Proteomic analysis of differentially expressed proteins between Xiangyou 15 variety and the mutant M15

doi:10.1007/s11515-014-1311-5

Front. Biol.

2014, Vol. 9

Issue (3) : 234-243 https://doi.org/10.1007/s11515-014-1311-5

RESEARCH ARTICLE

Proteomic analysis of differentially expressed proteins between Xiangyou 15 variety and the mutant M15

Zhen-Qian ZHANG^1,²,Gang XIAO^1,²,Rui-Yang LIU¹,Tai-Long TAN^1,²,Chun-Yun GUAN^1,^2,^*(

),Guo-Huai WANG^1,²,She-Yuan CHEN¹,Xian-Meng WU¹,Mei GUAN¹,Qin LI²

1. Hunan Branch of National Oilseed Crops Improvement Centre, Hunan Agricultural University, Changsha, Hunan 410128, China
2. Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization,Changsha, Hunan 410128, China

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Abstract

A high oleic acid rapeseed material M15 (derived from Xiangyou 15 variety) has been received more attention for its significant effect for human health. And it has almost the same physiological characteristic with Xiangyou 15 variety. To find out the difference between high oleic acid rapeseed material and Xiangyou 15 seedling, a comparative proteomic approach based on 2-DE and mass spectrometry was adopted. A total of 277 protein spots showed a significant change in intensity by more than 2.0-fold from M15 compared with Xiangyou 15 variety. Among them, 48 spots that changed at least 3.0-fold were excised from gels and successfully identified by MALDI-TOF/TOF MS. The identified proteins involved in metabolism of carbohydrate and energy (75%), stress and defense (8.3%), photosynthesis (6.3%), protein metabolism (2.1%) and other functions (8.3%). Then real-time quantitative PCR (qPCR) analysis was used to verify the expression levels of differentially expressed proteins, but the results did well agree with the proteomic results. In this work, most of the proteins involved in metabolism of carbohydrate and energy have higher expression in M15, which may reveal M15 has higher metabolism ability. These results provided much information to understand the differences between high oleic acid rapeseed material and Xiangyou 15 variety, which will be useful to screen high oleic rapeseed materials in seedling period.

Keywords proteomic analysis high oleic acid 2-D electrophoresis real-time quantitative PCR

Corresponding Author(s): Chun-Yun GUAN

Issue Date: 24 June 2014

Cite this article:

Tai-Long TAN,Chun-Yun GUAN,Guo-Huai WANG, et al. Proteomic analysis of differentially expressed proteins between Xiangyou 15 variety and the mutant M15[J]. Front. Biol., 2014, 9(3): 234-243.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-014-1311-5
https://academic.hep.com.cn/fib/EN/Y2014/V9/I3/234

Fig.1 The 15-day-seedling of different rapeseed. (A) The 15-day-seedling of Xiangyou 15 variety. (B) The 15-day-seedling of M15

Tab.1 Running conditions of isoelectric focusing

Tab.2 Primer sequences used in qPCR

Tab.3 The fatty acid content of rapeseed

Fig.2 Two-dimensional electrophoresis gel of separated proteins. Proteins were separated in an IPG strip (24 cm, pH 4-7) and in the second dimension on a 12% gel. (A) Xiangyou 15 variety, (B) M15.

