<i>FABP2</i>基因在不同比例花椒籽饲喂育肥猪的组织差异表达、克隆及生物信息学分析

doi:10.7668/hbnxb.20193156

摘要/Abstract

摘要：

旨在分析不同比例花椒籽替代部分玉米饲喂三元杂交育肥猪不同组织FABP2的差异表达,寻找肌内脂肪沉积的关键基因;同时克隆三元杂交育肥猪FABP2编码区序列,并用生物信息学方法对其进行分析。选取288头90日龄杜×长×大三元杂交育肥猪(33 kg),随机分为4组,每组4个重复,每个重复18头猪;对照组饲喂基础日粮,试验组分别用花椒籽代替饲粮中2.5%(试验Ⅰ组),5.0%(试验Ⅱ组),7.5%(试验Ⅲ组)的玉米,预试验7 d,正试期100 d。采用qRT-PCR检测试验组不同组织中FABP2的相对表达量,同时克隆三元杂交育肥猪FABP2编码区,利用生物信息学分析软件,预测FABP2蛋白理化性质、结构域和磷酸化激酶位点等。结果表明:FABP2在各组织中均有不同程度表达,其中空肠和肝脏中FABP2显著高表达。与对照组相比,各试验组心脏、肺脏、肾脏、十二指肠、空肠和盲肠组织中FABP2显著下调表达;试验Ⅱ组和试验Ⅲ组脾脏和回肠组织中FABP2显著上调表达;试验Ⅰ组肝脏、结肠、直肠和背肌组织中FABP2显著上调表达,说明饲粮中添加花椒籽对三元杂交育肥猪FABP2基因的表达谱具有显著影响。成功克隆并鉴定出三元杂交育肥猪FABP2编码区全长(399 bp),编码132个氨基酸,存在2处错义突变;FABP2蛋白是一种酸性稳定的非分泌蛋白,二级结构主要由延伸链和无规则卷曲构成,含有一个Lipocalin-FABP2超家族保守结构域和3个无序化区域。

关键词: 三元杂交育肥猪, 花椒籽, FABP2基因, 组织表达, 克隆, 生物信息学分析

Abstract:

The purpose of this study was to analyze the differential expression of FABP2 in different tissues of DLY fattening pigs fed different proportions of Zanthoxylum seeds instead of some corn,and to find the key gene for intramuscular fat deposition;at the same time,the sequence of the coding region of FABP2 in DLY fattening pigs was cloned and analyzed by bioinformatics methods.A total of 288,90-day-old Duroc Landrace Yorkshire fattening pigs(33 kg)were randomly divided into 4 groups with 4 replicates and 18 pigs in each replicate;the control group was fed the basal diet and the test group was fed Zanthoxylum seeds instead of corn in the diet at 2.5%(test group Ⅰ),5.0%(test group Ⅱ)and 7.5%(test group Ⅲ),respectively,for 7 d in the pretest and 100 d in the test period.PCR was used to detect the relative expression of FABP2 in different tissues of the test groups,and the coding region of FABP2 in DLY fattening pigs was also cloned,and bioinformatics analysis software was used to predict the physicochemical properties,structural domains and phosphorylation kinase sites of FABP2 protein.The results showed that FABP2 was differentially expressed in all tissues,with significantly higher expression in jejunum and liver.Compared with the control group,FABP2 was significantly down-regulated in heart,lung,kidney,duodenum,jejunum and cecum tissues in each test group;FABP2 was significantly up-regulated in spleen and ileum tissues in test group Ⅱ and test group Ⅲ;FABP2 was significantly up-regulated in liver,colon,rectum and dorsal muscle tissues in test group Ⅰ,indicating that the addition of Zanthoxylum seeds in the diet had a significant effect on the expression profile of FABP2 gene in DLY fattening pigs.In this experiment,the full length(399 bp)of the coding region of FABP2 was successfully cloned and identified,encoding 132 amino acids,with two missense mutations;FABP2 protein was an acidic stable non-secretory protein with a secondary structure consisting mainly of extended chains and random coils,containing a Lipocalin-FABP2 superfamily conserved structural domain and three disordered regions.

