Comparative Analysis of the Effects of Different Precipitation Agents on Extraction of Plant Protein by Phenol Extract

doi:10.3969/j.issn.1000-2561.2021.10.035

Abstract

Abstract:

The borax/PVPP/phenol (BPP) protocol is a widespread protein extraction method in plant proteome research. However, BPP extraction of plant protein, combined with different precipitation agents, the precipitation time, yield and purity of extracted protein is strikingly different. In this study, Sesuvium portulacastrum and Para-rubber tree leaves, and rubber latex were used as the research materials. The plant protein was extracted and analyzed with precipitating agents of supersaturated potassium ammonium sulfate alcohol solution, ammonium acetate methanol solution and acetone solution. Using the methods of SDS-PAGE, gel electrophoresis image quality, mass spectrum identification, settling duration and comparative analysis of protein yield, it was showed that the protein bands extracted by the ammonium acetate precipitation method were not evident. Hevea leaves and latex showed a higher yield of protein in acetone solution. S. portulacastrum leaves had a higher yield of protein in supersaturated potassium ammonium sulfate alcohol solution. The clear background of gel electrophoresis figures was found for the supersaturated potassium ammonium sulfate ethanol solution and acetone solution. The comprehensive protein sequence information identified by mass spectrometry had a higher quality of precipitated protein. The research results can optimize the method of extracting high-quality plant protein, and it will be expected to provide references for recalcitrant plant taxa proteomics research in the future.

Key words: protein extraction, Hevea brasiliensis, Sesuvium portulacastrum, precipitant, polypropylene gel electrophoresis, mass spectrometry

CLC Number:

TQ936

HUO Yuxin, GAN Panlu, WANG Yujing, ZHANG Xueyan, WANG Xuchu, XIE Quanliang. Comparative Analysis of the Effects of Different Precipitation Agents on Extraction of Plant Protein by Phenol Extract[J]. Chinese Journal of Tropical Crops, 2021, 42(10): 3008-3016.

Figures/Tables 6

References 31

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样品 Samples	条带数 Number of protein bands	样品 Samples	条带数 Number of protein bands	样品 Samples	条带数 Number of protein bands
SAM-HL	41±1.41	SAM-LA	35±0.63	SAM-SL	42±1.41
AA-HL	29±2.45	AA-LA	28±2.42	AA-SL	31±1.50
AS-HL	37±3.90	AS-LA	34±2.45	AS-SL	38±3.44

样品 Samples	条带数 Number of protein bands	样品 Samples	条带数 Number of protein bands	样品 Samples	条带数 Number of protein bands
SAM-HL	41±1.41	SAM-LA	35±0.63	SAM-SL	42±1.41
AA-HL	29±2.45	AA-LA	28±2.42	AA-SL	31±1.50
AS-HL	37±3.90	AS-LA	34±2.45	AS-SL	38±3.44

编号^a Number	蛋白名称^b Protein name	登陆号^c Accession number	等电点/分子量（实验值/理论值）^dpI/M_w (experimental/theoretical value)	匹配肽段数/覆盖率^eMatching peptide number/coverage rate/%	分数^fScore
A1-1	Ribulose bisphosphate arboxylase	ref\|YP_004327670.1	6.09/53.0; 6.32/50.2	6/22; 28%	390
A1-2	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.49/48.9	6/47; 29%	225
A1-3	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.06/52.1	6/48; 28%	463
A2-1	Small rubber particle protein	ref\|XP_021653597.1	4.80/22.0; 4.71/25.0	13/62; 21%	94
A2-2	Small rubber particle protein	ref\|XP_021653597.1	4.80/22.0; 4.88/24.0	14/37; 38%	161
A2-3	Small rubber particle protein	ref\|XP_021653597.1	4.80/22.0; 4.65/23.0	13/62; 21%	94
A3-1	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 4.95/12.0	10/33; 30%	125
A3-2	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.08/15.0	10/33; 30%	126
A3-3	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.02/13.0	10/33; 30%	125
A4-1	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.8; 6.69/55.5	4/12; 33%	366
A4-2	Ribulose bisphosphate carboxylase	ADG02945.1	6.26/52.9; 6.89/56.3	3/9; 27%	277
A4-3	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.8; 6.64/60.2	3/9; 33%	264
B1-1	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.45/53.1	10/10; 26%	542
B1-2	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.05/52.9	6/24; 25%	497
B1-3	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.08/53.0	8/48; 28%	809
B2-1	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.61/12.8	4/12; 61%	455
B2-2	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.61/12.8	5/12; 55%	410
B2-3	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.33/20.6	8/57; 58%	558
B3-1	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.28/19.6	12/21; 34%	494
B3-2	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.21/25.3	3/18; 31%	483
B3-3	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 4.8/22.3	5/14; 38%	319
B4-1	Ribulose bisphosphate carboxylase	AFY17050.1	6.70/53.2; 6.75/55.2	9/21; 26%	663
B4-2	Ribulose bisphosphate carboxylase	ADD12830.1	6.16/52.6; 3.52/57.5	6/25; 26%	781
B4-3	Ribulose bisphosphate carboxylase	CAA04981.1	6.12/52.1; 6.89/39.7	3/13; 23%	127
C1-1	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1\|	6.09/53.0; 6.16/53.2	7/19; 26%	481
C1-2	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.42/51.5	6/23; 26%	484
C1-3	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.01/53.0	6/24; 25%	441
C2-1	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.58/21.5	10/21; 74%	887
C2-2	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.8/22.3	10/21; 74%	739
C2-3	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.64/22.3	5/35; 71%	830
C3-1	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.28/19.6	5/27; 41%	519
C3-2	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.04/14.7	5/14; 46%	451
C3-3	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.04/14.7	5/14; 46%	433
C4-1	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.0; 6.03/50.6	7/42; 24%	374
C4-2	Ribulose bisphosphate carboxylase	ADG02945.1	6.26/53.0; 6.06/52.1	6/25; 17%	250
C4-3	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.0; 6.06/52.1	6/48; 17%	388