Spot No.	Protein name	Accession no.	The species category	Protein MW	Protein PI	Fold change(M15/Xiangyou 15 variety)
Carbohydrate and energy proteins
8510	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	0.32
7513	Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit	gi\|27752813	Arabidopsislyrata subsp. lyrata	47433.9	6.13	3.45
2115	Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit	gi\|21634119	Jacquemontia reclinata	49905.1	6.71	0.31
3111	Ribulose 5-bisphosphate carboxylase large subunit	gi\|11990183	Diodia sarmentosa	51527.9	5.96	0.24
8613	Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit	gi\|52001695	Tristiropsis acutangula	50582.4	6	0.33
8408	Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit	gi\|34015063	Tristiropsis acutangula	44167.2	6.12	3.44
1106	Ribulose-bisphosphate carboxylase large subunit	gi\|17154704	Tigridia pavonia	49200.7	6.34	3.91
609	Ribulose bisphosphate	gi\|266893	Cucumber	45681.2	7.57	4.04
3205	Ribulose-1,5-bisphosphate carboxylase/oxygenase	gi\|12274881	Huperzia billardierei	49929.2	6.73	3.62
9019	Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit	gi\|13548758	Dacrycarpus imbricatus	47995.2	6.6	3.41
4107	Ribulosebiphosphate carboxylase large subunit	gi\|1079594	Pentodon	53300.7	6.22	3.06
1111	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	4.54
1003	Rubisco ssu precursor	gi\|17855	Brassica napus	20301.1	7.63	3.5
4002	Rubisco ssu precursor	gi\|17855	Brassica napus	20301.1	7.63	3.18
9702	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	0.24
4304	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	0.33
4313	Ribulose 1,5-bisphospate carboxylase	gi\|14670019	Synandrodaphne paradoxa	43687	6.25	3.87
6516	Ribulose-1,5-bisphosphate carboxylase	gi\|1050738	Pentodon pentandrus	51722	6.04	3.68
6002	Rubisco ssu precursor	gi\|17855	Brassica napus	20301.1	7.63	3.77
4003	Rubisco ssu precursor	gi\|17855	Brassica napus	20301.1	7.63	4.76
5305	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	4.21
9405	Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit	gi\|34015063	Tristiropsis acutangula	44167.2	6.12	0.25
6709	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	4.4
1617	Rubisco activase	gi\|23320705	Medicago sativa	29999.2	5.63	0.25
8414	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	0.31
7403	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	3.76
7201	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	3.66
10	Ribulose bisphosphate carboxylase	gi\|1346967	Chloroplast Brassica oleracea	52922.5	5.88	2.01
5504	Ribulose bisphosphate	gi\|266893	Cucumber	45681.2	7.57	4.09
809	ATP synthase CF1 alpha subunit	gi\|7525018	Arabidopsis thaliana	55294	5.19	3.97
3802	ATP synthase subunit β-1	gi\|26391487	Arabidopsis thaliana	59634	6.18	3.74
1710	ATP synthase subunit beta	gi\|75336517	Brassica napus	53682.9	5.21	4.05
1610	ATP synthase subunit beta	gi\|75336517	Brassica napus	53682.9	5.21	3.01
3113	Fructose-bisphosphate aldolase	gi\|42573227	Arabidopsis thaliana	41318.5	9.07	0.24
8706	β-glucosidase	gi\|1732572	Brassica nigra	50676	6.27	0.26
8705	β-glucosidase	gi\|1732572	Brassica nigra	50676	6.27	0.28
Stress and defense proteins
9701	Myrosinase	gi\|127733	Brassica napus	62695.9	6.62	0.28
3816	Myrosinase	gi\|56130949	Brassica rapa subsp. pekin	62926	6.65	4.39
5806	Myrosinase	gi\|127733	Brassica napus	62695.9	6.62	3.36
7808	Myrosinase	gi\|127733	Brassica napus	62695.9	6.62	3.38
Photosynthesis
3204	Chlorophyll a/b binding protein	gi\|18266039	Brassica oleracea	28184.1	5.17	4.13
1205	Chlorophyll a/b binding protein type I	gi\|7271945	Asarina barclaiana	18689.6	5.6	3.08
2403	HCF136 (High chlorophyll fluorescence 136)	gi\|15237225	Arabidopsis thaliana	44076.4	6.79	3.76
Protein metabolism
1511	Glutamine synthetase, chloroplastic	gi\|12643761	Brassica napus	47314.9	6.16	3.54
Other proteins
2605	Actin	gi\|32186916	Upland cotton	41700.9	5.31	4.43
8608	Putative chloroplast translation elongation factor EF-Tu	gi\|23397095	Arabidopsis thaliana	51623.6	5.84	0.31
6815	OSJNBa0042N22.23	gi\|32492228	Oryza sativa	84497	9.17	4.08
2002	Unknow protein	gi\|55168344	Oryza sativa	123805.3	7.02	4.19

Tab.4 Proteins identified by MS/MS

Fig.3 Real-time PCR analysis of the transcript levels of differentially expressed proteins.

Spot 8706, 8705, β-glucosidase(gi1732572),

Spot 809, ATP synthase CF1 alpha subunit(gil7525018), Spot 9701, 5806, 7808,, Myrosinase (gi|127733),

Spot 1511, Myrosinase(gi|12643761),

Spot 7513, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit(gi|27752813),

Spot 609 and 5504, Ribulose bisphosphate(gi|266893),.

Spot 1003, 4002, 6002 and 4003, rubisco ssu precursor(gi|17855),

Spot 1710, 1610, ATP synthase subunit beta(gi|75336517),

Spot 3113, fructose-bisphosphate aldolase(gi|42573227),

Spot 8608, putative chloroplast translation elongation factor EF-Tu(gi|23397095),

Spot 2605, actin(gi|32186916),

Spot 1617, rubisco activase(gi|23320705),

Spot 2403, High chlorophyll fluorescence 136(gi|15237225),

Spot 3204, chlorophyll a/b binding protein(gi|18266039),

Spot 1205, chlorophyll a/b binding protein type I, (gi|7271945).

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