Effects of Dietary Protein Deficiency Followed by Realimentation on the Antioxidation of Lamb Based on Transcriptomics Analysis

doi:10.13560/j.cnki.biotech.bull.1985.2021-0224

Abstract

Abstract:

This study is aimed to investigate the effects of dietary protein deficiency followed by realimentation on the antioxidation of lamb based on RNA-seq analysis at transcriptome level. Thirteen-two Hu twin lambs weaned on 15 d were selected and randomly divided into two groups. The control group was fed milk replacer(CP 25%)and starter(CP 21%)of normal protein level(NPL),and the other group fed milk replacer(19%)and starter(15%)of low protein level(LPL). Milk replacer was fed from weaning to d 60,and starter was freely taken. LPL group of 60-90 d freely took the starter of normal protein level(CP 21%). Two 60 and 90 d lambs in each group were selected and slaughtered for tissue collection. RNA-seq technology was used to identify the difference expression genes(DEGs)in liver tissue under dietary protein deficiency followed by realimentation,to screen pathways closely related to antioxidant function through bioinformatics analysis,and to verify the expressions of genes of antioxidant enzymes using qRT-PCR method. Low level protein diet led to 302 differentially expressed genes(DEGs)in the sheep liver and 23 down regulated genes involved in antioxidant process. GO analysis showed that DEGs were mainly involved in oxidoreductase activity(GO:0016491),glutathione transferase activity(GO:0004364)and oxidation-reduction process(GO:0055114). KEGG analysis discovered that antioxidant related genes were enriched in metabolism of xenobiotics by cytochrome P450 and glutathione metabolism. The expression of genes involved in cytokine-cytokine receptor interaction was significantly up-regulated. Compared with NPL group,the expressions of genes related to antioxidation showed no significant difference after protein realimentation for 30 d. In conclusion,protein deficiency led to the changes of liver gene expression,decreased the expressions of genes related to antioxidation. The DEGs affect the antioxidation and immune function of liver via pathways of xenobiotics metabolism,glutathione metabolism,and cytokine-cytokine receptor.

Key words: protein realimentation, lamb, RNA-seq, antioxidation

KANG Ling-yun, CHEN Jian-sheng, GAN Han-ling, HAN Lu-lu, FENG Hai-xia, DIAO Qi-yu, XING Kai, CUI Kai. Effects of Dietary Protein Deficiency Followed by Realimentation on the Antioxidation of Lamb Based on Transcriptomics Analysis[J]. Biotechnology Bulletin, 2021, 37(6): 171-180.

