| Lipid metabolism has an important effect on carcass quality and meat quality.Studies have shown that SCD1(stearoyl coenzyme A desaturase 1),LIPIN1(phosphatidic acid phosphatase 1)and DGAT2(acyl CoA acyltransferase 2)are key enzymes of lipid metabolism.SCD1 plays an important role in the formation of the double bond of palmitic acid and oleic acid,and it also provides acyl coenzyme A for the synthesis of triglyceride.LIPIN1 can provide diacylglycerol for the final step of the synthesis of triglyceride,which is catalyzed by DGAT2.At the same time,LIPIN1 is a transcription cofactor of some downstream genes.Previous studies in our laboratory found that there were significant differences in fat deposition between Landrace and Lantang pigs,and there were significant differences in the expression of these three genes too.So far,the study of lipid metabolism in pigs is mainly concentrated in the related gene expression,but their promoters are rarely investigated.Promoter sequences of the three genes is unclear so far,whereas promoter plays an important role in regulation of gene expression.In order to reveal the molecular mechanism of significant differences between the fat deposition of pigs and Chinese pig breeds,the promoter of SCD1,DGAT2 and LIPIN1 of Landrace and Lantang pig were cloned in this study,and were compared between varieties.Further,we constructed two typs of reporter gene vector of Lantang and Landrace LIPIN1 promoter,which the 5' ends are sequentially deleted,to analyzed the influence of sequence differences in LIPIN1 gene transcription activity.The results are as follows:1.This study had cloned the promoter of SCD1,DGAT2 and LIPIN1 which are related to lipid metabolism of Landrace and Chinese Lantang pig.Respectively sequenced the 1700 bp long promoter of SCD1,the 2100 bp long promoter of DGAT2 and the 2900 bp long promoter of LIPIN1.We found that,between Landrace and Lantang pigs,in DGAT2 promoter,we only found 2 single nucleotide polymorphism(SNP)and a different length of a poly A domain.In SCD1 promoter,we found 8 SNP and a different length of a poly A domain.2.Through bioinformatics analysis,we found that the LIPIN1 promoter is not so conservative,and only the 3' end and the 5' end is homologous in different species,and the 3' end is GC-riched.The sequence of 5' end(-2100 ~-1800 bp)in LIPIN1 promoter is exactly same between Landrace and Lantang pig with some binding site of lipid metabolism related transcription factors(TF).Compare to Landrace,Lantang pigs has a longer GC-rich domain in the 3' end(-100 ~ +305 bp)which can be bound with some core TF.Between two conservative domains,we found other 21 SNPs and some insertion,deficiency and replacement of motif.3.In order to demonstrate whether the high GC region is the core promoter of LIPIN1 gene and to reveal the effect of sequence differences on the transcriptional activity of LIPIN1 gene,we constructed two types of recombined luciferase report vector with 5' deficiency promoter,ones with GC-rich domain in them,the other ones without GC-rich domain in them.The promoter activity of LIPIN1 varies as the 5' end become more integrated when GC-rich domain exists.When transfected to 3T3-L1,the promoter activity become weaker as the 5' end become longer.While in HepG2,the promoter activity become stronger as the 5' end become longer but drop down when the section of-1820 ~-1308 bp.No matter in 3T3-L1 or in HepG2,the Landrace's LIPIN1 promoter activity is higher than Lantang pig's when the section of-1308 ~-709 bp is added.While the promoter activity disappeared in all sections when the GC-rich domain is absent.We amplified the GC-rich domain of LIPIN1 promoter in some other varieties for agarose gel electrophoresis and sequencing,and found that the length of GC-rich domain in LIPIN1 promoter varies in different varieties.In summary,the difference of LIPIN1 promoter sequence between different varies can affect gene transcription activity,which may cause the differences in fat deposition between different varieties. |
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