Cloning and identification of chicken CDS2 splice variants
Based on the predicted chicken CDS2 sequence (XM_417669), four CDS2 transcripts alternatively spliced at the 3'-untranslated region (UTR) were cloned by combining conventional PCR amplification (LP1–LP5, Fig. S1), 5' RACE (Fig. 1A), and 3' RACE (Fig. 1B) and named CDS2-1 (4643 bp, GenBank accession no. KC886604), CDS2-2 (4770 bp, GenBank accession no. KC886602), CDS2-3 (4893 bp, GenBank accession no. KC886601), and CDS2-4 (5545 bp, GenBank accession no. KC886603). The sequence of the longest transcript CDS2-4 was consistent with the predicted CDS2 (accession no. XM_417669). Cloned CDS2 transcripts containing part of the 5′-UTR, 1348 bp coding sequence, and a long 3'-UTR ranged from 3265 to 4167 bp with a polyA signal (AATAAA) and polyA tail, with differences at 4310–5210 bp compared with the longest transcript CDS2-4, and were predicted to encode proteins of 448 amino acids. The sequence in the alternative region of chicken CDS2 was predicted to contain multiple binding sites of microRNA (miRNA) sequences including gga-miR-12243-5p and gga-miR-12215-5p (http://www.mirbase.org/search.shtml), as well as gga-miR-1680-5p, gga-miR-1751-5p, gga-miR-301b-5p, and gga-miR-6561-3p (http://www.mirdb.org/custom.html).
Chicken CDS2 is located at chr.22, spanning approximately 23 kb of the genome (Table S1). The longest splice form (CDS2-4) contains 13 exons with the exon–intron boundary (Table S1) abiding by the GT-AG rule; while CDS2-1, CDS2-2, and CDS2-3 contain 14 exons. The exon–intron boundary of intron 13 abides by AC-AG for CDS2-3, GC-CA for CDS2-2, and CT-AG for CDS2-1 (http://genome.ucsc.edu/cgi-bin/hgBlat, Table S1).
Conservation of CDS2 sequences among species
We investigated the sequence similarity of the coding sequence, predicted AA sequence, and the 3′-UTR of CDS2 among species. The chicken CDS2 coding sequence shared 96% similarity with turkey (XM_021375090.1), 81.0% with human (NM_003818.3), 81.2% with mouse (NM_138651.6), 81.1% with cattle (NM_001078046.1), 76.5% with frog (NM_001126503.1), and 78.7% with zebrafish (NM_201186.1). The deduced amino acid sequence of chicken CDS2 shared 99.1% homology with turkey (XP_010721376.1), 91.4% with human (NP_003809.1), 93.0% with mouse (NP_619592.1), 91.0% with cattle (NP_001071514.1), 85.9% with zebrafish (NP_957480.1), and 87.7% with frog (NP_001119975.1) (Fig. S2). The 3′-UTR sequence of chicken CDS2 (KC886603) was also conserved among birds, which shares 87.7% identity with quail (NC_029537.1), 75.3% with duck (NC_045585.1), and 75.2% with goose (NW_013185922.1). The 3′-UTR of CDS2 among mammals was also conserved and the 3′-UTR of CDS2 in humans (NM_003818.3) shared more than 93% similarity with monkey (XM_012052138.1), 71.7% with horse (XM_005604492.3), and 67.6% with seal (XM_027118224.1) while no identical sequences were identified in the 3′-UTR of CDS2 between birds and mammals.
Syntenic analysis revealed that chicken CDS2 was located between PCNA and ARHGAP25/BMP10 on chr.22, close to the chromosomal breakage/fusion point of the mammalian/bird evolutionary event. The upstream chromosomal region of CDS2 in birds containing the CDS2-PCNA-TMEM230-SVCT gene is homologous to chr.20 in human and chr.2 in mouse while the downstream chromosomal region containing ARHGAP25-BMP10-GKN2-GKN1 was homologous to chr.2 in human and chr.6 in mouse (Fig. 2). The amino acid phylogenetic tree of CDS2 (Fig. S3) reflects the evolutionary relationship among species similar to the syntenic analysis of chromosomes (Fig. 2).
Tissue expression profile of chicken CDS2
We conducted semi-quantitative reverse transcription PCR (RT-PCR) and qPCR to investigate the tissue expression pattern of chicken CDS2 in 49 day-old Silky chickens with CP2, AS1, and QP2 primer sets (Table S2).
Amplification with the CP2 primer set located in the coding region of the CDS2 gene reflects the total expression level of CDS2 (referred to as total CDS2). Total CDS2 was extensively expressed in all detected tissues (Fig. 3A). The qPCR revealed that total CDS2 was relatively highly expressed in adipose tissues including sebum (at the tail root), abdominal fat, and neck fat, followed by heart and leg muscle, and weakly expressed in the liver and pectoralis (P = 0.0528, Fig. 3B).
