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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (01): 31-39.doi: 10.3724/SP.J.1006.2020.94036

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

Screening of NFR1α-interactive proteins in soybean using yeast two hybrid system

KE Dan-Xia(),PENG Kun-Peng   

  1. College of Life Sciences, Xinyang Normal University/Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang 464000, Henan, China
  • Received:2019-03-07 Accepted:2019-08-09 Online:2020-01-12 Published:2019-09-02
  • Contact: Dan-Xia KE E-mail:kdx_029@163.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31400213);Science and Technology Research Projects of Henan Province(182102110448);Nanhu Scholars Program for Young Scholars of XYNU

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Abstract:

Soybean is the important plant protein crop and grain-bean intercropping crop. Exploring the biological nitrogen fixation potential of soybean is of far-reaching significance to promote the sustainable development of ecological agriculture. GmNFR1α, a soybean nod factor receptor protein, is very important for nodulation, but its specific regulatory mechanism is still unclear. Soybean mRNA was used as template to amplify the kinase domain of GmNFR1α protein (GmNFR1α-pk) by RT-PCR method and the pGBKT7-GmNFR1α-pk bait plasmid was constructed. Seventy-two positive clones interacted with GmNFR1α-pk were isolated through yeast two hybrid screening of soybean nodule AD-cDNA library from the library. Among them, 12 proteins including calcium ion binding chiral protein, hemoglobin, nodulin Nod44 and other proteins interacted with GmNFR1α-pk were screened by sequencing and homology analysis. The interaction between soybean hemoglobin and bait protein was verified by re-transforming in yeast and BiFC in tobacco. Comparison of homologous proteins and phylogenetic tree analysis were also done at the same time. The important function of GmLbc2 in nodulation process was clarified by hairy root transformation technology in Lotus japonicus. The results further complement and improve the signal transduction pathway mediated by GmNFR1α, and provide new molecular evidence for the symbiotic interaction mechanism between soybean and rhizobia.

Key words: soybean, symbiotic nitrogen fixation, nod factor receptor protein, yeast two-hybrid, soybean hemoglobin

Fig. 1

Construction and auto-activation test of the bait plasmid pGBKT7-GmNFR1α-pk (A) M: trans 2K Plus II DNA marker; 1: isolation of GmNFR1α-pk cDNA; 2: pGBKT7-GmNFR1α-pk digested by EcoR I and Sal I; 3: pGBKT7 digested by EcoR I and Sal I. (B) positive control (pGBKT7-53 and pGADT7-T). (C) negative control (pGBKT7-lam and pGADT7-T). (D) pGBKT7-GmNFR1α-pk and pGADT7."

Fig. 2

Screening of yeast two-hybrid cDNA library (A) positive clones on QDO/X/A plate; (B) PCR detection of inserts from yeast two-hybrid cDNA library."

Fig. 3

Interaction between GmNFR1α and GmLbc2 (A) yeast two-hybrid analysis of interaction between GmNFR1α-pk and GmLbc2. 1: positive control (pGBKT7-53 and pGADT7-T); 2: negative control (pGBKT7-lam and pGADT7-T); 3: pGBKT7-GmNFR1α-pk and pGADT7-GmLbc2. (B) BiFC analysis of interaction between GmNFR1α and GmLbc2."

Fig. 4

Amino acid sequence alignment analysis of GmLbc2 and its homologous proteins in some other plants Gm: Glycine max; Gs: Glycine soja; Cc: Cajanus cajan; Va: Vigna angularis; Vr: Vigna radiata; Mt: Medicago truncatula; Lj: Lotus japonicus; As: Astragalus sinicus."

Fig. 5

Phylogenetic tree of GmLbc2 and its homologs The scale represents genetic similarity, indicating the proximity relationships among species. Abbreviations are the same as those given in Fig. 4."

Fig. 6

Effect of GmLbc2 overexpression on nodulation in L. japonicus (A) phenotype of hairy roots expressing p1301U (CK). (B) phenotype of hairy roots overexpressing GmLbc2 (GmLbc2-OX); photographs were taken at 30 d after inoculation, Bars = 5 mm. (C) mean number of nodules per plant with standard deviation (SD) of L. japonicus expressing empty vector pU1301 (CK) of GmLbc2-OX at 30 days after inoculation with M. loti, **P<0.01. (D) transcript levels of GmLbc2, NIN, Enod40-1, and Enod40-2 in CK and GmLbc2-OX hairy roots detected by qRT-PCR."

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