Key words: DLY fattening pigs, Zanthoxylum seeds, FABP2 gene, Tissue expression, Cloning, Bioinformatics analysis

李杰, 黄晓宇, 尚学峰, 杨姣姣, 张娟丽, 谢开会, 张亚丽, 闫尊强, 王鹏飞, 高小莉, 杨巧丽, 马艳萍, 滚双宝. FABP2基因在不同比例花椒籽饲喂育肥猪的组织差异表达、克隆及生物信息学分析[J]. 华北农学报, 2022, 37(6): 228-238. doi: 10.7668/hbnxb.20193156.

LI Jie, HUANG Xiaoyu, SHANG Xuefeng, YANG Jiaojiao, ZHANG Juanli, XIE Kaihui, ZHANG Yali, YAN Zunqiang, WANG Pengfei, GAO Xiaoli, YANG Qiaoli, MA Yanping, GUN Shuangbao. Differential Expression,Cloning and Bioinformatics of FABP2 Gene in Tissues of Fattening Pigs Fed with Different Proportions of Zanthoxylum Seeds[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 228-238. doi: 10.7668/hbnxb.20193156.

http://www.hbnxb.net/CN/Y2022/V37/I6/228

图/表 18

表1 引物信息

Tab.1 Primers information

基因名 Gene name	转录本 Transcript No.	引物序列(5'—3') Primer sequences (5'—3')	扩增长度/bp Length	用途 Purpose
FABP2	NM_001031780.1	F: GGTGCCTGGAAGATAGACCG	224	荧光定量
		R: AGTTCAGTTCCGTCTGCGAG
FABP2	NM_001031780.1	F: GCTGGAGACTATGGCATTTGAT	427	PCR扩增
		R: GTGCTCCAAGAACAATGCTCAAT
GAPDH	NM_001206359.1	F: AGTATGATTCCACCCACGGC	139	荧光定量
		R: TACGTAGCACCAGCATCACC

表2 生物信息学分析软件

Tab.2 Bioinformatics analysis software

软件Software	功能用途Function	网址Website
Protparam	氨基酸理化性质分析	https://web.expasy.org/protparam/
Sopma	蛋白的二级结构预测	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
Phyre2	蛋白三级结构模型建立	http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index
ExPASy-Protscale	蛋白亲疏水性分析	https://web.expasy.org/protscale/
TMHMM Server.v.2.0	跨膜螺旋结构预测	http://www.cbs.dtu.dk/services/TMHMM-2.0/
PSORT Ⅱ Prediction	蛋白亚细胞定位	https://psort.hgc.jp/form2.html
SignalP 4.1	信号肽裂解位点预测	http://www.cbs.dtu.dk/services/SignalP/
NetPhos 3.1 Server	激酶磷酸化位点分析	http://www.cbs.dtu.dk/services/NetPhos/
NetNGlyc 1.0	N端糖基化定位	http://www.cbs.dtu.dk/services/NetNGlyc/
Conserved domains (CDD)	蛋白保守结构域预测	https://www.ncbi.nlm.nih.gov/cdd
FoldIndex	蛋白折叠无序化分析	http://bioportal.weizmann.ac.il/fldbin/findex

图1 FABP2在三元杂交育肥猪不同组织中的差异表达不同小写字母表示差异显著(P<0.05)。图2同。

Fig.1 Differential expression of FABP2 in different tissues of DLY fattening pigs Different lowercase letters indicate significant difference(P<0.05).The same as Fig.2.

图2 FABP2基因在不同比例花椒籽饲喂三元杂交育肥猪的组织差异表达

Fig.2 FABP2 gene was differentially expressed in tissues of DLY fattening pigs fed with different proportions of Zanthoxylum seed

图3 三元杂交育肥猪FABP2基因PCR扩增 M.DNA Marker DL2000;1,2.FABP2基因PCR扩增产物。

Fig.3 The PCR amplification of FABP2 gene in DLY fattening pigs M.DNA Marker DL2000 ;1,2.PCR amplification product of FABP2 gene.

