山羊CIDE家族基因克隆分析及互作蛋白预测鉴定

doi:10.7668/hbnxb.20192543

摘要/Abstract

摘要：

旨在获得CIDEB、CIDEC基因的CDS,检测该家族基因在山羊不同组织中的表达量,预测其互作蛋白,为进一步揭示CIDE家族基因在脂代谢中的调控网络提供参考。主要利用RT-PCR克隆获得山羊CIDEB、CIDEC基因序列并利用在线工具分析CIDE家族蛋白的生物学特性,并预测其互作蛋白。利用实时荧光定量PCR(RT-qPCR)检测了CIDE家族基因在山羊不同组织中的表达水平,并检测各互作蛋白在其表达水平最高的组织中的表达量,利用SPSS分析其表达量之间的相关性。克隆获得了CIDEB基因CDS区660 bp,共编码219个氨基酸,CIDEC基因CDS区714 bp,编码237个氨基酸。进化树分析显示,山羊CIDEA、CIDEB、CIDEC均与绵羊亲缘关系最近。RT-qPCR检测发现CIDEA、CIDEB、CIDEC基因分别在山羊瘤胃、肝脏、小肠中表达量最高。CIDEA与DFFB和ELOVL3在瘤胃中的表达量具有极显著相关性(P<0.01),与DIO2、TMEM26、PRDM16、PLIN1具有显著相关性(P<0.05)。CIDEB与DFFA和SDR39U1在肝脏中的表达量具有显著相关性(P<0.05)。CIDEC与PLIN2、GPLD1、ADIPOQ在小肠中的表达量具有显著相关性(P<0.05)。通过分析发现了与CIDE家族表达量具有相关性的基因,可为进一步揭示CIDE基因在脂质代谢中的作用及其调控网络提供理论基础,且CIDE家族蛋白均为脂滴相关蛋白,也可为进一步研究精确的脂滴形成机制提供参考。

关键词: 山羊, CIDE家族, RT-qPCR, 脂滴, 基因克隆

Abstract:

The aim of this study was to obtain the CDS of CIDEB and CIDEC gene,detect the expression of CIDEB and CIDEC in different tissues of goats,predict their interaction proteins.It would provide basial data for revealing the regulatory role of CIDEs in lipid metabolism.The CDS of CIDEB and CIDEC gene were cloned by RT-PCR,and the biological characteristics and interaction proteins of CIDE proteins were analyzed by online tools.Real-time quantitative PCR(RT-qPCR)was used to detect the expression of CIDEs and the genes,encoding the interaction proteins,in different tissues of goats,following the correlation analyses by a SPSS software.The CDS of CIDEB was 660 bp,encoding 219 amino acids,while the CDS of CIDEC was 714 bp,encoding 237 amino acids.Phylogenetic tree analysis showed that goat CIDEA,CIDEB and CIDEC were closely related to corresponding genes from sheep.RT-qPCR detection showed that CIDEA,CIDEB and CIDEC were highly expressed in the rumen,liver and small intestine of goat,respectively.The expression of CIDEA was significantly correlated with the expression of DFFB (P<0.01),ELOVL3 (P<0.01),DIO2 (P<0.05),TMEM26 (P<0.05),PRDM16 (P<0.05)and PLIN1 (P<0.05)in rumen.The expression of CIDEB was significantly correlated with that of DFFA and SDR39U1 in liver(P<0.05).The expression of CIDEC was obviously correlated with that of PLIN2,GPLD1 and ADIPOQ in small intestine(P<0.05).Through the analysis,we found genes related to the expression level of CIDEs,which could provide a theoretical basis for further revealing the role of CIDE gene in lipid metabolism and its regulatory network.Moreover,the proteins of CIDEs were all lipid-droplet related proteins,and could also provide a reference for further studying the precise mechanism of lipid droplet formation.

Key words: Goat, CIDEs, RT-qPCR, Lipid droplets, Gene clone

李琪, 杨昌恒, 王永, 林亚秋, 向华, 朱江江. 山羊CIDE家族基因克隆分析及互作蛋白预测鉴定[J]. 华北农学报, 2022, 37(1): 211-221. doi: 10.7668/hbnxb.20192543.

LI Qi, YANG Changheng, WANG Yong, LIN Yaqiu, XIANG Hua, ZHU Jiangjiang. Cloning and Analysis of Goat CIDEs and Identification of Predictive Interaction Proteins[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 211-221. doi: 10.7668/hbnxb.20192543.

http://www.hbnxb.net/CN/Y2022/V37/I1/211

图/表 13

参考文献 39

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基因 Gene	序列(5'—3') Sequence (5'—3')	退火温度/℃ Tm	产物长度/bp Products length
CIDEB	S: TCCAGGCTTGACTCATCCC	56	923
	A: GGGAGATCAGGTGAGTTAGCC
CIDEC	S: CCAAAACTATCCAGTTGACGA	59	1 043
	A: GGCAGTGTAATGAAAGTGTAGC

