Jebunnahar Khandakar
Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Md. Abdul Muktadir
Pulses Research Centre, Bangladesh Agricultural Research Institute, Gazipur, Bangladesh
Md. Shafiqul Islam
Department of Physical Sciences, Independent University, Dhaka Bangladesh.
Kenichi Yamaguchi
Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan and Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Tatsuya Oda
Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Yoshie Kitamura
1Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan and 3Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-
8521, Japan
2DE, Hyoscyamus albus, Sequential subproteome fractionation, TRIzol, MALDI-QIT-TOF MS
Pulses Research Centre, Bangladesh Agricultural Research Institute, Gazipur, Bangladesh
Development of Host and Medicinal Plants
Medicinal Plants
Root culture and sample Collection: The hairy roots of Hyoscyamus albus L have been used in this study were cultured accordingly Higa et al. al., 2008). Twelve days old root tips (2 cm in length) were collected that was pre cultivated in the standard liquid B5 medium Gamborg et al. al., 1968 containing one (1) % sucrose for two (2) weeks and then the medium was exchanged for the same medium, followed by further culture for 5 days. A 100 mL conical flask containing 25 mL of liquid medium was used for maintaining the culture, which was incubated on a rotary shaker at 25 °C with agitation at 80 rpm. Finally, root tips were harvested and kept at 80 °C until used. Extraction of protein Frozen root tips (100 mg fresh weight) with or without 20% w/w DTT (Bio-Rad, Hercules, CA, USA) and 10% w/w polyvinylpolypyrrolidone (PVPP, Sigma Aldrich, St. Louis, M O, USA) were disrupted for 1 min using a bead-beating apparatus (ShakeMaster Auto ver 1.5, BMS, Tokyo, Japan) equipped with pre-chilled bullet-shaped stainless bead crushers (SK 100 D10, Tokken, Inc., Chiba, Japan) and 2 mL stainless tubes (Tokken, Inc., C hiba, Japan), that were set in an aluminum block (25 well Master Rack for 1.5/2.0 mL tubes, BMS, Tokyo, Japan). After the cell disruption, 1 mL of TRIzol reagent (Invitrogen, Boston, MA, USA) with or without 20% w/w DTT and 10% w/w PVPP was mixed to the tube. The tube was shaken vigorously by hand for 10 20 seconds and incubated at room temperature for 5 mins, and then 0.2 mL of chloroform was added. After that, the tube was again strongly agitated for 15 seconds and allowed 2 3 min for incubation at ambient temperature, then centrifuged (12,000 x g, 15 min, at 4 °C ) to differentiated into a lower organic phenol-chloroform phase, interphase, and an upper aqueous phase. The upper watery phase was collected to a new tube. Ethanol (0.3 fold volumes) was added to the remaining interphase and organic phase, mixed by inversion for 3 5 times, incubated at room temperature for 2 3 min and centrifuged (2,000 x g, 5 min, at 4 °C ) separating the DNA pellet and the phenol ethanol supernatant. The phenol ethanol supernatant was dispensed to 1.5 mL low binding PTX tubes (Progress Tube, IEDA Trading Corp., Tokyo, Japan). Then, 1.5 fold volume of isopropanol was added to each tube and incubated for 10 min at ambient temperature. The mixture was centrifuged (12,000 x g, 10 min, at 4 °C ) separating the protein pellet and the phenol ethanol/isopropanol supernatant. For SDS PAGE analysis of proteins in the aqueous phase, DNA pellets, and phenol ethanol/isopropanol supernatant were separately transferred to new tubes. Each protein pellet was incubated with 1 mL of 300 mM guanidium hydrochloride in 95% v/v ethanol for 10 min at room temperature, and centrifuged (12,000 x g, 10 min, at 4 °C). This washing step was repeated three times.
J Bangladesh Agril Univ 17(4): 430–436, 2019
Journal