Figures/Tables 9

References 30

[1]	Pamplona R, Barja G. Mitochondrial oxidative stress, aging and caloric restriction:The protein and methionine connection[J]. Biochim et Biophys Acta BBA Bioenerg, 2006, 1757(5/6):496-508.
[2]	徐少庭, 徐晨晨, 罗海玲. 饲粮抗氧化剂对肌肉嫩度的影响及作用机制[J]. 动物营养学报, 2017, 29(8):2676-2680.
	Xu ST, Xu CC, Luo HL. Effects and mechanisms of dietary antioxidants on meat tenderness[J]. Chin J Animal Nutr, 2017, 29(8):2676-2680.
[3]	Sohal RS, Ku HH, Agarwal S, et al. Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse[J]. Mech Ageing Dev, 1994, 74(1/2):121-133. doi: 10.1016/0047-6374(94)90104-X URL
[4]	He ZX, Sun ZH, Tan ZL, et al. Effects of maternal protein or energy restriction during late gestation on antioxidant status of plasma and immune tissues in postnatal goats[J]. J Anim Sci, 2012, 90(12):4319-4326. doi: 10.2527/jas.2012-5088 pmid: 22952363
[5]	张帆, 崔凯, 王杰, 等. 妊娠后期母羊饲粮营养水平对产后羔羊生长性能、器官发育和血清抗氧化指标的影响[J]. 动物营养学报, 2017, 29(2):636-644.
	Zhang F, Cui K, Wang J, et al. Effects of maternal dietary nutrient level in late gestation on growth performance, organ development and serum antioxidant capacity of postpartum lambs[J]. Chin J Animal Nutr, 2017, 29(2):636-644.
[6]	谷春梅, 施用晖, 乐国伟. 高蛋白日粮对小鼠胰腺活性氧自由基产生的影响[J]. 吉林农业大学学报, 2007, 29(6):679-682.
	Gu CM, Shi YH, Le GW. Effect of high protein diet on generation of reactive oxygen species in pancreas of mice[J]. J Jilin Agric Univ, 2007, 29(6):679-682.
[7]	李东东, 李宗锐, 丁雪梅, 等. 不同粗蛋白质水平饲粮添加外源蛋白酶对肉鸡生产性能、血清生化指标和抗氧化功能的影响[J]. 动物营养学报, 2015, 27(9):2820-2831.
	Li DD, Li ZR, Ding XM, et al. Effects of different crude protein levels diet supplemented with exogenous protease on performance, serum biochemical indices and antioxidant function of broilers[J]. Chin J Animal Nutr, 2015, 27(9):2820-2831.
[8]	耿红红, 张敬旸, 李莲, 等. RNA-seq转录组测序分析不同季节对槟榔江水牛精液品质的影响[J]. 畜牧兽医学报, 2016, 47(7):1373-1380.
	Geng HH, Zhang JY, Li L, et al. Semen quality analysis of betelnut-Jiang buffalo in different seasons by RNA-seq[J]. Chin J Animal Vet Sci, 2016, 47(7):1373-1380.
[9]	孟宪然, 杜琛, 王静, 等. 基于RNA-Seq识别山羊肉品质候选基因[J]. 畜牧兽医学报, 2015, 46(8):1300-1307.
	Meng XR, Du C, Wang J, et al. RNA-seq approach for identifying candidate genes of meat quality in goats[J]. Chin J Animal Vet Sci, 2015, 46(8):1300-1307.
[10]	崔凯, 吴伟伟, 刁其玉. 转录组测序技术的研究和应用进展[J]. 生物技术通报, 2019, 35(7):1-9.
	Cui K, Wu WW, Diao QY. Application and research progress on transcriptomics[J]. Biotechnol Bull, 2019, 35(7):1-9.
[11]	屠焰, 刁其玉, 岳喜新. 一种0-3月龄羔羊的代乳品及其制备方法:CN102894218B[P], 2013-01-30.
	Tu Y, Diao QY, Yue XX. Milk replacer of 0-3 month lambs and production method thereof:CN102894218B[P], 2013-01-30.
[12]	王桂秋. 营养水平对羔羊物质消化的影响及羔羊早期断奶时间的研究[D]. 北京:中国农业科学院, 2005.
	Wang GQ. Study on nutrition utilization of lambs when fed milk replacers of different nutrition levels and early-weaned time of lambs[D]. Beijing:Chinese Academy of Agricultural Sciences, 2005.
[13]	中华人民共和国农业部. 中华人民共和国农业行业标准:肉羊饲养标准 NY/T 816—2004[S]. 北京: 中国农业出版社, 2004.
	Ministry of Agriculture of the People’s Republic of China. Agriculture Standard of the People’s Republic of China:Feeding standard of meat-producing sheep and goats. NY/T 816—2004[S]. Beijing: Chinese Agriculture Press, 2004.
[14]	Trapnell C, Roberts A, Goff L, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks[J]. Nat Protoc, 2012, 7(3):562-578. doi: 10.1038/nprot.2012.016 pmid: 22383036
[15]	Wang L, Feng Z, Wang X, et al. DEGseq:an R package for identifying differentially expressed genes from RNA-seq data[J]. Bioinformatics, 2010, 26(1):136-138. doi: 10.1093/bioinformatics/btp612 URL
[16]	Trapnell C, Williams BA, Pertea G, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J]. Nat Biotechnol, 2010, 28(5):511-515. doi: 10.1038/nbt.1621 pmid: 20436464
[17]	Young MD, Wakefield MJ, Smyth GK, et al. Gene ontology analysis for RNA-seq:accounting for selection bias[J]. Genome Biol, 2010, 11(2):R14. doi: 10.1186/gb-2010-11-2-r14 URL
[18]	Mao X, Cai T, Olyarchuk JG, et al. Automated genome annotation and pathway identification using the KEGG Orthology(KO)as a controlled vocabulary[J]. Bioinformatics, 2005, 21(19):3787-3793. doi: 10.1093/bioinformatics/bti430 URL
[19]	Betteridge DJ. What is oxidative stress?[J]. Metab:Clin Exp, 2000, 49(Suppl 1):3-8.
[20]	Schwerin M, Kurts-Ebert B, Beyer M, et al. Temporary consumption of diet with unbalanced amino acid pattern affects long-lasting growth retardation correlated with oxidative stress response associated gene expression in juvenile pigs[J]. Clin Nutr, 2008, 27(5):781-789. doi: 10.1016/j.clnu.2008.06.010 pmid: 18692284
[21]	Lykkesfeldt J, Svendsen O. Oxidants and antioxidants in disease:oxidative stress in farm animals[J]. Vet J, 2007, 173(3):502-511. pmid: 16914330
[22]	Benz CC, Yau C. Ageing, oxidative stress and cancer:paradigms in parallax[J]. Nat Rev Cancer, 2008, 8(11):875-879. doi: 10.1038/nrc2522 URL
[23]	Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress[J]. Curr Biol, 2014, 24(10):R453-R462. doi: 10.1016/j.cub.2014.03.034 URL
[24]	肖文霞, 马秀菊. 氧化应激与妊娠[J]. 国外医学妇幼保健分册, 2005, 16(3):142-144.
	Xiao WX, Ma XJ. Oxidative stress and pregnancy[J]. Foreign Med Sci Sect Matern Child Heal, 2005, 16(3):142-144.
[25]	Kurata M, Suzuki M, Agar NS. Antioxidant systems and erythrocyte life-span in mammals[J]. Comp Biochem Physiol B, 1993, 106(3):477-487. doi: 10.1016/0305-0491(93)90121-K URL
[26]	Miller JK, Brzezinska-Slebodzinska E, Madsen FC. Oxidative stress, antioxidants, and animal function[J]. J Dairy Sci, 1993, 76(9):2812-2823. pmid: 8227685
[27]	Wang J, Chen L, Li P, et al. Gene expression is altered in piglet small intestine by weaning and dietary glutamine supplementation[J]. J Nutr, 2008, 138(6):1025-1032. doi: 10.1093/jn/138.6.1025 URL
[28]	Thakare VN, Aswar MK, Kulkarni YP, et al. Silymarin ameliorates experimentally induced depressive like behavior in rats:Involvement of hippocampal BDNF signaling, inflammatory cytokines and oxidative stress response[J]. Physiol Behav, 2017, 179:401-410. doi: S0031-9384(16)30738-7 pmid: 28711395
[29]	姚金晶, 陈宜涛. Th1/Th2平衡调节与疾病发生的研究进展[J]. 现代生物医学进展, 2009, 9(13):2597-2600.
	Yao JJ, Chen YT. Advances of regulation Th1/Th2 type cytokines balance in human diseases[J]. Prog Mod Biomed, 2009, 9(13):2597-2600.
[30]	De la Fuente M. Effects of antioxidants on immune system ageing[J]. Eur J Clin Nutr, 2002, 56(Suppl 3):S5-S8. doi: 10.1038/sj.ejcn.1601476 URL

