Cloning And Activity Analysis Of Promoter Of Chicken MTNR1A Gene

MTNR1A gene encoding melatonin receptor 1A, is a candidate gene of chicken egg production traits. Activity analysis of MTNR1 A gene promoter region may help us understand MTNR1 A regulatory sequences and transcriptional regulatory mechanisms at the molecular level, also contributes to learning more about melatonin involvement in reproductive control mechanisms.In this experiment, Hy-Line Brown Layers for the study, was amplified by PCR chicken MTNR1 A gene 5 'regulatory region of 205 bp to 3134 bp different lengths, seven fragments of the same downstream primer, be cloned, sequenced, build the MTNR1 A promoter report gene vectors, transiently transfected chicken theca cells, the application of the dual-luciferase reporter assay system measures the relative luciferase activity, and an effect section were biological information analysis. Meanwhile, PCR amplification of 31 chickens MTNR1 A gene 5 'regulatory region of-941~+107 was sequenced to find its SNPs loci of SNP loci for genetic analysis and gene sequence analysis of biological information before and after the mutation. The results are as follows:(1) The segment of-243~-39 can express MTNR1 A gene promoter basal activity. Bioinformatics analysis showed that the region contains a TATA box, CCAAT box and other typical core promoter elements. Therefore, the segment of-243~-39 plays an important role in the expression of MTNR1 A gene.(2) The promoter analysis carrier removed by-553~-39 to-243~-39, the luciferase activity decreased significantly, indicating that there are enhanced components of gene expression between-553~-243. Several SP1 binding sites and USF binding sites presence in the region suggesting that the two binding sites played an important role for-553~-243 activity enhancing.(3) The promoter analysis carrier removed by-1963~-39 to-1525~-39, the luciferase activity decreased significantly,indicating that there are enhanced components of gene expression between-1963~-1525. It speculated that AP-1 binding site, HNF-1 binding site, USF binding site and Arnt trans-acting factors play a role in reducing promoter activity.(4) The promoter analysis carrier removed by-2373~-39 to-1963~-39, the luciferase activity decreased significantly, indicating that there are enhanced gene expression of components between-2373 ~-1963. Presumed TATA box, AP-1 binding sites, c-Myb binding sites, USF binding site and Oct-1 trans-acting factors in the region played an important role for-2373 ~-1963 activity enhancing.(5) Six SNPs were detected in MNTR1 A gene 5 'regulatory region of-941 ~ +107, respectively is :-34(C?T),- 100(T?G),- 110(T?C),- 126(C?T),- 151(G?A),- 538(T?G). SNPs were accord with Hardy Weinberg equilibrium state. Among them, two mutations may cause change of transcription factor binding sites.In summary, this study proved the segments of-553~-39 and-2373~-1525 are important sections to regulate the expression of chicken MTNR1 A gene; 5 'regulatory region of MTNR1 A has rich polymorphism. 2 mutations in 5 'regulatory region of-941~+107 may result in transcription factor binding sites' change.