Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (1): 14-26.doi: 10.3864/j.issn.0578-1752.2016.01.002

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

Identification of Interaction Domain of SRK-ARC1-Exo70A1 and Interaction Strength Analysis in Brassica oleracea var. capitata L.

SHI Song-mei1,2, GAO Qi-guo1,2, LIAN Xiao-ping2, BI Yun-long1, LIU Xiao-huan2, PU Quan-ming2, LIU Gui-xi1,    LIU Jing1, REN Xue-song2, YANG Xiao-hong1, ZHU Li-quan1, WANG Xiao-jia2   

  1. 1Faculty of Agriculture, Southwest University, Chongqing 400715
    2Key Laboratory in Olericulture of Chongqing, Chongqing 400715
  • Received:2015-07-13 Online:2016-01-01 Published:2016-01-01

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Abstract: 【Objective】 The study was for further study on the mechanism of mutual recognition among S receptor kinase (SRK), ARM-repeat containing 1 (ARC1), and Exo70A1, which were the key signal elements in self-incompatible response, and to identify the interaction domains and compare their interaction strength.【Method】According to a bioinformatics analysis, we obtained the different functional domains of three proteins. And on this basis, the truncated fragments containing different functional domains were amplified from Brassica oleracea var. capitata L. E3. Then the encoding sequences of SRKj with subfragments (SRKjΔ1-SRKjΔ4) and the full length Exo70A1 with subfragments (Exo70A1Δ1-Exo70A1Δ3) were separately subcloned into the vector pGADT7 to generate the AD recombinant plasmids, after which the encoding sequence of ARC1 with subfragments (ARC1Δ1-ARC1Δ3) were respectively constructed into vector pGBKT7 to generate the BD recombinant bait plasmids. These recombinant plasmids were respectively cotransformed into the yeast competent cells of strain AH109, and then were planted on SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT nutritional media to detect the growth and color change. A β-galactosidase assay was conducted. Finally, anin vitro binding assay was performed to confirm the interaction between SRK-ARC1 and ARC1-Exo70A1.【Result】A DNA sequence and restriction enzyme analysis suggested the recombinant plasmids were correct. The yeast AH109 cells were without an autonomous activation effect on the reporter gene MEL1. In the combinations of SRKj with ARC1Δ4, ARC1Δ8, and ARC1, the yeast AH109 cells could grow and turn blue on SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT nutritional media with a transcription activation of the reporter genes HIS3, ADE2 and MEL1. With the extension of the amino acid sequence, a greater β-galactosidase activity was induced, in which the combinations of SRKj with ARC1 showed relatively high levels of β-galactosidase activity (15.98). In the combinations of ARC1-Exo70A1, Exo70A1Δ3 could interact with ARC1Δ1-ARC1Δ3. Their yeast cells could grow and turn blue on SD/-Leu-Trp-His–Ade/ X-a-gal/25 mM 3-AT nutritional media, activating the reporter genes HIS3, ADE2, and MEL1. With the extension of truncated ARC1 or Exo70A1, β-galactosidase activity showed a lower trend after the first increase. Meanwhile, ARC1Δ2 with Exo70A1Δ3 exhibited the highest levels of β-galactosidase activity in all combinations (25.07). In vitro binding assay further showed that SRKj could interact with ARC1Δ4, and ARC1Δ2 also could interact with Exo70A1Δ3. 【Conclusion】The kinase domain of SRK(SRKj) and the C-terminal ARM of ARC1 were the core interaction domains of SRK-ARC1. No interactions were detected for either truncating SRKj functional domains or ARM of ARC1. The Leucine zipper with coiled-coil of ARC1 and N terminal of Exo70A1 mediated the interaction between ARC1 and Exo70A1. The interaction strength of SRK-ARC1 was less than that of ARC1-Exo70A1.

Key words:  Brassica oleracea var. capitata L., self-incompatible, SRK, ARC1, Exo70A1, yeast two-hybrid, truncation

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