Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4274-4285.doi: 10.3864/j.issn.0578-1752.2021.20.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Effects of PvEG261 Gene on the Fusarium Wilt and Drought- Resistance in Common Bean

XUE RenFeng1(),FENG Ming1,HUANG YuNing1,Matthew BLAIR2,Walter MESSIER3(),GE WeiDe1()   

  1. 1Crop Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China
    2Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville 37209, TN USA
    3Evolutionary Genomics, Inc., Lafayette 80501, LA USA
  • Received:2021-04-06 Accepted:2021-05-27 Online:2021-10-16 Published:2021-10-25
  • Contact: MESSIER Walter,WeiDe GE E-mail:xuerf82@163.com;wmessier@evolgen.com;snowweide@163.com

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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

Table 1

Primers used in this research"

引物名称 Primer name 引物序列 Primer sequence (5′-3′)
EG261-F GTTGTGGGAAGTGCTGAA
EG261-R CCCTGGCAAATCTGAATA
ACT-F GAAGTTCTCTTCCAACCATCC
ACT-R TTTCCTTGCTCATTCTGTCCG
OE-F GCTCTAGAATGTCCTTAAGCTACAAGAA
OE-R GCGAGCTCTTAGTGGTAAACGTAGACGT
GS-F GCGGATCCCTTAGCCACTTCAGGTTCT
GS-R GCATCGATTCGTGTTGTTCCTCGTTT

Fig. 1

Sequence analysis of PvEG261 A: Sequence analysis of PvEG261 protein; B: Multiple sequence alignment of PvEG261 and the dirigent proteins from other plants. Green box indicated no less than 50% in the sequence identity; Pink box indicated no less than 75% in the sequence identity; Black box indicated 100% in the sequence identity; C: Phylogenetic analysis of PvEG261 and the dirigent proteins from other plants. CaDIR22: Coffea arabica, XP_027102329.1; LaDIR22: Lupinus angustifolius, XP_019462472.1; MpDIR3: Mucuna pruriens, RDX73385.1; ApDIR22: Abrus precatorius, XP_027337739.1; GmDIR22: Glycine max, XP_006576480.1; GsDIR: Glycine soja, KHN26491.1; VaDIR22: Vigna angularis, XP_017441835.1; VrDIR22: Vigna radiata, XP_014492698.1; VuDIR22: Vigna unguiculata, XP_027906892.1"

Fig. 2

Expression analysis of PvEG261 A: Tissue specific expression analysis of PvEG261; B: PvEG261 expression analysis induced by FOP-DM01; C: PvEG261 expression analysis induced by PEG6000. Different lowercase letters indicate significant difference at the 0.05 probability level. The same as below"

Fig. 3

qRT-PCR identify of the PvEG261 overexpressed and silenced plants A: qRT-PCR identify of the PvEG261 overexpressed plants; B: qRT-PCR identify of the PvEG261 silenced plants"

Fig. 4

Disease assessment of the PvEG261 overexpressed and silenced plants A: Disease characteristics of the PvEG261 overexpressed plants at 2 weeks post inoculation; B: Disease scores of the PvEG261 overexpressed plants; C: Disease characteristics of the PvEG261 silenced plants at 2 weeks post inoculation; D: Disease scores of the PvEG261 silenced plants. NT: Non-transformed plants; EV: Empty vector plants; OE: PvEG261 hair-root transgenic plants; GS: PvEG261 gene silencing plants. The same as below"

Fig. 5

Drought resistance of the PvEG261 overexpressed and silenced plants A: Phenotypic characteristics of the PvEG261 overexpressed plants at 2 weeks under drought stress; B: Root length of the PvEG261 overexpressed plants under drought stress; C: Root fresh weight of the PvEG261 overexpressed plants under drought stress; D: Phenotypic characteristics of the PvEG261 silenced plants at 2 weeks under drought stress; E: Root length of the PvEG261 silenced plants under drought stress; F: Root fresh weight of the PvEG261 silenced plants under drought stress"

Fig. 6

Analysis of H2O2 contents, SOD activity, POD activity and MDA contents in roots of common beans induced by F. oxysporum f. sp. phaseoli and drought stress A: H2O2 contents of PvEG261 overexpressed plants; B: SOD activity of PvEG261 overexpressed plants; C: POD activity of PvEG261 overexpressed plants; D: MDA contents of PvEG261 overexpressed plants;E: H2O2 contents of PvEG261 silenced plants; F: SOD activity of PvEG261 silenced plants; G: POD activity of PvEG261 silenced plants; H: MDA contents of PvEG261 silenced plants"

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