Identification of the members in the CDK family
To identify the CDK family members, 59 verified CDK amino acid sequences from cattle (Bos taurus, 8), human (Homo sapiens, 26) and mouse (Mus musculus, 25) were used as the query for genome-wide detection of the homologous sequences in Bos taurus, Bos indicus, Bos grunniens, Hybrid-Bos Indicus, Hybrid-Bos taurus, Bos mutus, Bison bison bison, and Bubalus bubalis. As a result, 25 non-redundant CDK protein sequences including CDK1-10,CDK11B, CDK12-20, CDKL1-5 were identified in Bos taurus (Table 1). In parallel, 22, 21, 22, 24, 22, 25 and 24 CDK family proteins were recognized in Bos grunniens, Hybrid-Bos Indicus, Hybrid-Bos taurus, Bos mutus, Bison bison bison, Bos indicus and Bubalus bubalis, respectively (Additional file 1) and the protein sequences of all the CDKs were provided in Additional file 2. Transcript ENSBIXT00000049337 is a newly identified member of CDK family in Hybrid-Bos taurus, which is named CDK20 according to the sequence similarity and collinearity.
Table 1
Details of Genome-wide identified CDK family members in Bos taurus
Protein Name | gene ID | transcript ID | pI | Mw/Da | Amino acids | description |
CDK1 | ENSBTAG00000010109 | ENSBTAT00000013337 | 8.38 | 34025.40 | 297 | cyclin dependent kinase 1 |
CDK2 | ENSBTAG00000004021 | ENSBTAT00000005252 | 8.79 | 33873.46 | 298 | cyclin dependent kinase 2 |
CDK3 | ENSBTAG00000010509 | ENSBTAT00000013885 | 8.13 | 34805.48 | 305 | cyclin dependent kinase 3 |
CDK4 | ENSBTAG00000007160 | ENSBTAT00000009420 | 6.51 | 33646.73 | 303 | cyclin dependent kinase 4 |
CDK5 | ENSBTAG00000007766 | ENSBTAT00000010212 | 7.57 | 33288.47 | 292 | cyclin dependent kinase 5 |
CDK6 | ENSBTAG00000044023 | ENSBTAT00000061349 | 6.22 | 37014.40 | 326 | cyclin dependent kinase 6 |
CDK7 | ENSBTAG00000011046 | ENSBTAT00000014667 | 8.67 | 38946.26 | 346 | cyclin dependent kinase 7 |
CDK8 | ENSBTAG00000016737 | ENSBTAT00000022252 | 8.72 | 53282.71 | 464 | cyclin dependent kinase 8 |
CDK9 | ENSBTAG00000004695 | ENSBTAT00000006162 | 9.04 | 42747.58 | 372 | cyclin dependent kinase 9 |
CDK10 | ENSBTAG00000033333 | ENSBTAT00000047400 | 9.16 | 41046.93 | 361 | cyclin dependent kinase 10 |
CDK11B | ENSBTAG00000010737 | ENSBTAT00000014227 | 5.34 | 89901.85 | 771 | cyclin dependent kinase 11B |
CDK12 | ENSBTAG00000013238 | ENSBTAT00000002005 | 9.54 | 140641.60 | 1264 | cyclin dependent kinase 12 |
CDK13 | ENSBTAG00000001528 | ENSBTAT00000002003 | 9.71 | 164717.14 | 1512 | cyclin dependent kinase 13 |
CDK14 | ENSBTAG00000048664 | ENSBTAT00000068321 | 9.06 | 53169.98 | 470 | cyclin dependent kinase 14 |
CDK15 | ENSBTAG00000055073 | ENSBTAT00000086547 | 6.68 | 45011.42 | 405 | cyclin dependent kinase 15 |
CDK16 | ENSBTAG00000016769 | ENSBTAT00000022303 | 7.23 | 55758.68 | 496 | cyclin dependent kinase 16 |
CDK17 | ENSBTAG00000001510 | ENSBTAT00000077282 | 9.1 | 59563.16 | 523 | cyclin dependent kinase 17 |
CDK18 | ENSBTAG00000012673 | ENSBTAT00000085187 | 9.26 | 54126.19 | 471 | cyclin dependent kinase 18 |
CDK19 | ENSBTAG00000007288 | ENSBTAT00000009583 | 8.66 | 56685.13 | 500 | cyclin dependent kinase 19 |
