Screening And Validation Of Differential Mirna In Spleen Between Queshan Black Pig And Yorkshire

Alongwith depth study of miRNA,it provided a new perspective for the immune-related research with miRNA-mediated transcription level,and also it provided a new research direction,which was disease resistance of breeding,to breed a new pig breed with strong disease-resistant ability,high immune performance and healthy meat.The Queshan black pig was a good local varieties in Henan province,it had some good characters,such as the strong fertility,strong adaptability,good meat quality and stable genetic performance,etc.This studywas carried out on different pig breeds(Large white and Queshan black pig)and in different growth stages(3-day-old piglets and 6 months of age large white)of spleen by the method of Solexa sequence.Then differentially expressed miRNAwhich immune-related was analyzed and selected.And it provide reference for the subsequent utilization of miRNA and its target genes for disease resistance breeding.The results of this study are as follows:1.The total RNAs was extracted which belonged to the spleens of six-month Yorkshire,three-day Yorkshire and six-month Queshan black pig,and also the libraries of small RNAs were constructed,respectively.293?305 and 295 known miRNAs were found,and 166?244 and 160 novel miRNAs were found as well as by Solexa sequence.2.In the group of six-month old and three-day piglets Yorkshire,85 differentially expressed known miRNAs were seleted,and 55 known miRNAs were up-regulation expression in six-month Yorkshire,30 known miRNAs were down-regulation expression.By the same way,20 differentially expressed known miRNAs were seleted in the group of six-month Yorkshire and Queshan black pig,12 known miRNAs were up-regulation expression in Queshan black pig,and 8was down-regulation expression.Then the results of high throughput sequencing were validation by the method of Q-PCR,which including the 4miRNAs of the first group,which were ssc-miR-122?ssc-miR-1?ssc-miR-451 and ssc-miR-486,and the 4miRNAs of the second group,which were ssc-miR-4334-3p?ssc-miR-27a?ssc-miR-4331 and ssc-miR-1271,respectively,the Q-PCR results indicatedthey were consistent with the results of Solexa sequencing.3.After verification of miRNA,the study predicted the target genes for the known miRNAs in three samples,which including 26508?26600 and 26567,respectively.At the same time,23694 and 16481 target genes were found for the differentially expressed known miRNAs of the two groups.Then,ADD2,TRIM58,CD3 E and COL4A1 gene were selected and validated by the method of Q-PCR,and the resultsshowed that miRNAs had been as negative regulatory roles to their target genes.The analysis of extensive tissue expression profile showed that expressions of ADD2,CD3E and TRIM58 were highest in spleen,and the expression of COL4A1 gene was highest in adipose tissue.4.The results of the GO function annotations for the five differentially expressed miRNAs,which were ssc-miR-1?ssc-miR-122?ssc-miR-451?ssc-miR-486 and ssc-miR-27 a,indicated that these target genes were mainly enriched in hemopoiesis,leukocyte differentiation,T cell activation,regulation of T cell activation and activation of immune response,etc.KEGG pathway analysis showed that 13 KEGG regulatory pathways were significantly enriched,in which the tumor pathway and c GMP-PKG signaling pathway were the most enriched two pathways.The research for miRNA provided new ideas for used in the actual production of pig farms,andand for further studies on the molecular mechanism of miRNAs related to pig immune and reveal porcine miRNA mediated regulation function of the foundation,and also provided theoretical basis for breeding varieties with high disease resistance.