Analysis Of Mirnas Profiling And The Related Mirnas Of Splice Variants Of Bola-DQA2Gene In Mammary Gland Of Dairy Cattle

Mastitis, which is a common and complex inflammatory disease, caused genetic contagious and environmental factor in the modern dairy cow industry. It could lead to significant economic loss for dairy cow industry. There are many related immune genes were involved in the process of mastitis. So far, the resistance molecular mechanism of mastitis is still unknown, especially resistant related gene expressed regulation mechanism is not clear. MicroRNAs, approximately22nts in size, are class endogenous noncoding RNAs that can negatively regulate gene expression posttranscriptionally and play an important in the immune response, and splice variant is a mechanism of gene regulation posttranscriptionall-y and plays a control biological function. In order to explore the mechanism of miRNA and splice variant regulation on the immunologically relevant gene BOLA-DQA2and improve the mastitis resistance of dairy cow, this research preliminarily researched the relationships between miRNAs, splice variant and mastitis and is described in the following three sections.Section1:miRNAs play crucial role in the regulation of gene expression in eukaryotic cells. In recent years, many miRNAs has found in the mammals. In order to expand the amount of host miRNA and to inspect relationship between miRNA expression and mastitis, using Solexa sequencing method.One small RNA library was prepared from matistis-infect-ed mammary gland derived from the adult cow, then bioinformatics approach was used to distinguish authentic muture miRNA sequences from other classes of small RNAs. Using this method we detectde the375miRNAs, including the known230miRNAs in the miRNA database and145novel miRNAs.In additon, we also identified a new mirton and a bidirectionaly transcribed miRNA. These novel miRNAs may be specific expression in mastitis-effected mammary gland and play an important in the process of mastitis. Futhermore, the results of this study will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.Section2:Major histocompatibility complex, class Ⅱ, DQ alpha2, also named BOLA-DQA2,belongs to the Bovine Leukocyte Antigen (BOLA) class Ⅱ genes which are involved in the immune response. To explore the variability of the BOLA-DQA2gene and resistance to mastitis in cows, the splice variants (SV) targeted miRNAs and SNPs were identified in this study. A new splice variant (BOLA-DQA2-SV1) lacking of partial exon3(195bp) and two3’-untranslated regions (52bp+167bp) of the BOLA-DQA2gene, was found in the healthy and mastitis-infected mammary gland tissues.4of13new SNPs and multiple-nucleotide polymorphisms (MLNPs) resulted in amino acid changes in the putative domain of the protein and SNP (c.+1283C>T) may affect the binding to the seed sequence of bta-miR-2318. Furthermore, the relative expression of two BOLA-DQA2transcripts was detected in the mammary gland tissues in dairy cattle by using the quantitative real-time PCR. The result showed that expression of the BOLA-DQA2-SV1mRNA was significantly up-regulated2.67folds (P<0.05) in mastitis-infected mammary tissues (n=5) as compared with the healthy mammary gland mammary tissues (n=5). Our findings reveal that BOLA-DQA2-SV1may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2gene or their associations with mastitis resistance are unknown and warrant further investigation.Section3:miRNA prediction software was used to analysis the combination of miRNA binding to two splice variants of BOLA-DQA2gene and5candidated microRNAs binding to the3’-UTR of BOLA-DQA2two transcripts in the mammary gland tissues in dairy cattle by using the stem-loop q-PCR. The results displayed that the number of miRNAs binding to BOLA-DQA2-SV1were less than BOLA-DQA2-complete and except for the bta-miR-1777a, three miRNAs (bta-miR-296, miR-2430and miR-671) expression was significantly upregulated1.75to2.59folds (P<0.05), while the miR-2318was down-regulated in the mastitis cows. The results revealed that BOLA-DQA2-SV1may be regulated by miRNA, but also need to be studied further.