Association Analysis Of Polymorphisms In The Promoter Region Of LncRNA NORHA And Reproductive Traits Of Yorkshire

Reproductive trait is a low fertility trait,and molecular breeding based on genetic markers is an important method to accelerate its breeding progress.At present,a large number of candidate genes and mutation sites related to sow reproductive traits have been identified,but these sites are mainly focused on protein-coding genes,and few reports have been made on non-coding genes.Our group previously identified a Yorkshire lncRNA NORHA that is associated with follicular atresia and granulosa cell apoptosis through RNA sequencing,and this gene is located in a QTL locus related to the reproductive traits on chromosome 7 of Yorkshire,suggesting that NORHA may be a potential candidate gene for reproductive traits.However,it is not clear whether there is a mutation site related to the reproductive traits in NORHA of Yorkshire.In this article,NORHA as the research object,using the luciferase report system and so on appraisal the core promoter region,using DNA sequencing technology to screen the mutation sites in NORHA core promoter region of Yorkshire and polymorphism analysis of mutations and the relationship between the litter size traits.We Using Ch IP and other techniques to analyze the molecular mechanism of polymorphic sites affecting sow litter size,To evaluate the feasibility of NORHA as a candidate gene for litter size traits in Yorkshire.The research results are as follows:(1)Identification and Sequence Analysis of NORHA Core Promoter Region in YorkshireThe promoter region of NORHA in-1581 bp was obtained by PCR amplification and sequencing.Sequence analysis revealed a cis-acting element,TATA-box,at the transcription start site-890 nt.In addition,there was a Cp G island in the promoter region of-675 ～-546 nt with a length of 129 bp.Four deletion vectors were constructed,and Dual-luciferase activity analysis showed that the luciferase activity in the-279 ～-128 bp region was significantly increased(P<0.01),is the core promoter region of NORHA.The base composition of the sequence was 24.6% base A,16.7% base T,36.7% base C and 22%base G,among which GC content was 58.7% and AT content was 41.3%.In addition,there are some transcription factor binding sites,such as HAND2,SP1,WT1 and ESR2,which associated with reproduction.(2)Association Analysis of NORHA Core Promoter Mutation Polymorphism and Reproductive Traits in YorkshireThrough pool DNA sequencing,a C>G mutation was found at-246 nt in the core promoter region of NORHA,named g.-246C>G.Genotyping of the g.-246C>G locus of the NORHA in Yorkshire population(n=313)by direct sequencing revealed two genotypes CC and CG,with genotype frequencies of 0.952(CC)and 0.048(CG),the allele frequencies are 0.976(C)and 0.024(G),respectively.The mixed linear model was used to analyze the effect of NORHA g.-246C>G on the total number of piglets born(TNB),the number of born alive(NBA),the number of strong piglets(NSP),the number of weak piglets(NWP),the number of stillborn(NSB)and the litter weight(LW).The results showed that the total litter size(TNB)of CC sows was significantly higher than that of CG sows(P<0.05),but other reproductive traits were not significant in difference.These results indicate that the NORHA is a candidate gene that affects the litter size traits of Yorkshire.(3)The effect of g.-246C>G site on the activity of NORHA promoter regionTo analyze the effect of g.-246C>G mutation in the promoter region on the transcriptional activity of NORHA,two different allelic promoter reporter vectors p GL3-C and p GL3-G were constructed and transfected into Yorkshire ovarian granulosa cells.The Dual-luciferase activity analysis revealed that the luciferase ctivity of p GL3-G vector was significantly higher than that of p GL3-C(P<0.01).Using an online tool JASPAR to predict the specific transcription factor binding sites in two different allelic promoter regions,the results found that the transcription factor KLF4 can bind to the G allelic promoter region.After interference with KLF4,it was found that the luciferase activity of p GL3-C and p GL3-G vectors were significantly down-regulated(P<0.05),but the down-regulation ratio of p GL3-G vector was significantly higher than that of p GL3-C,indicating that g.-246C>G affected the transcriptional activity of KLF4 on NORHA.The Ch IP assay confirmed that the transcription factor KLF4 can directly bind to the core promoter region of NORHA.In addition,the expression of NORHA in Yorkshire ovarian granulosa cells was significantly down-regulated after interference with KLF4(P<0.01),indicating that KLF4 is a transcriptional activator of NORHA in Yorkshire ovarian granulosa cells.These results indicate that g.-246C>G mutation affects the transcriptional activity of NORHA gene by affecting the binding of transcriptional activator KLF4 to the promoter region of NORHA.(4)KLF4 regulates NORHA-mediated apoptosis of Yorkshire ovarian granulosa cellsKLF4 was significantly up-regulated in Yorkshire atretic follicles(P<0.05),and it was significantly positively correlated with the expression of the pro-apoptotic factor NORHA in Yorkshire granulosa cells(r=0.8733,P<0.01).To analyze the function of KLF4 in Yorkshire follicular atresia and granulosa cell apoptosis,RNAi technology was used to interfere with the expression of KLF4 in Yorkshire granulosa cells.The results showed that after interference with KLF4,the apoptosis rate of granulosa cells was significantly down-regulated(P<0.05),the expression level of pro-apoptotic factor BAX was significantly reduced(P<0.05),and the ratio of BCL2/BAX was significantly increased(P<0.05).It shows that KLF4 can promote the apoptosis of Yorkshire ovarian granulosa cells.Co-transfection experiments found that overexpression of NORHA can reverse the inhibitory effect of KLF4-si RNA on granulosa cell apoptosis,indicating that KLF4 can regulate granulosa cell apoptosis through NORHA.In addition,the expression level of Fox O1,a downstream target of NORHA,was significantly down-regulated(P<0.05)in granulosa cells after interference with KLF4,while overexpression of NORHA could reverse this process.These results indicate that in porcine ovarian granulosa cells,KLF4 regulates NORHA-mediated Yorkshire granulosa cell apoptosis and Fox O1 expression.In conclusion,our findings confirms that NORHA is a candidate lncRNA for litter size traits in Yorkshire,and reveals the molecular mechanism of the mutation g.-246C>G affecting the reproductive performance of Yorkshire,and provides potential molecular markers and targets for molecular breeding of reproductive traits in Yorkshire.