CDK20 | ENSBTAG00000015171 | ENSBTAT00000020188 | 6.06 | 38546.53 | 346 | cyclin dependent kinase 20 |
CDKL1 | ENSBTAG00000004780 | ENSBTAT00000036046 | 9.08 | 40735.16 | 352 | cyclin dependent kinase like 1 |
CDKL2 | ENSBTAG00000014038 | ENSBTAT00000031574 | 8.76 | 64289.09 | 569 | cyclin dependent kinase like 2 |
CDKL3 | ENSBTAG00000010979 | ENSBTAT00000014574 | 9.37 | 67477.82 | 591 | cyclin dependent kinase like 3 |
CDKL4 | ENSBTAG00000024044 | ENSBTAT00000033135 | 8.88 | 39465.72 | 342 | cyclin dependent kinase like 4 |
CDKL5 | ENSBTAG00000007428 | ENSBTAT00000076996 | 9.56 | 107236.16 | 960 | cyclin dependent kinase like 5 |
Mw: molecular weight, pI: isoelectric point |
The length of amino acid sequences of 25 cattle CDK proteins ranged from 292 (CDK5) to 1512 (CDK13), and their molecular weight (Mw) was 33288.47-164717.14 Da, which correlated well with the protein length. The isoelectric points (pI) of most CDK family proteins was higher than 8.0, which containing more basic amino acids than acidic amino acids, except for 2 neutral proteins(CDK5 and CDK16), whose pI are 7.57 and 7.23, respectively, and 5 acidic proteins (CDK4, CDK6, CDK11B, CDK15 and CDK20), whose pI is between 5.34 and 6.68. Moreover, we detected all the 25 CDK proteins contained the Serine/Threonine Kinase conserved domain (Additional file 3).
Structural features of bovine CDK family members
To explore the structural characteristics of bovine CDK proteins and genes, the conserved motifs and gene structures were projected based on their phylogenetic relationships (Fig. 1). Results showed the CDKs of cattle initially categorized into three main subfamily according to the evolutionary clades. Among 25 bovine CDK family genes, the first subfamily contains 6 members including CDKL1, CDKL2, CDKL3, CDKL4, CDKL5 and CDK20. The second subfamily possesses CDK10 and CDK11B, and the other members belongs to the third subfamily. Six conserved domains(Motif 1, 3, 5, 6, 7, and 9), containing 29, 21, 21, 21, 21, and 21 amino acids respectively, were shared among all the CDK family proteins (Additional file 4). As a small branch in the third subfamily, CDK16, CDK17 and CDK18, have all of the ten motifs. CDK4, CDK15 and CDK20 all consists of eight motifs, while CDK4 lacks of Motif 4 and Motif 10, CDK15 is short for Motif 2 and Motif 10, and CDK20 is without Motif 4 and Motif 10. The rest CDK proteins comprise nine motifs lacking of CDK10, which indicates they all have the same conserved patterns.
The items of introns, coding sequences (CDS) and untranslated region (UTR) were various among CDK family genes, for instance, the gene length CDKs ranged from 3599nt (CDK4) to 678562nt (CDK14), which is mainly due to the variation in intron. The number of CDS varied from 7 to 17 and the length and layout of 3’UTR and 5’UTR were also various in the noncoding areas. Although CDS, introns and UTRs varied greatly, analysis discovered that CDK family members in the same evolutionary branch tend to show similar gene structures and semblable conserved patterns in motifs.