图4 测序结果分析

Fig.4 Analysis of sequencing results

图5 氨基酸序列比对

Fig.5 Alignment of amino acid sequences

表3 不同物种间遗传距离

Tab.3 Genetic distance between different species

物种Species	1	2	3	4	5	6	7	8	9
三元杂交育肥猪DLY fattening pigs
绵羊Ovis aries	0.13
马Equus caballus	0.25	0.20
牛Bos taurus	0.14	0.01	0.21
犬Canis lupus familiaris	0.11	0.11	0.20	0.11
智人Homo sapiens	0.15	0.15	0.23	0.16	0.14
猕猴Macaca mulatta	0.14	0.14	0.21	0.14	0.13	0.05
小鼠Mus musculus	0.22	0.20	0.30	0.21	0.19	0.22	0.23
大鼠Rattus norvegicus	0.18	0.17	0.27	0.17	0.14	0.18	0.17	0.08
鸡Gallus gallus	0.27	0.27	0.33	0.27	0.23	0.30	0.27	0.22	0.22

图6 不同物种FABP2基因系统进化树分析

Fig.6 Phylogenetic tree analysis of FABP2 gene in different species

图7 氨基酸组成及频率

Fig.7 Amino acid composition and frequency

图8 三元杂交育肥猪FABP2 蛋白二级结构 h.α-螺旋;t.β-转角;c.无规则卷曲;e.延伸链。

Fig.8 Secondary structure of FABP2 protein in DLY fattening pigs h.α-helix;t.Beta angle;c.Random crimp;e.Extension chain.

图9 不同物种三级结构 A.三元杂交育肥猪;B.绵羊;C.鸡。

Fig.9 Tertiary structure of different species A.DLY fattening pigs ;B.Sheep ;C.Chicken.

图10 FAPB2亲疏水性分析(A)和跨膜螺旋结构域预测(B)

Fig.10 FAPB2 hydrophobicity analysis(A)and transmembrane helical structure domain prediction(B)

图11 蛋白亚细胞定位(A)和信号肽预测(B)

Fig.11 Protein subcellular localization(A)and signal peptide prediction(B)

图12 激酶磷酸化位点(A)和N-端糖基化位点预测(B)

Fig.12 Kinase phosphorylation site(A)and N-terminal glycosylation site prediction(B)

表4 三元杂交育肥猪FABP2蛋白可能的磷酸化位点

Tab.4 Possible phosphorylation sites of FABP2 protein in DLY fattening pigs

位点 Site	氨基酸 Name	激酶 Kinase	得分 Score
15	Y	Unsp	0.788
42	T	DNAPK	0.573
49	T	PKC	0.817
49	T	Unsp	0.779
53	S	Unsp	0.692
53	S	PKA	0.503
54	S	Unsp	0.993
55	T	PKC	0.662
72	S	Unsp	0.502
80	T	CKI	0.508
103	S	CKII	0.520
105	T	Unsp	0.855
105	T	CKII	0.507

图13 三元杂交育肥猪FABP2蛋白的保守结构域

Fig.13 Conserved domain of FABP2 protein in DLY fattening pigs

图14 折叠无序化分析 A.三元杂交育肥猪;B.鸡;C.绵羊。

Fig.14 Folding disordering analysis A.DLY fattening pigs;B.Chicken;C.Sheep.

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	Chen Y T. Correlation between serum Lipocalin-2 level and obesity and type 2 diabetes mellitus[D]. Suzhou: Soochow University, 2011.

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FABP2基因在不同比例花椒籽饲喂育肥猪的组织差异表达、克隆及生物信息学分析

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FABP2基因在不同比例花椒籽饲喂育肥猪的组织差异表达、克隆及生物信息学分析

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