Screening for the Interacting Proteins with GhENODL6 in Gossypium hirsutum by Yeast Two-Hybrid System

doi:10.7668/hbnxb.20194603

Abstract

Abstract:

GhENODL6 has been discovered to be involved in the resistance of Gossypium hirsutum to Verticillium dahliae.To further reveal its role in cotton resistance against Verticillium wilt,GhENODL6 open reading frame was inserted into the yeast expression vector pNC-GBKT7 using Nimble Cloning technology to construct the recombinant plasmid pNC-GBKT7-ENODL6.The resulting plasmid was successfully transferred into yeast Y2HGold and exhibited normal growth on DDO medium,while failing to grow on QDO/X/A medium,indicating that the GhENODL6 protein did not exhibit toxicity towards the yeast host and lacked autoactivating activity.Subsequently,the Y2HGold yeast carrying the pNC-GBKT7-ENODL6 decoy vector was subjected to hybridization with a cDNA library and screened for colonies displaying blue coloration.Through PCR amplification,a 526 bp non-vector insert was obtained in these blue yeasts,which showed high sequence consistency with the WRKY47 gene within G.hirsutum genome.By employing homologous amplification techniques,we successfully cloned the WRKY47 open reading frame from upland cotton,which spanned 1 587 bp in length and encoded 528 amino acid residues.Furthermore,a yeast two-hybrid assay confirmed an interaction between WRKY47 and GhENODL6.To summarize our findings,the study constructed the recombinant vector pNC-GBKT7-ENODL6 and identified WRKY47 as an interacting protein of GhENODL6.

Key words: Gossypium hirsutum, Verticillium wilt, Yeast two-hybrid, Interaction protein, Screen

ZHENG Xinxin, ZHANG Yan, XIE Meixia, ZHANG Dongmei, WU Liqiang, WANG Xingfen, YANG Jun. Screening for the Interacting Proteins with GhENODL6 in Gossypium hirsutum by Yeast Two-Hybrid System[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 173-178. doi: 10.7668/hbnxb.20194603.

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Figures/Tables 6

Fig.1 Construction of bait vector pNC-GBKT7-ENODL6 for protein interaction screening A.Schematic illustration of the construction of recombinant plasmid pNC-GBKT7-ENODL6;B.Detection of inserted fragments from GhENODL6 by PCR.M.DNA Marker DL2000.

Fig.2 The growth of yeast containing plasmids pNC-GBKT7-ENODL6

Fig.3 The observation on the hybridization that occurs between yeast Y2HGold and Y187 The red arrows indicate the yeasts that are hybridizing.

Fig.4 The growth of hybrid yeasts on QDO medium

Fig.5 The growth of positive yeasts and negative controls on QDO/X/A medium

Fig.6 Cloning of the gene coding of protein interacting with GhENODL6 A.Detection of the inserted fragment using pNC-GBKT7 vector specific primers;B. Detection of the inserted fragment using pNC-GADT7 vector specific primers;C.Full-length amplification of WRKY47 ORF.

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