Primer pair AS1, which can simultaneously amplify four splicing forms, was used to observe the tissue expression pattern of multiple transcripts by semi-quantitative RT-PCR (Figs. 4A and S4). The amplified lengths of the CDS2-1, CDS2-2, CDS2-3, and CDS2-4 transcripts should be 245, 379, 504, and 1142 bp, respectively. QP2 is located in the specific region of the longest transcript CDS2-4, which could specifically detect expression of the CDS2-4 variant. CDS2-4 was extensively expressed, and predominantly present in nearly all detected tissues (Figs. 4 and S4). Expression of CDS2-3 was detected in almost all tissues, while the short transcripts CDS2-1 and CDS2-2 were undetected with the AS1 primer set in two separate experiments (Figs. 4A and S4).
The qPCR with the QP2 primer revealed that CDS2-4 showed a similar expression pattern as total CDS2, with a relative high mRNA level in adipose tissues including sebum, abdominal fat, and neck fat, followed by heart, testis, brain, and leg muscle, and low levels in the liver and pectoralis (Fig. 5). Compared with the tissue expression pattern of total CDS2 detected with the CP2 primer set (P = 0.0528, Fig. 3B), the level of CDS2-4 showed greater tissue fluctuation (P < 0.0001, Fig. 5). CDS2-4 exhibited similar mRNA levels in heart and adipose tissues including sebum, abdominal fat, and neck fat, while CDS2-4 levels in abdominal fat were significantly higher than those of other tissues (P < 0.01). The CDS2-4 level in adipose tissue was approximately four-fold that in the testis and 120-fold that in the liver (Fig. 5).
The spatio-temporal expression patterns of total CDS2 and CDS2-4
We further investigated the spatio-temporal expression patterns of total CDS2 (with the CP2 primer set) and CDS2-4 (with the QP2 primer set) by qPCR. One-way ANOVA was used to analyze the effect of age and tissue on the expression of total CDS2 and CDS2-4 separately. Total CDS2 exhibited similar temporal expression patterns (Fig. 6A) in the brain (P = 0.1623), liver (P < 0.0001), and pectoralis (P = 0.1712). In general, the total CDS2 mRNA level was the highest in the later stage of embryogenesis (embryonic day 19 [E19]/ one-day-old [D1]), and the E19 level in the liver was significantly higher than that of the other time points (P < 0.05). In addition, the relative expression of total CDS2 in the brain was significantly (P < 0.05) higher than that in the liver (9-fold) and pectoralis (3-fold) at 49 days (Fig. 6A).
Unlike total CDS2, the mRNA level of the CDS2-4 variant presented a distinct temporal change in pattern in the brain, liver, and pectoralis (Fig. 6B). CDS2-4 levels peaked at 21 days in the brain (P = 0.1336) and pectoralis (P = 0.0104). The level of CDS2-4 in the pectoralis at 21 days was significantly higher than that at the other ages tested, with the exception of 1 day (P < 0.05); whereas hepatic CDS2-4 levels presented a decreasing trend with development, with highest levels at the early stage of hatching (E10), a weak decrease during embryogenesis (E14–D1), and a clear decrease with age after hatching (D21 and D49, P = 0.009). Hepatic CDS2-4 levels at E10 were significantly higher than at 21 days (P < 0.05) and 49 days (P < 0.01) (Fig. 6B). In addition, the brain had the highest level of CDS2-4 at all time points among the three tissues; brain CDS2-4 levels at 21 days were significantly higher than those in the liver and pectoralis (P < 0.01, Fig. 6B).
Effect of exogenous insulin on chicken CDS2 expression
Exogenous insulin resulted to the rapid drop of blood glucose (until 120min) in both Silky chickens and broilers, and Silky chickens presented a more rapid recovery of blood glucose than broilers after 120 min (Fig. 7A and 7B). Exogenous insulin downregulated the expression of total CDS2 in the pectoralis of Silky fowl (p = 0.003, Fig. 7F), where the total CDS2 level at 240 min was significantly lower than at 0 and 120 min (Fig. 7F), and the total CDS2 level in the pectoralis of Silky chickens was lower than that of broiler chickens at 240 min after insulin injection (P < 0.05, Fig. 7H). The mRNA level of total CDS2 in the liver of Silky chickens was higher than that of broilers at the basal state and after insulin stimulation (P < 0.05, Fig. 7G), and exogenous insulin could weakly upregulate the mRNA level of total CDS2 at 240 min in the livers of broilers (P = 0.078, Fig. 7C).