Chinese Bulletin of Botany ›› 2016, Vol. 51 ›› Issue (6): 790-800.doi: 10.11983/CBB15226

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Optimization of Detection Methods for Zea mays ABA Receptor ZmPYL1 by Isothermal Titration Calorimetry

Ruixue Yang, Haiyang Liu, Shengli Liu, Tingqiao Yu, Yuzhen Chen*, Cunfu Lu*   

  1. 1College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
    2National Engineering Laboratory for Tree Breeding, Beijing 100083, China
    3Key Laboratory of Genetics and Breeding in Forest Tree and Ornamental Plants of Education Ministry, Beijing 100083, China
  • Received:2015-12-30 Accepted:2016-03-21 Online:2016-12-02 Published:2016-11-01
  • Contact: Chen Yuzhen,Lu Cunfu E-mail:chenyuzhen@bjfu.edu.cn;lucunfu@bjfu.edu.cn
  • About author:

    # Co-first authors

Abstract:

Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Identification of the ABA receptor has important theoretical and practical significance. Isothermal titration calorimetry (ITC) is one of the important techniques used to identify and select ABA receptors; however, high- quality receptor protein is needed in this method. In this study, we examined factors affecting the separation and purification of the Zea mays ABA receptor ZmPYL1 and the binding affinity of ZmPYL1 to ABA. ZmPYL1 was overexpressed in E. coli, and high-level ZmPYL1 was purified from sonicated E. coli cells. The optimized ultrasonification conditions were total time 15 min, single time 3 s, internal time 10 s, and concentration of bacterial suspension 100 mg·mL-1. The binding affinity reaction of (±)-ABA to ZmPYL1 was an endothermic process, so ZmPYL1 is a dimer receptor. ITC results showed Kd of 72.46 μmol·L-1 for the interaction between 4 mmol·L-1 (±)-ABA and 0.1 mmol·L-1 ZmPYL1. This study provides an important technical basis for screening and identifying plant ABA receptors.

Figure 1

Effect of total ultrasonic time on the OD600 and protein concentration of bacterial resuspension in lysis buffer"

Figure 2

Ultrasonic treatments for bacterial resuspension of different concentrations (A) A1-A4 indicate the comparison of 200, 100, 70 and 40 mg·mL-1 bacterial suspension before (left beaker) and after (right beaker) ultrasonic treatments; (B) Effect of bacterial resuspension of different concentrations on the OD600 before (0 min) and after (15 min) ultrasonic treatments; (C) Effect of bacterial resuspension of different concentrations on the protein content before (0 min) and after (15 min) ultrasonic treatments; (D) SDS-PAGE detection of protein content in sediment and supernatant of different bacterial concentration, lane 1, 3, 5, 7 represent the protein content in sediment of 200, 100, 70 and 40 mg·mL-1 bacterial suspension, respectively; and lane 2, 4, 6, 8 represent the protein content in supernatant of 200, 100, 70 and 40 mg·mL-1 bacterial suspension, respectively. Target protein band is showed by arrow."

Figure 3

Ultrasonic treatment of inclusion body ZmPYL4 (A) Effect of total ultrasonic time on the OD600 of bacterial resuspension and the protein content in supernatant; (B) Bacterial resuspension before (left beaker) and after (right beaker) ultrasonic treatment for 30 min; (C) SDS-PAGE detection of protein level in sediment and supernatant of bacterial suspension. Lane 1: The protein level in sediment before ultrasonic treatment. Lane 2: The protein level in supernatant before ultrasonic treatment. Lane 3: The protein level in sediment after ultrasonic treatment for 30 min. Lane 4: The protein level in supernatant after ultrasonic treatment for 30 min."

Figure 4

Effect of single ultrasonic time on the OD600 (A) and protein concentration (B) of bacterial resuspension in lysis buffer"

Figure 5

Effent of interval time on the OD600 (A) and protein concentration (B) of bacterial resuspension in lysis buffer"

Figure 6

SDS-PAGE detection of purified ZmPYL1 protein Lane 1: The protein level in sediment after ultrasonic treatment; Lane 2: The protein level in supernatant after ultrasonic treatment; Lane 3: The collecting liquid of the supernatant through Ni-NTA; Lane 4: The collecting liquid of wash buffer through Ni-NTA; Lane 5-8: The collecting liquid of elution buffer through Ni-NTA for the first, second, third and fourth time, respectively."

Table 1

The thermodynamic parameters of (±)-ABA and ZmPYL1 protein in different concentrations"

Thermodynamic parameters 2 mmol·L-1 (±)-ABA+
0.005 mmol·L-1 ZmPYL1
2 mmol·L-1 (±)-ABA+
0.1 mmol·L-1 ZmPYL1
4 mmol·L-1 (±)-ABA+
0.1 mmol·L-1 ZmPYL1
N (sites) 15.5±5.52 0.651±0.190 0.821±0.069
Kd (μmol·L-1) 135.32 72.99 72.46

Figure 7

The variation of differential power with time and the heat variation of injection with the molar ratio(A) 2 mmol·L-1 (±)-ABA was titrated into 0.05 mmol·L-1 ZmPYL1; (B) 2 mmol·L-1 (±)-ABA was titrated into 0.1 mmol·L-1 ZmPYL1; (C) 4 mmol·L-1 (±)-ABA was titrated into 0.1 mmol·L-1 ZmPYL1; (D) 4 mmol·L-1 (±)-ABA was titrated into HEPES solution without protein. The solid lines in bottom panels represent the fitting curves, and the closer the fitting curve is to experimental dots, the more credible the fitting result is."

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