基因 Gene	序列(5'—3') Sequence (5'—3')	退火温度/℃ Tm	产物长度/bp Products length
CIDEB	S: TCCAGGCTTGACTCATCCC	56	923
	A: GGGAGATCAGGTGAGTTAGCC
CIDEC	S: CCAAAACTATCCAGTTGACGA	59	1 043
	A: GGCAGTGTAATGAAAGTGTAGC

基因 Gene	序列(5'—3') Sequence(5'—3')	退火温度/℃ Tm	产物长度/bp Products length	基因序列号 GenBank ID
CIDEA	S: CAGGGGCAGAAATGGACACC	60	118	KP120985
	A:CAATGACATCCTTGGGGCTC
CIDEB	S:CAGCCGCTACCCGTCAGGAACT	60	233	本试验所得序列
	A:CTTGCTGTGCTTGGGCTTCTCC
CIDEC	S:GCTGCTTGGCACCTCCTGTTAC	60	107	本试验所得序列
	A:GCACCTTCAGACTGGTTGGGAT
DFFA	S:TGCTCATTGATGTCCCGTGT	60	157	XM_005690694.3
	A:TCCAAAGCCCGAAGGTAAAG
DFFB	S:AAAGAAGCGTGCGGTCGTG	60	159	XM_005690758.3
	A:CAGTGGAGTTTGTGGGTGGTC
基因 Gene	序列(5'—3') Sequence(5'—3')	退火温度/℃ Tm	产物长度/bp Products length	基因序列号 GenBank ID
HKDC1	S:TAAGGCGAGGGGAGTTCTGG	60	155	XM_018042596.1
	A:CAACTTCGCAGTAGGGATCGT
RUSC2	S:GTCCCTGGAGTTCTGGTTTA	67	235	XM_018052123.1
	A:CACTCGCAGCAATTCCTTGT
SDR39U1	S:TTCCAACCTCGTAACCAAATG	59	98	XM_018054653.1
	A:CTCCACGACCCAGCACAAC
HSL	S:TGCCCAAGACAGAGCCAATG	60	181	EU273879.1
	A:GCGGAGGAGCCGAGTATCT
API5	S:GGGACTTTAGGTGGCTTGT	60	175	XM_005690123.3
	A:CACCAGTCACATCTTCTAGCAC
BAD	S:GCCAGAGGATGATGAAGGGAC	62	187	XM_018042790.1
	A:CATTTGCGTTGCCGTTCC
CDIP1	S:GCTGCCCCCTACAGACATCG	60	254	XM_018040387.1
	A:GCACAGTCACGGTGGTAGCA
ATOH8	S:CTGGAAGACCGTGTGCGTGAAG	65	159	XM_018055369.1
	A:GTTCTGTCCCGCAGCCCGTT
PLIN4	S:ACCACGGACCTCTGTCAACT	60	299	XM_018050876.1
	A:CCAGACGCACGAAGTAGC
PLIN2	S:TTGCTGTTGCCAATACCT	60	284	NM_001285596.1
	A:CTGCATCATCCGACTTCC
PLIN1	S:CCCATTGCCAGCACTTCAGA	60	95	XM_018066567.1
	A:GCAGCGTACTCGGCAGTATCTC
PLIN3	S:GTCCGCTGACGAGACCGAA	60	319	NM_001285595.1
	A:CAGATTCTCCTCCAGTTTGTCC
PPAP2A	S:TGATTGTTGGAGAAACCCTG	60	165	XM_018065498.1
	A:GTCTGCCTATCGAATACTTGG
GPLD1	S:GTATGTGCCTGTTGAAGATC	60	155	XM_018061655.1
	A:CAGAATAAGTGGGATAAAGC
ADIPOQ	S:TACCGCTCAGCATTCAGTG	60	355	KF452236.1
	A:CGTTATCCGCATAGACCC
ACADL	S:CAGGGAGATGTATTGGTGC	60	170	XM_018059958.1
	A:TGTGACTGCTACGACGATC
NRIP1	S:GCACCAGGATTCTATTGTTCTA	60	91	XM_018052861.1
	A:AGACGCTTTGTCAACGGC
DIO2	S:CGCTCTATGACTCGGTCATTC	58	159	XM_018053898.1
	A:CACCCAGTTTCACCTGTTTGTA
TMEM26	S:GACCTGGCAGTACAGAATG	55	151	XM_018042612.1
	A:GTATGAGCCGCACAACAA
ELOVL3	S:CTGTCGGTATCCTGGCTTAT	55	151	XM_005698356.2
	A:GCTTTGATCTTGGGAATTATGT
PPARG	S:TCAAAGTGGAGCCTGTATC	60	309	NM_001285658.1
	A:TGATGGCGTTATGAGACA
UCP1	S:ACTGGGACTTACAATGCTTA	60	89	XM_018061376.1
	A:TTTCTTGTCAGATTGGGAGT
PRDM16	S:CAACAGTAGCCAGGGCACCA	60	220	XM_018060470.1
	A:GGTCTTGTCACGGTCACTATCC
UXT	S:GCAAGTGGATTTGGGCTGTAAC	60	180
	A: ATGGAGTCCTTGGTGAGGTTGT

