Isolation And Identification Of Buffalo SP1 And THRSP Genes And Their Functions In Milk Fat Synthesis

The content of dry matter,especially milk fat and milk protein in buffalo milk is higher than that in common cow milk,and the nutritional value of buffalo milk is much higher than that of dairy cattle milk.As an important component of milk,milk fat is an important index to evaluate the quality and processing characteristics of milk.In recent years,the study on milk fat traits of dairy cows has become a hot topic in the field of cow lactation biology,and the focus of main research in this field is the expression and regulation of genes related to milk fat synthesis,signal pathway,regulation network and regulation mechanism of milk fat synthesis.Previous studies in some mammals have shown that SP1 is the transcriptional regulator of many key genes of lipid metabolism,which is closely related to the process of lipid metabolism;THRSP gene is regulated by many transcriptional factors related to lipid synthesis at the transcriptional level,which can affect the expression of downstream genes related to lipid synthesis,and then play an important role in the process of lipid synthesis.However,the functional roles of these two genes in milk fat metabolism in buffalo and their interactions in this process is still unclear.Therefore,this study first isolated and identified the buffalo SP1 and THRSP genes,and then explored the SP1 transcripts that play a major role in the buffalo mammary gland by using RT-q PCR technology,and then detected the m RNA abundance of the main transcript of SP1 gene,and THRSP in multiple tissues of buffalo during lactation and non-lactation.The expression characteristics of these two genes in different tissues were determined,especially their association with the process of lactation.Further,by constructing the overexpression vectors of SP1 and THRSP and synthesizing their si RNAs,the effects under the overexpression and interference of SP1 and THRSP on the expression of genes related to milk fat synthesis and the content of triglyceride(TAG)were investigated in buffalo mammary epithelial cells(Bu MECs).In addition,the promoter sequence of buffalo THRSP gene was obtained by directed sequencing method of PCR products,and the transcriptional regulation mechanism of THRSP gene mediated by SP1 was preliminarily determined by bioinformatics techniques.The results were as follows:(1)Three alternative spliced transcripts of SP1 gene,namely SP1-1,SP1-2 and SP1-3,were cloned and identified in this study.The transcript SP1-2 is come from retaining a partial intron sequence between exon 1 and 2,which leads to a backward shift of the translation start site;The transcript SP1-3 was generated by the use of a 3’ alternative splice point in exon 3,which resulted in a 144 bp deletion of the coding sequence(CDS).Thus,compared with alternative splicing variant SP1-1,the SP1-2 and SP1-3 lack 7 and 48 amino acid residues,respectively,at their N-terminus.The m RNA expression level of SP1-1 was the highest in buffalo mammary tissues of both lactating and non-lactating period,and the expression level of SP1-1 in the lactating period was significantly higher than that in the non-lactating period(P<0.01),indicating that SP1-1 played an important regulatory role in the process of buffalo milk production,and was a transcript that played a major role in the mammary glands of buffalo.The SP1-1 displayed the highest expression level in the spleen and heart,followed by the muscle,liver and kidney,and was lower in the mammary gland,small intestine and lung,and the expression level was higher in the lactation than in the nonlactation.In the Bu MECs with the SP1 overexpression,the m RNA expression levels of SCD,THRSP,SREBP-1c,LXRα and FASN,which are closely related to milk fat metabolism,were significantly up-regulated compared with the control group(P<0.05),and the content of TAG in the cells was also significantly increased(P<0.05).In the Bu MECs that the SP1 gene were interfered,the m RNA expression levels of ACACA,PPARG,DGAT1,DGAT2,THRSP,SREBP-1c and TIP47 were significantly down-regulated compared with the control group(P<0.05).Although the expression of SCD gene with a tendency to decrease,there was no significant difference compared with the control group(P>0.05).Meanwhile,the TAG content in Bu MECs was also significantly decreased compared with the control group(P>0.05).These results suggested that SP1 gene could regulate the expression of genes related to buffalo milk fat synthesis,and was closely related to the TAG content in the Bu MECs,which was a key functional gene regulating buffalo milk fat synthesis.(2)The complete CDS of THRSP gene of buffalo was obtained,which was 471 bp and encoded a protein composed of 156 amino acid residues.The THRSP gene displayed the highest expression level in the liver and skin,followed by the kidney,spleen,heart and mammary gland,while the expression level of this gene was lower in the brain,muscle,rumen,lung and small intestine,and the m RNA expression in the liver,skin,kidney,mammary gland and muscle tissues was significantly higher in the lactation than in the nonlactation(P<0.05).Under the overexpression of THRSP gene,the m RNA expression levels of PPARG,SCD,ACACA,LXRα,SREBP-1c and SP1 genes which are closely related to milk fat synthesis and TAG content in the Bu MECs were significantly increased compared with the control group(P<0.05).However,the m RNA expression of ACACA,PPARG,SCD,LXRα and SREBP-1c and the content of TAG in the Bu MECs with the interference of THRSP gene were significantly decreased compared with the control group(P<0.05).The results here suggested that THRSP gene was involved in the milk fat synthesis of buffalo.(3)The promoter sequence of THRSP gene with 1844 bp in length was isolated from Binglang Jiang buffalo,including 1761 bp upstream of the transcription start site(counted as+1)and pre-82 bp of exon 1.Compared with the promoter sequence of buffalo THRSP gene reported in the NCBI database,there is only one base difference in this sequence.The promoter of THRSP gene isolated from buffalo contained three potentially active fragments(-659-609,-164-114 and-11 to +39)and two TATA-boxes(-1369-1358 and + 63-+ 74)without Cp G islands.It contains many binding sites for transcription factors such as SP1,C/EBPalp,C/EBPbeta and RXR-alpha,among which SP1 has the most number of binding sites(17).These results suggested that the expression of THRSP gene may be regulated by transcription factor SP1.In conclusion,both of the SP1 and THRSP are involved in the process of buffalo milk fat synthesis,and they interact with each other during this process.SP1 can regulate the transcriptional activity of THRSP gene,and THRSP can also regulates milk fat synthesis of buffalo by the mediation of SP1.The results of this study can provide a basis for further elucidating the molecular mechanism of buffalo milk fat trait formation and also lay a foundation for the molecular breeding of buffalo milk fat traits.