Screening Of Differentially Expressed Adipose Genes And Alternative Splicing In Ningxiang Pigs Based On Transcriptomics

Ningxiang pig is a famous local pig breed in China.Its meat quality is good,the intramuscular fat content is much higher than that of foreign breeds,and it is rich in nutrition.It is often used as female breeding to cross with other foreign breeds.In this study,transcriptome sequencing technology was used to sequence the adipose tissue of Ningxiang pigs,and using Suscrofa11.1(MJ20190122033 v1.0)as reference,bioinformatics software was used to analyze the function and alternative splices of differentially expressed genes in different periods of sequencing data.PCR and TA cloning and sequencing techniques were used to identify and analyze the genes screened from the results of alternative splicing analysis.The main results are as follows:Based on the reference genome of Ningxiang pigs,28010 genes and 45922 transcripts were identified from the transcriptome sequencing data of adipose tissue of Ningxiang pigs in three different periods.Differential expression analysis identified 5149(30 d VS 90 d),5786(30 d VS 210 d),and 3223(90 d VS 210 d)differentially expressed genes,which were mainly distributed in immune,biological metabolism,fat metabolism and other related GO terms and signaling pathways.The differential expression genes related to fat metabolism were screened and analyzed,and CD36 、 NCOR2 and APOC3 were selected as fat metabolism related candidate genes.Through the analysis of alternative splicing of fat tissue 282600 alternative splicing events were identified in Ningxiang pig.Alternative splicing genes were up to 22758,accounting for 81.2% of all genes,which were the highest proportion of exon jumping event,average for more than 75%,the lowest proportion of introns retain,average proportion was only 3%.Through PCR and TA cloning sequencing technology,the alternative splice transcript ADARB1-2 produced by ADARB1 was successfully identified from Ningxiang pigs for the first time.ADARB1-2 was caused by the fact that the intron remained in the transcript at47 bp due to the A-to-G editing of ADARB1 m RNA.The expression level of ADARB1-2 in liver was significantly higher than that in spleen(P<0.05),and in adipose tissue,the expression level of ADARB1-2 at 210 days of age was extremely significantly higher than that in other 3 days of age(P<0.01),and that at 30 days of age was extremely significantly higher than that in other 2 tissues(P<0.01).In addition to the expression trend of ADARB1-2 in lung,the expression trend of other tissues and adipose tissue at different days of age was similar to the expression trend of ADARB1-2,indicating that the editing level of this site was correlated with the expression level of ADARB1-2 to a certain extent.(5)By PCR and TA cloning sequencing technology for the first time successfully identified from Ningxiang pig NCOR2 produced gene transcription NCOR2-1 and NCOR2-2,NCOR2-2 on exons 40 for a period of sequence produced by jumping,found by qPCR NCOR2-1 the transcript in adipose tissue compared with liver,spleen,lung has a higher amount of expression which NCOR2-1 is likely to be involved in regulation of fatty deposits.