基于微观结构和蛋白质组学分析影响猪肉持水性的差异蛋白

杨波若; 李华健; 苏娅宁; 李霞; 瞿静; 陈韬

doi:10.13386/j.issn1002-0306.2020070148

基于微观结构和蛋白质组学分析影响猪肉持水性的差异蛋白

doi: 10.13386/j.issn1002-0306.2020070148

云南农业大学食品科学技术学院，云南昆明 650201

基金项目: 国家自然科学基金地区项目（31660441）

详细信息

作者简介:
杨波若（1994−），女，硕士研究生，研究方向：畜产品加工与品质控制研究，E-mail：yangboruo@163.com

通讯作者:
陈韬（1963−），男，博士，教授，研究方向：畜产品加工与品质控制研究，E-mail：chentao63@163.com

中图分类号: TS251.1
计量
- 文章访问数: 18
- HTML全文浏览量: 13
- PDF下载量: 3
- 被引次数: 0
出版历程
- 收稿日期: 2020-07-14
- 网络出版日期: 2021-01-28
- 刊出日期: 2021-04-01

Analysis of Different Proteins Affecting Water Holding Capacity of Pork Based on Microstructure and Proteomics

College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China

摘要

摘要: 为探究宰后初期生鲜猪肉肌细胞微观结构和蛋白质组变化对持水能力的影响，将猪背最长肌样品按照汁液流失的高低分为高汁液流失组（High drip loss group≥5.93%, H组, n=3）和低汁液流失组（Low drip loss group≤0.81%, L组, n=3），对两组样品的微观结构和蛋白质组进行比较。采用透射电镜（Transmission Electron Microscopy, TEM）观测细胞间隙，并用多肽体外标记技术（Tandem Mass Tag, TMT）鉴定高低汁液流失组间的差异蛋白。结果表明，宰后24 h时，H组的细胞外间隙极显著大于L组的细胞外间隙（P<0.01）。宰后肌肉中葡萄糖磷酸变位酶-1、热休克蛋白70（Heat shock protein 70, Hsp70）、锚蛋白、硒蛋白W和层黏连蛋白的表达量越高，汁液流失越低，持水性越好，而磷酸甘油变位酶和转酮醇酶的表达量越高，汁液流失越高，持水性越差。
- 猪肉 /
- 蛋白质组学 /
- 汁液流失 /
- 生物代谢 /
- 转酮醇酶
Abstract: In order to investigate the effects of microstructural and proteomic changes of fresh pork on their water holding capacity in the early postmortem period, the longissimus dorsi samples of pigs were divided into high drip loss group (H-group≥5.93%, n=3) and low drip loss group (L-group≤0.81%, n=3) according to the level of drip loss. The microstructure and proteomics of the two groups were compared. The difference proteins between the two groups were identified using Tandem Mass Tag (TMT) and intercellular spaces were observed using electron microscopy. The results showed that the extracellular space of H-group was significantly larger than that of L-group at 24 h after slaughter(P<0.01). The higher the expression level of glucose-phosphotransferase-1, heat shock protein 70 (Hsp70), ankyrin, selenoprotein W and laminin in postmortem muscle, the lower the drip loss and the better the water holding capacity, while the higher the expression level of phosphoglycerate mutase and transketolase, the higher the drip loss and the worse the water holding capacity.
- pork /
- proteomics /
- drip loss /
- biological metabolism /
- transketolase

HTML全文

图 1 宰后9 h和24 h时的两组不同汁液流失率肉样微观图片

注：A：宰后9 h的L组细胞间隙；B：宰后24 h的L组细胞间隙；C：宰后9 h的H组细胞间隙；D：宰后24 h的H组细胞间隙；A～D图中的箭头指向是肌细胞膜分离的位置；黑色的圆圈内为肌原纤维间。

Figure 1. Microscopic photos of two groups of meat samples with different drip loss at 9 h and 24 h after slaughter

下载: 全尺寸图片幻灯片

图 2 差异蛋白的GO功能分类图

Figure 2. Identification of the differential proteins by GO functional classification

下载: 全尺寸图片幻灯片

图 3 差异蛋白的KEGG通路的富集图

Figure 3. Identification of the differential proteins by KEGG pathway enrichment analysis

下载: 全尺寸图片幻灯片

图 4 宰后差异蛋白的互作网络图

Figure 4. Identification of the differential proteins by interaction network diagram

下载: 全尺寸图片幻灯片

表 1 宰后存放在4 ℃下9 和24 h的H组与L组的样品的肉质指标

Table 1. Meat quality indicators of samples in the H-group and L-group after slaughter at 4 ℃ for 9 and 24 h