Phylogenetic relationship of CDK proteins in different organisms
To assess evolutionary relationships of CDK proteins between cattle and other organisms, we conducted a phylogenetic analysis of animals in bovinae (Bos taurus, Bos indicus, Bos grunniens, Hybrid-Bos Indicus, Hybrid-Bos taurus, Bos mutus, Bison bison bison, and Bubalus bubalis). Besides, CDK proteins in Homo sapiens and Mus musculus were also included for they have been studied extensively as two model organisms. Accordingly, 236 amino acid sequences from 10 organisms were aligned to generate nonrooted Neighbor-Joining (NJ) tree (Fig. 2). Phylogenetic analyses revealed CDK family proteins were classified into eight major clades. Clade Ⅰ contained CDK4 and CDK6 and Clade Ⅱ included CDK5, CDK7 and CDK20. Then CDK14 and CDK15 coalesced into a single branch named Clade Ⅲ, CDK11A, CDK11B, CDK16, CDK17 and CDK18 got together named Clade Ⅳ, CDK1, CDK2 and CDK3 got together named Clade Ⅴ, CDK9 of the 10 species merged together named Clade Ⅵ, all the Cyclin Dependent Kinases Like proteins (CDKL1, CDKL2, CDKL3, CDKL4 and CDKL5) classified into a category named Clade Ⅶ, and the rest (CDK8, CDK10, CDK12, CDK13 and CDK19) clustered into a branch named Clade Ⅷ.
Chromosomal distribution and collinearity analysis of CDK genes
CDK family genes were mapped on the chromosomes of six bovinae species (Fig. 3). 25 bovine CDKs distribute on 16 chromosomes including Chr 2, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, Chr 10, Chr 11, Chr 12, Chr 16, Chr 18, Chr 19, Chr 20, Chr 28 and Chr X. Among them, the CDKs of cattle have a similar position distribution with Bos indicus, whereas the arrangement of a few genes on chromesomes are different between cattle and the other species. For example, the order of CDK14 (7.94–8.62 Mb), CDK6 (9.94–10.19 Mb), CDK13 (80.95–81.08 Mb) and CDK5 (113.630-113.634 Mb) in Bos taurus Chr 4 was opposite from that in Hybrid-Bos Indicus, Hybrid-Bos taurus and Bos grunniens, which is CDK5, CDK13, CDK6 and CDK14, respectively. CDK4, CDK2 and CDK7 were three tandem genes in Bos taurus at the location of 29.66–29.69 Mb, 29.66–29.69 Mb and 29.72–29.92 Mb on Chr 5, while the arrangement of these three genes were reversed in Bos grunniens, Hybrid-Bos Indicus Chr 5 and Bubalus Chr 4. In addition, compared with Bos taurus, Bos grunniens lacks of CDK7, CDK11B and CDK20, Hybrid-Bos Indicus lacks of CDK11B, CDK16, CDK20 and CDKL4, Hybrid-Bos taurus is without CDK11B, CDK20, CDKL4 and CDKL5, and Bubalus is short of CDK11B. What’s more, CDKL5 is located on chromosome X in Bos taurus, while on chromosome Y in Bos grunniens.
Collinearity analysis of the genome resulted in the identification of 31,691, 34,495, 33,570, 32,378 and 33,327 pairs of collinear genes between Bos taurus and Bos indicus, Hybrid-Bos Indicus, Hybrid-Bos taurus, Bos grunniens and Bubalus bubalis, respectively(Fig. 4). Results showed that there is a one-to-one correspondence between chromosomes of Bos taurus and Hybrid-Bos Indicus, Hybrid-Bos taurus, Bos indicus and Bos grunniens. A large chromosome homologous also existed between cattle (2 N = 60) and buffalo (2 N = 50), although the chromosome number is different of the two species.The syntenic blocks revealed that Chr1 of buffalo appears to be a fusion of cattle Chr1 and Chr 27, buffalo Chr 2 appears to be a combination of cattle Chr2 and Chr 23, buffalo Chr 3 amounts to cattle Chr 8 and Chr 19, buffalo Chr 4 equals cattle Chr 5 and Chr 28, and buffalo Chr 5 equals cattle Chr- 16 and Chr 29. The detailed syntenic relationships of CDK family genes between cattle and the other five species in bovinae was displayed in Table 2.
