The Reasearch Of Buffalo SCAP And SREBPl Gene Cloning,mRNA Expression And Ploymorhic Detection

Sterol regulatory element-binding protein cleavage-activating protein ?SCAP? is a membrane binding protein in the endoplasmic reticulum. It contains a sterol sensitive domain which can response to the changing level of intracellular cholesterol. Sterol regulatory element-binding protein-1 ?SREBP1? is a membrane binding transcription factors in the ER. It can activate a series of genes related to fatty acid synthesis when stimulated by SCAP. SCAP and SREBP1 work together to maintain intracellular cholesterol levels steady, which play an important role on lipid metabolism of animals. This research aimed to explore the relationship between the SCAP and SREBP1 genes and buffalo milk production trait. Firstly, the two genes were cloned to analyze the characteristics of their genetics. And then studied the two genes expression condition in the different tissues, the different lactation periods and the high and low yield buffalo milk respectly. Lastly, we studied the SCAP and SREBPl gene polymorphism by using DNA direct sequencing method, which would lay a foundation for finding the genetic SNP markers relating to buffalo milk production traits. The experimental main results were as follows:1. SCAP and SREBP1 gene cloningDesigning several pairs of primers to clone the buffalo SCAP and SREBP1 genes based on the bos taurus SCAP ?NC₀07320? and SREBP1 ?NC₀07317? gene sequence. After sequenced and blasted, we found that the length of SCAP gene cDNA was 4214 bp and the length of SREBP1 gene cDNA was 3961 bp. Their ORFs were 3837 bp and 3438 bp respectively. Then compared the SCAP and SREBP1 genetic homologies of buffalo with bos turaus, human, sos scrofa, ovis aries, equus caballus, mus muscalus, rattus norvegicus, oryctolagus and canis familiaris. The homologies were 99%,98%,89%,96%,89%,85%, 85%,87%,89%and 98%,86%,88%,96%,87%,79%,80%,82%, 86% respectively. And they revealed that the SCAP and SREBP1 genes were conservative in different species.2. SCAP and SREBP1 mRNA expressionusing the GAPDH gene as the reference genes, made the RNA extracted from the buffalo heart, lung, kidney, epithelium, spleen, ovarian, mammary gland, brain, large intestine, small intestine, rumen, abomasum, omasum, muscle, fat, lymph, liver and pancreas and mammary glands whose lactation periods were 7 day,50 day,280 day, and the high and low yield buffalo milk RNA as the templates for real-time fluorescent quantitative PCR. Results:?1? Among the different tissues, SCAP and SREBP1 genes both expressed highest in mammary gland, while expressed lowest in lymph and muscle. That revealed that SCAP, SREBP1 were associated with the buffalo mammary gland's function. ?2? Among the mammary gland which in different lactation period, SCAP genes expression were 7 d> 50 d>280 d, and SREBP1 gene expression were 50 d> 7 d> 280 d. These showed the SCAP gene can promote to buffalo launch galactosis, SREBP1 gene can facilitate milk syntheses and secretion. ?3? SCAP and SREBP1 genes both had a significantly higher expression in the high yield milk than the low yield milk. That could show that SCAP and SREBP1 gene effected on buffalo milk production. So we could infer that the SCAP and SREBP1 gene have a relationship with buffalo milk production.3. Buffalo SCAP and SREBP1 gene polymorphism detectionMade 18 buffalo blood genomic DNA as the templates for PCR. And the product were direct send to study gene polymorphism.32 SNPs of SCAP gene was obtained, among them there were 27 SNPs located on the intron and 5 SNPs on the exon that including one missense mutation and 4 synonymous mutations. And 23 SNPs of SREBP1 gene were detected, 14 of them found on the intron and the other 9 SNPs on the exon that containing 5 missense mutation and 4 synonymous mutations.