指标	汁液流失率
指标	L组（n=3）	H组（n=3）
汁液流失率(%)	0.81 ± 0.14	5.93 ± 0.22^**
肌间脂肪（%）	3.26 ± 0.06	3.01 ± 0.05^*
pH_{9 h}	5.74 ± 0.12	5.52 ± 0.01^*
pH_{24 h}	5.78 ± 0.07	5.62 ± 0.03
温度_{9 h}（℃）	7.03 ± 0.07	7.13 ± 0.86
温度_{24 h}（℃）	6.03 ± 1.35	7.08 ± 0.56^**
细胞内间隙_{9 h}	0.32 ± 0.10	0.39 ± 0.11
细胞内间隙_{24 h}	0.54 ± 0.26	0.66 ± 0.29
细胞外间隙_{9 h}	2.39 ± 1.25	2.93 ± 0.65
细胞外间隙_{24 h}	2.43 ± 0.74	5.21 ± 1.39^**
注：^表示L组与H组组间差异显著，P<0.05；^*表示L组与H组组间差异极显著，P<0.01；细胞内间隙和细胞外间隙单位为微米（μm）。

下载: 导出CSV

表 2 宰后背最长肌中细胞内外间隙与汁液流失率的相关性

Table 2. Correlation between intracellular and extracellular spaces and drip loss rate in the longissimus dorsi muscle

	细胞内间隙		细胞外间隙
时间（h)	9	24	9	24
汁液流失率（%）	0.536	0.804	0.598	0.882^*
注：^*表示在0.05 水平（双侧）上显著相关。

下载: 导出CSV

表 3 宰后9 h和24 h高低汁液流失组存在显著差异的57个蛋白

Table 3. 57 proteins with significant differences in high and low drip loss groups were identified at 9 h and 24 h postmortem