编号^a Number	蛋白名称^b Protein name	登陆号^c Accession number	等电点/分子量（实验值/理论值）^dpI/M_w (experimental/theoretical value)	匹配肽段数/覆盖率^eMatching peptide number/coverage rate/%	分数^fScore
A1-1	Ribulose bisphosphate arboxylase	ref\|YP_004327670.1	6.09/53.0; 6.32/50.2	6/22; 28%	390
A1-2	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.49/48.9	6/47; 29%	225
A1-3	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.06/52.1	6/48; 28%	463
A2-1	Small rubber particle protein	ref\|XP_021653597.1	4.80/22.0; 4.71/25.0	13/62; 21%	94
A2-2	Small rubber particle protein	ref\|XP_021653597.1	4.80/22.0; 4.88/24.0	14/37; 38%	161
A2-3	Small rubber particle protein	ref\|XP_021653597.1	4.80/22.0; 4.65/23.0	13/62; 21%	94
A3-1	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 4.95/12.0	10/33; 30%	125
A3-2	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.08/15.0	10/33; 30%	126
A3-3	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.02/13.0	10/33; 30%	125
A4-1	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.8; 6.69/55.5	4/12; 33%	366
A4-2	Ribulose bisphosphate carboxylase	ADG02945.1	6.26/52.9; 6.89/56.3	3/9; 27%	277
A4-3	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.8; 6.64/60.2	3/9; 33%	264
B1-1	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.45/53.1	10/10; 26%	542
B1-2	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.05/52.9	6/24; 25%	497
B1-3	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.08/53.0	8/48; 28%	809
B2-1	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.61/12.8	4/12; 61%	455
B2-2	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.61/12.8	5/12; 55%	410
B2-3	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.33/20.6	8/57; 58%	558
B3-1	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.28/19.6	12/21; 34%	494
B3-2	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.21/25.3	3/18; 31%	483
B3-3	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 4.8/22.3	5/14; 38%	319
B4-1	Ribulose bisphosphate carboxylase	AFY17050.1	6.70/53.2; 6.75/55.2	9/21; 26%	663
B4-2	Ribulose bisphosphate carboxylase	ADD12830.1	6.16/52.6; 3.52/57.5	6/25; 26%	781
B4-3	Ribulose bisphosphate carboxylase	CAA04981.1	6.12/52.1; 6.89/39.7	3/13; 23%	127
C1-1	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1\|	6.09/53.0; 6.16/53.2	7/19; 26%	481
C1-2	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.42/51.5	6/23; 26%	484
C1-3	Ribulose bisphosphate carboxylase	ref\|YP_004327670.1	6.09/53.0; 6.01/53.0	6/24; 25%	441
C2-1	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.58/21.5	10/21; 74%	887
C2-2	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.8/22.3	10/21; 74%	739
C2-3	Small rubber particle protein	ref\|XP_021653597.1	4.8/22.3; 4.64/22.3	5/35; 71%	830
C3-1	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.28/19.6	5/27; 41%	519
C3-2	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.04/14.7	5/14; 46%	451
C3-3	Rubber elongation factor	ref\|XP_021653602.1	5.04/15.0; 5.04/14.7	5/14; 46%	433
C4-1	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.0; 6.03/50.6	7/42; 24%	374
C4-2	Ribulose bisphosphate carboxylase	ADG02945.1	6.26/53.0; 6.06/52.1	6/25; 17%	250
C4-3	Ribulose bisphosphate carboxylase	AAA84059.1	6.26/53.0; 6.06/52.1	6/48; 17%	388