highest expression level in the roots. After inoculation with pathogen and drought treatment, the disease and drought-resistance of the overexpressed plants were significantly improved in comparison with the control, the plant disease scores and the wilting degree caused by water shortage were significantly reduced, and the H₂O₂ content, POD and SOD activity in the roots were all significantly higher than the control plant, while the MDA content was dramatically lower than the control plant. The disease and wilting degree of the gene silenced plants were significantly increased. The H₂O₂ content, POD and SOD activity in the roots were significantly lower than the control plant, and the MDA content was significantly higher than the control plants. 【Conclusion】 PvEG261 responded to Fusarium wilt pathogen infection and drought stress, and positively regulated the Fusarium wilt and drought-resistance in common beans.

Key words: common bean, PvEG261, Fusarium wilt, drought stress, response mechanism