项目Item	饲粮Diet
	开食料Starter		代乳粉Milk replacer
	NPL组	LPL组	NPL组	LPL组
成分Ingredients/%
玉米Corn	49.10	65.90	-	-
豆粕Soybean	28.90	12.10	-	-
麸皮Bran	8.00	8.00	-	-
苜蓿Alfalfa	10.00	10.00	-	-
预混料Premix	4.00	4.00	-	-
总计Total	100.00	100.00	-	-
营养水平Nutrient level/%
干物质DM	89.65	90.36	97.73	97.94
粗蛋白CP	21.08	15.02	25.08	19.23
代谢能DE(MJ·kg^-1)	13.06	13.06	18.38	18.32
粗脂肪EE	1.70	1.70	11.18	12.98
粗灰分Ash	7.40	6.50	5.29	4.85
中性洗涤纤维NDF	15.46	14.79	-	-
酸性洗涤纤维ADF	7.84	7.47	-	-
钙Ca	0.96	0.98	1.13	1.09
磷P	0.57	0.51	0.51	0.48

项目Item	饲粮Diet
	开食料Starter		代乳粉Milk replacer
	NPL组	LPL组	NPL组	LPL组
成分Ingredients/%
玉米Corn	49.10	65.90	-	-
豆粕Soybean	28.90	12.10	-	-
麸皮Bran	8.00	8.00	-	-
苜蓿Alfalfa	10.00	10.00	-	-
预混料Premix	4.00	4.00	-	-
总计Total	100.00	100.00	-	-
营养水平Nutrient level/%
干物质DM	89.65	90.36	97.73	97.94
粗蛋白CP	21.08	15.02	25.08	19.23
代谢能DE(MJ·kg^-1)	13.06	13.06	18.38	18.32
粗脂肪EE	1.70	1.70	11.18	12.98
粗灰分Ash	7.40	6.50	5.29	4.85
中性洗涤纤维NDF	15.46	14.79	-	-
酸性洗涤纤维ADF	7.84	7.47	-	-
钙Ca	0.96	0.98	1.13	1.09
磷P	0.57	0.51	0.51	0.48

