Identification and Transcriptional Activity Analysis of ATGL Gene Promoter in Dairy Goat

doi:10.7668/hbnxb.20192556

Abstract

Abstract:

Adipose triglyceride lipase(ATGL)is the main rate limiting enzyme in the process of fat hydrolysis.In order to analyze the structure and transcriptional regulation mechanism of ATGL promoter in dairy goat,the 5' flanking sequence of ATGL was amplified by PCR and analyzed by bioinformatics.Luciferase reporter gene vectors of five deletion fragments with different lengths of promoter were constructed and transfected into mammary epithelial cells of dairy goat.The cells were treated with rosiglitazone and T0901317 respectively to detect the effects of PPARG and SREBP1 on ATGL promoter activity.The results showed that the 5' flanking sequence of ATGL gene of dairy goat was 2 721 bp,including 2 024 bp upstream from the transcription start site.The ATGL promoter contained eukaryotic promoter elements TATA-box and CpG island.It was also found that there were transcription factors-binding sites of FOXO,SREBF1,PPARG,C/EBPα and E2F1 by the online software prediction.The core region of ATGL promoter was located at -256—+1,and there were negative regulatory elements at -527—-256.When cells were treated with rosiglitazone and T0901317,it was found that both of them could significantly up-regulate ATGL promoter activity.The response regions of rosiglitazone and T0901317 were -527—-256 and -882—-527,respectively,indicating that PPARG and SREBP1 could regulate ATGL promoter activity.

Key words: Dairy goat, Adipose triglyceride lipase, Promoter, Core region, Transcriptional activity

LI Jun, YAN Zhihao, JIA Wanli, XU Mengmeng, ZHANG Jingfeng, XU Qiuliang, HAN Haoyuan, QUAN Kai. Identification and Transcriptional Activity Analysis of ATGL Gene Promoter in Dairy Goat[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 223-230. doi: 10.7668/hbnxb.20192556.

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Figures/Tables 7

Tab.1 The sequences of primers used for cloning ATGL gene promoter of dairy goat

引物名称 Primer name	引物序列 Primer sequence	结合位点 Binding site	产物大小/bp Product length
APF	GGCAGAACTCGCAATCCTA	-2 024	2 721
APR	CTAGCACCCGGTCTTACA	+697
APS1	ATTACGCGTGGCAGAACTCGCAATCCTA	-2 024	2 240
APS2	ATTACGCGTCTCAGTCCTTACTTGAACCT	-1 399	1 615
APS3	ATTACGCGTTCAGAACAGAGACAGGACT	-882	1 098
APS4	ATTACGCGTGATTCTGTGATGAGTCTCG	-527	743
APS5	ATTACGCGTCCTGGCTACTGTCTTTGG	-256	472
APA	GGAAGATCTCACGCCGATATGGTAGAC	+216

Fig.1 The PCR amplification products of ATGL gene promoter

Fig.2 The partial nucleotide sequence and potential transcription factor binding sites of ATGL promoter
A.Schematic representation of ATGL 5'flanking region;B.Analysis of the putative transcription factor binding sites of the ATGL promoter(+1 is the first base at transcription start site).

Fig.3 Amplification products and vector construction of various deletion fragments of ATGL promoter
A.PCR amplification products of various deletion fragments:1.-2 024—+216;2.-1 399—+216;3.-882—+216;4.-527—+216;5.-256—+216;B.Restriction enzyme digestion of recombinant plasmids of various deletion fragments:1.pGL-APS1(-2 024—+216);2.pGL-APS2(-1 399—+216);3.pGL-APS3(-882—+216);4.pGL-APS4(-527—+216);5.pGL-APS5(-256—+216).

Fig.4 Relative activity of the various deletion fragments of ATGL promoter
pGL3-Basic.Luciferase reporter gene empty vector;LUC.Luciferase reporter gene vector with various deletion fragments;Different letters indicate significant differences(P<0.05).The same as Fig.5—6.

Fig.5 Effect of ROSI on the activity of ATGL promoter
A.Effect of ROSI on the activity of ATGL promoter in full length;B.Effect of ROSI on the activity of various deletion fragments of ATGL promoter.

Fig.6 Effect of T0901317 on the activity of ATGL promoter
A.Effect of T0901317 on the activity of ATGL promoter in full length;B.Effect of T0901317 on the activity of various deletion fragments of ATGL promoter.

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