蛋白检索号^a	蛋白质名称^a	基因检索号^a	物种^a	蛋白得分/肽段	质谱得分（%）	差异倍数
蛋白检索号^a	蛋白质名称^a	基因检索号^a	物种^a	蛋白得分/肽段	质谱得分（%）	9 h	24 h
F1SRI8	肌球蛋白结合蛋白C	MYBPC1	Sus scrofa	323.31/69	62.4	1.32^b	1.22^c
F1RZM4	层粘连蛋白亚基α4	LAMA4	Sus scrofa	52.118/5	0.042	1.08^c	1.21^c
B5KJG2	磷酸甘油酸酯变位酶	PGAM2	Sus scrofa	323.31/18	63.6	1.21^b	1.24^b
F1S814	葡萄糖磷酸变位酶1	PGM1	Sus scrofa	323.31/30	56.6	1.34^c	1.23^c
A0A287ARW1	肌钙蛋白T	TNNT3	Sus scrofa	11.937/21	45.1	1.23^c	1.35^b
A0A287BHG2	肌钙蛋白T	TNNT3	Sus scrofa	323.31/21	47.3	1.61^b	1.15^b
Q6S4N2	热休克蛋白70	HSPA1B	Sus scrofa	323.31/23	44.3	1.21^c	1.19^c
A0A287ANE3	LIM和衰老细胞含抗原样结构域蛋白	MYOM3	Sus scrofa	323.31/26	25.6	1.35^c	1.01^c
I3LIE7	结合蛋白H	MYBPH	Sus scrofa	323.31/16	50.7	1.72^c	1.10^c
F2Z5S8	微管蛋白α链	TUBA4A	Sus scrofa	278.04/15	47	1.25^c	1.11^c
P43368	钙蛋白酶-3	CAPN3	Sus scrofa	225.79/19	30.2	1.22^c	1.21^c
K7GQ48	巨球蛋白α2	A2M	Sus scrofa	170.36/17	15.1	1.22^b	1.14^b
I3LN42	维生素D结合蛋白	GC	Sus scrofa	267.86/11	28.5	1.24^b	1.04^b
A0A287BTD0	LIM和衰老细胞含抗原样结构域蛋白	LIMS1	Sus scrofa	25.352/3	11.4	1.33^c	1.03
P01846	链C区域	N/A	Sus scrofa	141.98/5	73.3	1.58^b	1.13^b
I3L9X0	冠蛋白	CORO6	Sus scrofa	80.9/5	14.3	1.29^b	1.01^c
F1S431	丙氨酰	AARS	Sus scrofa	25.266/4	4.9	1.21^b	1.02^b
F2Z4Y0	小核核糖核蛋白	SNRPD3	Sus scrofa	25.11/2	23.8	1.20^c	1.04^c
P36968	磷脂过氧化氢谷胱甘肽过氧化物酶	GPX4	Sus scrofa	19.056/3	14.7	1.49^c	1.66^c
Q29197	40号核糖体蛋白	RPS9	Sus scrofa	17.985/3	18.5	3.26^b	/
I3L804	酪氨酸- tRNA连接酶	YARS	Sus scrofa	17.626/3	7.2	1.20^b	1.12^b
I3LB80	溶质载体家族3成员2	SLC3A2	Sus scrofa	39.458/3	7.8	1.53^c	1.35^b
K7GRK7	腱生蛋白-X	TNXB	Sus scrofa	31.477/3	1.8	1.95^b	/
Q07717	微球蛋白β2	B2M	Sus scrofa	12.148/2	16.9	1.28^b	/
F1RQC1	具有序列相似性的家族114成员A2	FAM114A2	Sus scrofa	15.059/2	6.4	1.35^c	1.35^b
F2Z5N9	蛋白质回力球同族体	PELO	Sus scrofa	14.704/2	6.8	1.22^b	1.47^b
Q95KL4	硒蛋白W	SELENOW	Sus scrofa	11.99/2	24.1	3.55^c	2.74^c
A0A287A781	中心体蛋白85	CEP85	Sus scrofa	6.7513/1	1	1.73^b	/
A0A287A816	磷脂磷酸酶7（非活性）	PLPP7	Sus scrofa	6.4318/1	6.3	1.90^b	1.24^b
F1RN28	旁斑区域1	PSPC1	Sus scrofa	9.2318/1	5.9	1.46^b	1.47^b
I3LPU8	异质核核糖核蛋白L	HNRNPL	Sus scrofa	6.4381/1	4	1.38^b	1.08^c
F1SSL4	ATP结合区域	ABCF2	Sus scrofa	104.22/3	5.8	1.03^b	1.37^b
A0A287BI36	PDZ和LIM区域5	PDLIM5	Sus scrofa	59.067/7	38.3	1.07^b	1.37^c
F1SAW8	肌集钙蛋白	CASQ2	Sus scrofa	40.941/2	6.8	1.31^b	1.35^b
F1RYZ1	跨膜四蛋白	CD151	Sus scrofa	91.064/1	3.2	1.04^b	1.23^c
F2Z5N9	蛋白质回力球同族体	PELO	Sus scrofa	74.392/2	5	1.22^b	1.47^b
A0A286ZTL8	层粘连蛋白β2	LMNB2	Sus scrofa	56.139/5	9.1	1.02^c	1.26^c
P62844	40S核糖体蛋白	RPS1	Sus scrofa	45.28/2	9	1.13^b	1.78^b
A0A287B3D6	支链氨基酸氨基转移酶	BCAT2	Sus scrofa	125.22/2	9.1	1.08^b	1.30^b
A0A287ACR8	N(α)-乙酰转移酶50,NatE催化亚基	NAA50	Sus scrofa	34.773/2	14.3	1.02^b	1.39^b
F1RJ93	转凝蛋白	TAGLN2	Sus scrofa	90.15/3	18.1	1.06^b	1.06^b
D6QST6	2,4-dienoyl-CoA还原酶1	DECR1	Sus scrofa	58.699/2	5.18	1.25^b	1.34^b
F1SKI0	肌球蛋白-11	MYH11	Sus scrofa	58.433/5	3.2	1.07^c	1.49^c
A0A287AAD5	Alpha-1B-糖蛋白α1	A1BG	Sus scrofa	59.067/4	9.2	1.36^b	1.23^c
F1RLL9	IV型- 2型胶原链	COL4A2	Sus scrofa	49.715/4	3.1	1.05^b	1.26^b
A0A287B1F9	溶质载体家族12名成员2	SLC12A2	Sus scrofa	110.08/1	1	1.05^c	1.23^b
A0A286ZLW9	磷酸肌醇磷脂酶C	PLCL2	Sus scrofa	29.58/1	1.6	1.14^b	1.05^c
P43368	钙蛋白酶-3	CAPN3	Sus scrofa	96.299/9	14.3	1.22^c	1.21^b
A5A8V7	热休克蛋白70	HSPA1L	Sus scrofa	55.353/8	12.5	1.07^c	4.70^c
D2ST34	富亮氨酸重复蛋白4	FBXL4	Sus scrofa	446.001/1	1.6	/	2.73^b
F1SJ30	6-磷酸甘露糖异构酶	MPI	Sus scrofa	45.433/5	17.5	1.23^b	1.23^b
A0A287BEP2	锚蛋白1	ANK1	Sus scrofa	79.809/1	0.6	/	1.32^c
I3LB80	溶质载体家族3成员2	SLC3A2	Sus scrofa	48.091/2	5.2	2.64^b	3.30^c
A0A287A5C9	信号转换器和转录激活器	STAT1	Sus scrofa	46.88/2	4.3	2.59^b	2.77^c
A8U4R4	转酮醇酶	TKT	Sus scrofa	74.533/1	1.4	2.50^b	1.60^b
注：a: 从Uniprot数据库中提取蛋白序列、蛋白名称和基因；b: H组质谱峰面积度与L组质谱峰面积的比值，即该蛋白在H组中的表达量高；c: L组质谱峰面积度与H组质谱峰面积的比值，即该蛋白在L组中的表达量高。

