| Strong broodiness and high ability to store energy in liver are two characteristics of goose. miRNAs play a critical role in posttranscriptional regulation of gene expression. Zhedong-white goose is a domestic goose bred in the east of Zhejiang province and has strong broodiness. The French Landes Grey is famous for its ability to store high lipid levels in the liver. Our study identified miRNAs involved in the regulation of broodiness in hypothalamus and lipogenesis in liver by high-throughput sequencing analysis. And we analyzed the differential expressed genes between control and overfed geese liver by RNA-Seq. We cloned the mRNA sequences of some genes involved in fatty acids synthesis and identified the expression of these genes in different tissues. In addition, we examined whether the expression of these genes is regulated by insulin, glucose, and/or fatty acids in vitro.1. Identification of differentially expressed known and novel miRNAs in broodiness of goose.Broodiness is strictly controlled by the hypothalamic-pituitary-gonadal (HPG) axis. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in egg-laying and brooding behavior of geese in our study. We obtained a total of 11053784 and 8801405 sequences in the broody group and egg-laying group, respectively. In total sRNAs, the miRNA fragments were the most abundant sequences, with approximately 75.61% in the broody group and 79.82% in the egg-laying group. The most abundant size of goose hypothalamus sRNAs was 22 nt. Let-7 family had the most abundant reads in hypothalamus.38 up-regulated miRNAs and 14 down-regulated miRNAs with reads>1000 in at least one group and fold changes> 2.0 (P< 0.001) were identified in broody group. In these miRNAs, miR-30d, miR-30e, miR-148a-3p, miR-30a and miR-146c had high levels in two groups. In addition, we found a total of 158 novel miRNAs, including 114 novel miRNAs in the broody group and 94 in the egg-laying group. In these novel miRNAs,4 miRNAs were differentially expressed (reads>100 in at least one group, fold changes> 2.0 and P< 0.001).2. Identification of differentially expressed known and novel miRNAs in fatty liver of goose.The French Landes Goose was famous for its high capacity and susceptibility to fatty liver production. After 21-days overfeeding, the weight of liver increased 4-fold while the BW only increased 1.5-fold. And we can clearly observe a lot of lipid droplets in liver through histological examination. By high-throughput sequencing, we obtained 10396977 and 11321273 total sequences in control and overfed geese liver, respectively. In total sRNAs, the miRNA fragments were the most abundant sequences (88.92% in control and 86.98% in overfed). The most abundant size of goose liver sRNAs was 22 nt. The most frequently sequenced known miRNA was miR-122, which represented 70.34% and 72.01% of total miRNA reads in control and overfed group, respectively.22 up-regulated miRNAs and 12 down-regulated miRNAs were identified in fatty liver (expression> 100 in at least one group, fold changes> 2.0 and P< 0.001). And in these miRNAs, miR-222 and miR-30d had high level in two groups. In addition, we found a total of 67 novel miRNAs, including 2 up-regulated miRNAs and 1 down-regulated miRNA (expression> 10.0 in at least one group, fold changes> 2.0 and P< 0.001). We performed luciferase reporter assays to determine whether miR-125b specifically targets ACSL1 post-transcriptionally by binding to its 3’-UTR. Introduction of miR-125b mimic into cells resulted in a significant decrease (57.3%) in luciferase activity compared with control.3. Differential transcriptome analysis between control and overfed geese liverRNA-Seq and bioinformatics analysis were used to identify the differences in gene expression between control and overfed geese liver in our study. We obtained 50198914 and 49172954 total sequences, and mapped 52.85% and 48.74% of total reads to reference genome in control and overfed geese liver, respectively, while 36.61% and 32.88% of total reads mapped to reference gene in two groups. Alternative 3’splice site was the most frequently alternative splicing events and followed by alternative 5’splice site.602 up-regulated transcripts and 200 down-regulated transcripts were identified in fatty liver. We listed 33 up-regulated genes and 13 down-regulated genes involved in carbohydrate metabolism and lipid metabolism. In these genes, PDGDS, CYP2C45, FADS1, FADS1, Elovl6,17β-HSD, FAS, AACS and SCD1 had high levels in liver.4. cDNA sequences cloning of goose related fatty acid synthesis genes and studing on their tissue distributionThe full-length cDNA sequence goose ACSL1 is 3709 bp, comprising 119 bp 5’-UTR, 2097 bp CDS and 1493 bp 3’-UTR. The predicted amino acid sequence is 699 AA in length, which has 98.14% identity with duck and 93.71% with chicken. The expression of ACSL1 decreased in liver and kidney but increased in abdominal fat and heart after overfeeding. The mRNA expression levels of SCD1 increased in liver, crureus, pactorales and abdominal fat. The cDNA sequence goose Elovl6 with incomplete 3’-UTR is 2782 bp, comprising 74 bp 5’-UTR,789 bp CDS and 1909 bp 3’-UTR. The predicted amino acid sequence is 266 AA in length, which has 98.49% identity with chicken. After overfeeding, the expression of Elov16 was increased 2.06-fold in liver and 6.02-fold in spleen. The cDNA sequence goose FADS1 is 3147 bp, comprising 314 bp 5’-UTR,1083 bp CDS and 1750 bp 3’-UTR. The predicted amino acid sequence is 361 AA in length, which has 98.61% identity with duck and 79.14%with chicken. After overfeeding, the mRNA level of FADS1 increased 2.61-fold in liver and 2.89-fold in spleen. The CDS sequence of FADS2 is 1335 bp in length. The predicted amino acid sequence is 455 AA in length, which has 97.07% identity with duck and 93.69%with chicken. After overfeeding, the expression of FADS2 was up-regulated 3.09-fold.5. The role of fatty acids, glucose and insulin in regulation of goose hepatic fatty acid synthesisIn the primary hepatocytes, low concentrations of SA and LA (50 μM and 100 μM) induced lipid accumulation, while 200 μM concentration had no effect on lipid accumulation. Low glucose and insulin levels had a minimal effect, while 100 mM glucose and 200 nM of insulin had a significantly excitatory effect on intracellular lipid accumulation. Differential concentrations of SA increased the mRNA levels of FAS, FADS1 and Elovl5.100μM SA can induce the expression of ACSL1. SCD1 was up-regulated in 150 μM SA. Differential concentrations of LA increased the expression of FAS, FADS1, Elovl5 and ACSL1 while 50μM also induce FADS2. Differential concentrations of glucose increased the expression of FAS and Elovl5. Further, we found that high glucose level induced gene expressions, except that of ACSL1 and FADS2. And the expression ofACSLl was down-regulated in 50 mM glucose. A high level of insulin increased the mRNA levels of FAS, SCD1, Elovl6, Elovl5 and ACSL1. |
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