分组 Group	样品名 Sample	原始序列 Raw reads	过滤后序列 Clean reads	过滤后碱基数量Clean bases	Q20比例Q20 ratio /%	Q30 比例Q30/%	GC含量 GC content /%
NPL60	NP1	59304426	57538562	8.63G	96.40	91.31	49.26
NPL60	NP2	52921108	50561072	7.58G	95.47	89.64	47.87
LPL60	LP1	55080900	53703926	8.06G	96.61	91.75	50.04
LPL60	LP2	41189720	40057846	6.01G	96.33	91.07	48.88
NPL90	NP3	62798110	60592144	9.09G	96.13	90.98	48.40
NPL90	NP4	42917164	41617614	6.24G	96.41	91.44	48.88
LPL90	LP3	54828428	53056652	7.96G	96.65	91.91	49.26
LPL90	LP4	60109450	58311854	8.75G	96.23	91.03	49.35

分组 Group	样品名 Sample	原始序列 Raw reads	过滤后序列 Clean reads	过滤后碱基数量Clean bases	Q20比例Q20 ratio /%	Q30 比例Q30/%	GC含量 GC content /%
NPL60	NP1	59304426	57538562	8.63G	96.40	91.31	49.26
NPL60	NP2	52921108	50561072	7.58G	95.47	89.64	47.87
LPL60	LP1	55080900	53703926	8.06G	96.61	91.75	50.04
LPL60	LP2	41189720	40057846	6.01G	96.33	91.07	48.88
NPL90	NP3	62798110	60592144	9.09G	96.13	90.98	48.40
NPL90	NP4	42917164	41617614	6.24G	96.41	91.44	48.88
LPL90	LP3	54828428	53056652	7.96G	96.65	91.91	49.26
LPL90	LP4	60109450	58311854	8.75G	96.23	91.03	49.35

基因ID Gene ID	基因名称 Gene symbol	基因描述 Gene description	差异倍数 Fold change	P
ENSOARG00000000042	CYP2E1	Cytochrome P450,family 2,subfamily E,polypeptide 1	-1.75	0.0424
ENSOARG00000000591	LOC101105314	Cytochrome P450 2C42-like	-6.74	0.0180
ENSOARG00000004562	ZADH2	Zinc binding alcohol dehydrogenase domain containing 2	-1.77	0.0433
ENSOARG00000005352	-	-	-1.91	0.0314
ENSOARG00000005747	-	-	-3.17	0.0032
ENSOARG00000007128	CYP2A6	Cytochrome P450 2A6	-2.59	0.0010
ENSOARG00000007880	SOD1	Superoxide dismutase 1	-2.69	0.0267
ENSOARG00000009277	LOC101107119	Prostaglandin F synthase 1-like	-2.61	0.0009
ENSOARG00000011087	LOC101109111	Dihydrodiol dehydrogenase 3	-2.02	0.0140
ENSOARG00000011663	--	-	-3.26	0.0001
ENSOARG00000011769	PYROXD2	Pyridine nucleotide-disulphide oxidoreductase domain 2	-2.32	0.0085
ENSOARG00000012581	TM7SF2	Transmembrane 7 superfamily member 2	-2.50	0.0013
ENSOARG00000012705	ALDH1A1	Aldehyde dehydrogenase 1 family member A1	-2.40	0.0113
ENSOARG00000013364	HHIPL2	HHIP like 2	-2.72	0.0433
ENSOARG00000013700	LOC101114408	Carbonyl reductase[NADPH]1	-1.90	0.0276
ENSOARG00000014759	GSTA1-1	Microsomal glutathione-S-transferase 1-1	-2.15	0.0082
ENSOARG00000017584	GSTA1	Glutathione S-transferase alpha 1	-3.14	0.0022
ENSOARG00000019285	LOC101107831	Glutathione S-transferase Mu 1-like	-4.75	1.73E-05
ENSOARG00000019297	LOC101108092	Glutathione S-transferase Mu 1	-3.13	0.0010
ENSOARG00000020412	PHGDH	Phosphoglycerate dehydrogenase	-2.19	0.0054
ENSOARG00000020582	MGST1	Microsomal glutathione S-transferase 1	-1.89	0.0254
Novel00632	-	-	-3.51	0.0033
Novel02088	-	-	-2.02	0.0425