下载: 导出CSV

参考文献(38)

[1]	Diniz W J, Banerjee P, Regitano L C, et al. Cross talk between mineral metabolism and meat quality: A systems biology overview[J]. Physiological Genomics,2019,51(11):529−538. doi: 10.1152/physiolgenomics.00072.2019
[2]	肖智超, 王桂瑛, 王雪峰, 等. 云南盐津乌骨鸡与武定鸡肌肉蛋白质组学差异研究[J]. 食品工业科技,2019,40(16):102−106, 117.
[3]	马静, 武开乐, 库西塔别克·买买提依不拉音, 等. iTRAQ技术在动物生产中的应用[J]. 中国细胞生物学学报,2017,39(12):1599−1604.
[4]	金绍明, 宁霄, 曹进, 等. 非标记蛋白定量方法在掺假肉鉴别中的应用[J]. 食品安全质量检测学报,2019,10(1):71−74. doi: 10.3969/j.issn.2095-0381.2019.01.012
[5]	牟永颖, 顾培明, 马博, 等. 基于质谱的定量蛋白质组学技术发展现状[J]. 生物技术通报,2017,33(9):73−84.
[6]	赵雅娟, 苏琳, 尹丽卿, 等. 蛋白质组学技术在肉品质中的研究进展[J]. 食品工业,2016,37(4):233−236.
[7]	Lee S H, Kim J M, Ryu Y C, et al. Effects of morphological characteristics of muscle fibers on porcine growth performance and pork quality[J]. Korean Journal for Food Science of Animal Resources,2016,36(5):583−593. doi: 10.5851/kosfa.2016.36.5.583
[8]	杨汝男, 李燕清, 陈韬. 宰后猪最长肌踝蛋白降解与汁液流失的关系[J]. 食品工业科技,2018,39(20):12−17.
[9]	Wang Z, He F, Rao W, et al. Proteomic analysis of goat longissimus dorsi muscles with different drip loss values related to meat quality traits[J]. Food Science & Biotechnology,2016,25(2):425−431.
[10]	Pas M F W T, Kruijt L, Pierzchala M, et al. Identification of proteomic biomarkers in longissimus dorsi as potential predictors of pork quality[J]. Meat Science,2013,95(3):679−687. doi: 10.1016/j.meatsci.2012.12.015
[11]	Luca A D, Mullen A M, Elia G, et al. Centrifugal drip is an accessible source for protein indicators of pork ageing and water-holding capacity[J]. Meat Science,2011,88(2):261−270. doi: 10.1016/j.meatsci.2010.12.033
[12]	Gap-Don, Kim, Jin-Yeon, et al. Differential abundance of proteome associated with intramuscular variation of meat quality in porcine longissimus thoracis et lumborum muscle[J]. Meat Science,2019:85−95.
[13]	Sandberg A, Brance R M, Lehtio J, et al. Quantitative accuracy in mass spectrometry based proteomics of complex samples: The impact of labeling and precursor interference[J]. Journal of Proteomics,2014:133−144.
[14]	Warner R D, Kaufman R G, Greaser M L. Muscle protein changes post mortem in relation to pork quality traits[J]. Meat Science,1997,45(3):339−352. doi: 10.1016/S0309-1740(96)00116-7
[15]	Florowski T, Pisula A, Rola M, et al. Comparison of meatiness and the technological quality of pork from Polish Pulawy breed and its crosses with Polish Large White and Polish Landrace pigs[J]. Meat Science,2008,64(5):673−676.
[16]	Honikel K O. Reference methods for the assessment of physical characteristics of meat[J]. Meat Science,1998,49(4):447−457. doi: 10.1016/S0309-1740(98)00034-5
[17]	Luo X, Zhu Y, Zhou G. Electron microscopy of contractile bands in low voltage electrical stimulation beef[J]. Meat Science,2008,80(3):948−951. doi: 10.1016/j.meatsci.2008.03.017
[18]	吴霜, 陈韬, 张冬怡, 等. 猪屠宰后正常肉与PSE肉中整联蛋白变化与持水性的相关性[J]. 食品工业科技,2015,36(21):64−67, 72.
[19]	Schäfer A, Rosenvold K, Purslow P. Physiological and structural events post mortem of importance for drip loss in pork[J]. Meat Science,2002,61(4):355−366. doi: 10.1016/S0309-1740(01)00205-4