基因 Gene	序列(5'—3') Sequence(5'—3')	退火温度/℃ Tm	产物长度/bp Products length	基因序列号 GenBank ID
CIDEA	S: CAGGGGCAGAAATGGACACC	60	118	KP120985
	A:CAATGACATCCTTGGGGCTC
CIDEB	S:CAGCCGCTACCCGTCAGGAACT	60	233	本试验所得序列
	A:CTTGCTGTGCTTGGGCTTCTCC
CIDEC	S:GCTGCTTGGCACCTCCTGTTAC	60	107	本试验所得序列
	A:GCACCTTCAGACTGGTTGGGAT
DFFA	S:TGCTCATTGATGTCCCGTGT	60	157	XM_005690694.3
	A:TCCAAAGCCCGAAGGTAAAG
DFFB	S:AAAGAAGCGTGCGGTCGTG	60	159	XM_005690758.3
	A:CAGTGGAGTTTGTGGGTGGTC
基因 Gene	序列(5'—3') Sequence(5'—3')	退火温度/℃ Tm	产物长度/bp Products length	基因序列号 GenBank ID
HKDC1	S:TAAGGCGAGGGGAGTTCTGG	60	155	XM_018042596.1
	A:CAACTTCGCAGTAGGGATCGT
RUSC2	S:GTCCCTGGAGTTCTGGTTTA	67	235	XM_018052123.1
	A:CACTCGCAGCAATTCCTTGT
SDR39U1	S:TTCCAACCTCGTAACCAAATG	59	98	XM_018054653.1
	A:CTCCACGACCCAGCACAAC
HSL	S:TGCCCAAGACAGAGCCAATG	60	181	EU273879.1
	A:GCGGAGGAGCCGAGTATCT
API5	S:GGGACTTTAGGTGGCTTGT	60	175	XM_005690123.3
	A:CACCAGTCACATCTTCTAGCAC
BAD	S:GCCAGAGGATGATGAAGGGAC	62	187	XM_018042790.1
	A:CATTTGCGTTGCCGTTCC
CDIP1	S:GCTGCCCCCTACAGACATCG	60	254	XM_018040387.1
	A:GCACAGTCACGGTGGTAGCA
ATOH8	S:CTGGAAGACCGTGTGCGTGAAG	65	159	XM_018055369.1
	A:GTTCTGTCCCGCAGCCCGTT
PLIN4	S:ACCACGGACCTCTGTCAACT	60	299	XM_018050876.1
	A:CCAGACGCACGAAGTAGC
PLIN2	S:TTGCTGTTGCCAATACCT	60	284	NM_001285596.1
	A:CTGCATCATCCGACTTCC
PLIN1	S:CCCATTGCCAGCACTTCAGA	60	95	XM_018066567.1
	A:GCAGCGTACTCGGCAGTATCTC
PLIN3	S:GTCCGCTGACGAGACCGAA	60	319	NM_001285595.1
	A:CAGATTCTCCTCCAGTTTGTCC
PPAP2A	S:TGATTGTTGGAGAAACCCTG	60	165	XM_018065498.1
	A:GTCTGCCTATCGAATACTTGG
GPLD1	S:GTATGTGCCTGTTGAAGATC	60	155	XM_018061655.1
	A:CAGAATAAGTGGGATAAAGC
ADIPOQ	S:TACCGCTCAGCATTCAGTG	60	355	KF452236.1
	A:CGTTATCCGCATAGACCC
ACADL	S:CAGGGAGATGTATTGGTGC	60	170	XM_018059958.1
	A:TGTGACTGCTACGACGATC
NRIP1	S:GCACCAGGATTCTATTGTTCTA	60	91	XM_018052861.1
	A:AGACGCTTTGTCAACGGC
DIO2	S:CGCTCTATGACTCGGTCATTC	58	159	XM_018053898.1
	A:CACCCAGTTTCACCTGTTTGTA
TMEM26	S:GACCTGGCAGTACAGAATG	55	151	XM_018042612.1
	A:GTATGAGCCGCACAACAA
ELOVL3	S:CTGTCGGTATCCTGGCTTAT	55	151	XM_005698356.2
	A:GCTTTGATCTTGGGAATTATGT
PPARG	S:TCAAAGTGGAGCCTGTATC	60	309	NM_001285658.1
	A:TGATGGCGTTATGAGACA
UCP1	S:ACTGGGACTTACAATGCTTA	60	89	XM_018061376.1
	A:TTTCTTGTCAGATTGGGAGT
PRDM16	S:CAACAGTAGCCAGGGCACCA	60	220	XM_018060470.1
	A:GGTCTTGTCACGGTCACTATCC
UXT	S:GCAAGTGGATTTGGGCTGTAAC	60	180
	A: ATGGAGTCCTTGGTGAGGTTGT

蛋白 Protein	其他结构 Other (likelihood)	信号肽 Signal peptide	线粒体转运肽 Mitochondrial transfer peptide
CIDEA	0.982 9	0.003 9	0.013 2
CIDEB	0.990 8	0.001 5	0.007 7
CIDEC	0.914 5	0.007 5	0.078 0

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