基于转录组学技术分析蛋白质限制与补偿对羔羊肝脏抗氧化性能的影响

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

摘要/Abstract

摘要：

本试验旨在通过RNA-seq技术在转录组水平分析早期蛋白质限制与后期补偿对羔羊抗氧化性能的影响。试验选取15日龄断母乳湖羊公羔32只,随机分为两组,一组饲喂正常蛋白水平的代乳粉(CP为25%)和开食料(CP为21%),记为对照组(normal protein level,NPL);另一组饲喂蛋白水平分别为19%的代乳粉和15%的开食料,记为试验组(low protein level,LPL)。人工饲喂相应的代乳粉至60日龄,开食料自由采食。61-90 d所有试验羊均自由采食正常蛋白水平(CP为21%)的开食料。分别在羔羊60和90日龄,每组随机选取2只羔羊进行屠宰,采用RNA-seq技术鉴定蛋白质限制与补偿条件下肝脏组织差异表达基因(DEGs),通过生物信息学分析,筛选与抗氧化功能密切相关的通路,利用qRT-PCR的方法验证抗氧化酶系基因表达水平。低蛋白水平日粮引起羔羊肝脏组织差异表达基因有302个,其中23个差异表达基因参与机体抗氧化调节过程,且表达水平呈显著下调。GO富集分析发现差异表达基因参与氧化还原酶活性(GO:0016491)、谷胱甘肽转移酶活性(GO:0004364)和氧化还原过程(GO:0055114)。KEGG分析发现,抗氧化相关基因主要富集到P450介导的外源性物质代谢和谷胱甘肽代谢通路;细胞因子受体作用通路相关基因表达水平显著提高。恢复蛋白水平30 d后,抗氧化相关基因表达水平与对照组无差异。日粮蛋白质水平与机体抗氧化功能密切相关。蛋白质限制导致肝脏基因表达发生改变,降低了抗氧化相关基因的表达水平,DEGs可通过外源性物质代谢、谷胱甘肽代谢、细胞因子受体作用通路等信号通路影响肝脏抗氧化和免疫功能。

关键词: 蛋白质补偿, 羔羊, RNA-seq, 抗氧化

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

康凌云, 陈建胜, 甘瀚凌, 韩露露, 冯海霞, 刁其玉, 邢凯, 崔凯. 基于转录组学技术分析蛋白质限制与补偿对羔羊肝脏抗氧化性能的影响[J]. 生物技术通报, 2021, 37(6): 171-180.

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.

图/表 9

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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