Table 2
Syntenic relationships of CDK family genes between cattle and the other five species
Gene | Bos indicus | Hybrid-Bos taurus | Hybrid-Bos Indicus | Bos grunniens | Bubalus bubalis |
CDK1 | Y | Y | Y | Y | Y |
CDK2 | Y | Y | Y | Y | Y |
CDK3 | Y | Y | Y | Y | Y |
CDK4 | Y | Y | Y | Y | Y |
CDK5 | Y | Y | Y | Y | Y |
CDK6 | Y | Y | Y | Y | Y |
CDK7 | N | Y | Y | - | Y |
CDK8 | Y | Y | Y | Y | Y |
CDK9 | Y | Y | Y | Y | Y |
CDK10 | Y | Y | Y | Y | Y |
CDK11B | N | - | - | - | - |
CDK12 | Y | Y | Y | Y | Y |
CDK13 | Y | Y | Y | Y | Y |
CDK14 | Y | Y | Y | Y | Y |
CDK15 | Y | Y | Y | Y | Y |
CDK16 | Y | Y | - | Y | Y |
CDK17 | Y | Y | Y | Y | Y |
CDK18 | Y | Y | Y | Y | Y |
CDK19 | Y | Y | Y | Y | Y |
CDK20 | Y | Y | - | - | Y |
CDKL1 | Y | Y | Y | Y | Y |
CDKL2 | Y | Y | Y | Y | Y |
CDKL3 | Y | Y | Y | Y | Y |
CDKL4 | Y | - | - | Y | Y |
CDKL5 | Y | - | Y | Y | Y |
‘Y’ represents the synteny of genes between two species, while ‘N’ means not and ‘-’ means lacking of the gene. |
The expression analysis of CDKs in different tissue
The expression pattern of genes could provide important references for their function. To explore the expression pattern of the CDK gene family during adipogenic differentiation, we investigated the relative expression level in 163 samples of 60 tissue types including heart, liver, spleen, lung, kidney, muscle, fat, etc. The results showed that CDKs displayed differential expression patterns in diverse tissues (Fig. 5a), which could be classified into 5 groups (A to E). As a marker gene for adipocyte differentiation, PPARγ had a high expression in Group B including omental fat, intramuscular fat, subcutaneous fat and mammary gland fat, indicating that the results is reliable. The 25 CDKs could be grouped into 4 categories according to their expression patterns and they all expressed in 60 tissues, suggesting that they may play a broad regulatory role in life activities. Group Ⅰ(CDK4, CDK9 and CDK11B) showed the highest expression levels, followed by Group Ⅲ(CDK3, CDK5, CDK7, CDK8, CDK10, CDK18, and CDK20) and Group Ⅳ (CDK1, CDK2, CDK6, CDK12, CDK13, CDK14, CDK16, and CDK17). Group Ⅱ comprised the rest members of CDKs, whose expression level was the lowest. Further analysis of the five different fat tissues revealed that CDK9 was highly expressed in all the fat tissues and its expression pattern was similar to PPARγ (Fig. 5b).
Expression analysis of CDKs in preadipocytes and differentiated adipocytes by RNA-seq
Transcriptome analysis of 25 CDKs in preadipocytes and differentiated adipocytes revealed that CDKs showed a up-regulation trend in preadipocytes compared with differentiated adipocytes except for CDK1, CDK3, CDK6, CDK19, CDKL1 and CDKL4 (Fig. 6). CDK7 displayed a significant high expression, whereas CDK1 showed a significant low expression in preadipocytes within the 95% confidence interval. And CDK4, CDK8, CDK9 and CDK14 all displayed a significant high expression in preadipocytes within the 99% confidence interval.
Expression analysis of CDKs during adipocyte differention by qPCR
To further explore the expression pattern of CDK family genes, preadipocytes collected from perirenal adipose tissue of premature calves were induced differentiation. The results of oil red O staining showed that lipid droplet accumulation was significantly increased in adipocytes induced for 10 days compared to preadipocytes (Additional file 5), indicating that the induction and differentiation was successful. And we conducted qPCR to detect the expression of CDKs at 0, 2, 4, 6 and 10 days during adipocytes differentiation(Fig. 7). Results suggested that CDKs showed a relatively high expression in preadipocytes and then decreased as differentiation process went on in addition to CDK1, CDK15, CDK18, CDKL3 and CDKL5. The three members, CDK1, CDKL3 and CDKL5, all had the highest expression on the second day of differentiation and the lowest expression points were on the 6, 8 and 6 day, respectively. The expression level of CDK15 and CDK18 increased with adipocyte differentiation and reached the peak on the fourth day, then decreased.