[20]	雄安秀. 锚蛋白研究进展[J]. 国外医学(儿科分册),2003(3):159−161.
[21]	Aslan O, Hamill R M, Mullen A M, et al. Association between promoter polymorphisms in a key cytoskeletal gene (Ankyrin 1) and intramuscular fat and water-holding capacity in porcine muscle[J]. Molecular Biology Reports,2012,39(4):3903−3914. doi: 10.1007/s11033-011-1169-4
[22]	王州. 基于重测序研究贵州地方住猪种基因组拷贝数变异[D]. 贵阳: 贵州大学, 2019.
[23]	Ceasar A, Regitano L C, Koltes J E, et al. Putative regulatory factors associated with intramuscular fat content[J]. Plos One,2015,10(6):1−21.
[24]	Watanabe G, Motoyama M, Nakajima I, et al. Relationship between water-holding capacity and intramuscular fat content in Japanese commercial pork loin[J]. Asian Journal of Animal Sciences,2017,31(6):914−918.
[25]	宋林霞, 徐振彪. 磷酸甘油酸变位酶[J]. 生命的化学,2011(1):90−93.
[26]	Koning D, Harlizius B, Rattink A P, et al. Detection and characterization of quantitative trait loci for meat quality traits in pigs[J]. Journal of Animal Science,2001,79(11):2812−2819. doi: 10.2527/2001.79112812x
[27]	Yang H, He J, Wei W, et al. The c. −360 T>C mutation affects PGAM2 transcription activity and is linked with the water holding capacity of the longissimus lumborum muscle in pigs[J]. Meat Science,2016,122(12):139−144.
[28]	Prejano M, Medinal F E, Fernandes P A, et al. The catalytic mechanism of human transketolase[J]. Chem Phys Chem,2019,20(21):2881−2886. doi: 10.1002/cphc.201900650
[29]	Kochetov G A. et al. Structure and functioning mechanism of transketolase[J]. Proteins & Proteomics,2014,61(12):59−68.
[30]	谢华. 冷却猪肉汁液流失及控制研究[D]. 杨凌: 西北农林科技大学, 2006.
[31]	Trocino A, Zomeno C, Birolo M, et al. Impact of pre-slaughter transport conditions on stress response, carcass traits, and meat quality in growing rabbits[J]. Meat Science,2018:68−74.
[32]	Hu H, Chen L, Dai S, et al. Effect of glutamine on antioxidant capacity and lipid peroxidation in the breast muscle of heat-stressed broilers via antioxidant genes and Hsp70 pathway[J]. Animals,2020,10(3):404. doi: 10.3390/ani10030404
[33]	Wei Y, Li X, Zhang D, et al. Comparison of protein differences between high-and low-quality goat and bovine parts based on iTRAQ technology[J]. Food Chemistry,2019,289(AUG. 15):240−249.
[34]	Traore S, Aubry L, Gatellier P, et al. Higher drip loss is associated with protein oxidation[J]. Meat Science,2011,90(4):917−924.
[35]	Liu Z, Xiong Y L, Chen J. Protein oxidation enhances hydration but suppresses water-holding capacity in porcine longissimus muscle[J]. Journal of Agricultural & Food Chemistry,2010,58(19):10697−10704.
[36]	Jeong D W, Kim E H, Kim T S, et al. Different distributions of selenoprotein W and thioredoxin during postnatal brain development and embryogenesis[J]. Molecules and Cells,2004,17(1):156−159.
[37]	Li Jungang. Enhanced water-holding capacity of meat was associated with increased Sepw1 gene expression in pigs feed selenium-enriched yeast[J]. Meat Science,2011,13(1):101−112.
[38]	Zhang M, Wang D, Xu X, et al. Comparative proteomic analysis of proteins associated with water holding capacity in goose muscles[J]. Food Research International,2019:354−361.