| Gene alternative splicing is an important mechanism to regulate gene expression and produce protein diversity,and it is also an important reason for the large difference in the number of genes and proteins in eukaryotes.Variable splicing is a research hotspot in the field of gene expression regulation.However,there are few reports on the mechanism of action and regulation of isomers produced by variable splicing in the process of liver lipid metabolism in poultry.In this study,Nanopore real-time single molecule sequencing technology was used to sequence the full-length transcriptome of liver tissue of Lushi green-shell-egg chicken in pre-laying period(20W)and peak-laying(30W)hens.The change of gene variable splicer in liver tissue with significant difference in lipid metabolism was analyzed at the level of full transcriptome.The key gene AGPAT2 for triglyceride synthesis was analyzed by qRT-PCR.The results showed that the function of AGPAT2 variable splicer in the synthesis of PA and TG was verified by the function acquisition experiment.Finally,the function of AGPAT2 variable splicer in the process of liver lipid synthesis was clarified.The main results are as follows:1.Full length transcriptome analysis of chicken liver during pre-laying period and peak-egg hens.Three liver tissues of LuShi green-shell-egg chicken were collected in pre-laying period(20W)and peak-laying(30W)hens,respectively.The full-length transcriptome sequencing was carried out by using Nanopore three generation sequencing technology platform.The clean date of each sample was 16.87GB.The analysis of alternative splicing data showed that the number of alternative splicing events increased significantly in pre-laying period(20W)and peak-laying(30W)hens.Exon Skipping is a widely existing regulatory mechanism in these two physiological stages;1482 transcripts were specifically expressed at 20W,2248 at 30W;Go functional enrichment analysis showed that 20W was similar to the first 16 items of 30W specific transcripts,but the number of 30W specific transcripts was significantly higher than that of 20W.Further analysis of the transcripts of the two common genes identified 1106 differentially expressed transcripts,828 up-regulated and 278 down-regulated compared with pre-laying.Through the analysis of pathway enrichment,we found that the differentially expressed transcripts were mainly enriched in the lipid metabolism pathway.2.Bioinformatics analysis of variable spliceosome of AGPAT2 gene.By analyzing the differentially expressed transcripts,AGPAT2,a key candidate gene enriched in fatty acid metabolic pathway and highly expressed in 30W liver tissue sequencing data,was screened.Through further analysis of AGPAT2 gene,five alternative splicing isomers were found,which were named AGPAT2a,AGPAT2b,AGPAT2c,AGPAT2d and AGPAT2e,respectively.The analysis of functional domains showed that AGPAT2(a-e)had typical PlsC functional domains,and the transcripts AGPAT2a had signal peptide and transmembrane domains.3.Analysis of expression characteristics of AGPAT2 variable spliceosome.qRT-PCR was used to detect the expression patterns of AGPAT2 gene spliceosome in the liver of 20W and 30W Lushi green-shell-egg chicken.The results showed that the expression levels of AGPAT2a and AGPAT2b increased significantly at peak-laying(30W)hens,while the expression levels of AGPAT2(c-e)were lower and the difference not significant in two periods.Compared with AGPAT2(c-e),AGPAT2a may play an important role in lipid synthesis.The results of estrogen treated individual and cell experiments showed that the expression of AGPAT2a was consistent in chicken liver tissue and chicken embryo liver primary cells,while the expression of AGPAT2b was significant only in liver tissue of high concentration treated group,but there was no significant change in cells.It is suggested that the expression of AGPAT2a gene splice is regulated by estrogen,and AGPAT2b needs further study.4.Subcellular localization of AGPAT2 variable splices.PcDNA3.1-AGPAT2a/AGPAT2b-EGFP overexpression vector was constructed successfully.AGPAT2a and AGPAT2b were co-transfected with ER-DsRed-λ,which were the marker proteins of ER-dsred-λ,and the locations of AGPAT2a and AGPAT2b in the cell were determined under the confocal laser microscope.The results showed that AGPAT2a protein and ER-dsred-λ protein were fused by fluorescence,but AGPAT2b protein was not fused with ER-dsred-λ,indicating that AGPAT2b was not located in the endoplasmic reticulum.5.Study on the function of alternative splicing isomer of AGPAT2 gene.Over-expression of AGPAT2a and AGPA T2b,respectively,detected the content of PA and TG in chicken.The results showed that after over-expression of AGPAT2a,the content of PA and TG increased significantly(P ≤ 0.01),while over-expression of AGPAT2b had no effect on the production of direct product PA(P≥0.05),but promoted the increase of TG content in final product(P ≤ 0.01).Therefore,AGPAT2a gene played a major role in the synthesis of chicken PA,and both of them can promote the formation of TG.In summary,the number of variable splicing events at 30W was significantly higher than 20W.Exon jumping is a widely existed regulatory mechanism in these two physiological stages.20W was similar to the first 16 GO entries with significant enrichment of 30W specifically expressed transcripts,and the number of 30W enriched transcripts in each entry was significantly higher than that of 20W.AGPAT2 gene has 5 splisomers,only AGPAT2a,the expression level of AGPAT2b gene in the liver of hens at the peak of egg production(30W)increases to different degrees,and AGPAT2a is significantly higher than AGPAT2b.AGPAT2a expression is regulated by estrogen.AGPAT2a locates in the endoplasmic reticulum and participates in the assembly and transport of phospholipids in the endoplasmic reticulum.AGPAT2b may be located in the endoplasmic reticulum.Overexpression of AGPAT2a can significantly increase intracellular PA and TG content,while AGPAT2b gene only promotes the production of final product TG. |
