Open Access

Cloning and differential expression analyses of Cdc42 from sheep

Yong-Jie Yang
Yong-Jie Yang
, 
Zeng-Shan Liu
Zeng-Shan Liu
, 
Shi-Ying Lu
Shi-Ying Lu
, 
Pan Hu
Pan Hu
, 
Chuang Li
Chuang Li
, 
Waqas Ahmad
Waqas Ahmad
, 
Yan-Song Li
Yan-Song Li
, 
Yun-Ming Xu
Yun-Ming Xu
, 
Feng Tang
Feng Tang
, 
Yu Zhou
Yu Zhou
 and 
Hong-Lin Ren
Hong-Lin Ren
   | Mar 30, 2018
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Published Online: Mar 30, 2018
Page range: 113 - 119
Received: Sep 07, 2017
Accepted: Feb 14, 2018
DOI: https://doi.org/10.2478/jvetres-2018-0016
Keywords
© 2018 Y.-J. Yang1et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Sequence analyses of OaCdc42. A. The full-length cDNA and deduced amino acid sequences of OaCdc42. GenBank accession number for OaCdc42 cDNA was KC425615. The conserved domain (CD) was in the frame. Double asterisks (∗∗) represent stop codon (TGA). Primers for 3′-, 5′-RACE are marked with underlined arrows. “S” in bold and italics indicates the predicted phosphorylated sites. Residue numbers of secondary structure are coloured per domain as in panel. B. The three-dimensional structure of OaCdc42. Each of α- helices and β-sheet is indicated by a different colour
Sequence analyses of OaCdc42. A. The full-length cDNA and deduced amino acid sequences of OaCdc42. GenBank accession number for OaCdc42 cDNA was KC425615. The conserved domain (CD) was in the frame. Double asterisks (∗∗) represent stop codon (TGA). Primers for 3′-, 5′-RACE are marked with underlined arrows. “S” in bold and italics indicates the predicted phosphorylated sites. Residue numbers of secondary structure are coloured per domain as in panel. B. The three-dimensional structure of OaCdc42. Each of α- helices and β-sheet is indicated by a different colour

Fig. 2

Multiple alignments of Cdc42 between sheep and other species. The amino acid sequence of OaCdc42 is underlined. (∗) – 100% identical; (:) – highly conserved; (.) – semi-conserved
Multiple alignments of Cdc42 between sheep and other species. The amino acid sequence of OaCdc42 is underlined. (∗) – 100% identical; (:) – highly conserved; (.) – semi-conserved

Fig. 3

Phylogenetic analysis of Cdc42 from sheep compared with other species. The phylogenetic tree was constructed by MAGA5.1 software using neighbour-joining (NJ) method. The numbers on the nodes reveal percentage frequencies in 1,000 bootstrap replications. The scale bar indicates 0.005 substitutions per site
Phylogenetic analysis of Cdc42 from sheep compared with other species. The phylogenetic tree was constructed by MAGA5.1 software using neighbour-joining (NJ) method. The numbers on the nodes reveal percentage frequencies in 1,000 bootstrap replications. The scale bar indicates 0.005 substitutions per site

Fig. 4

Tissue distribution of OaCdc42 mRNA from healthy sheep. The relative value of OaCdc42 mRNA was calculated using 2-ΔΔCt method and β-actin as the reference gene. Data were presented as mean ±SD (n = 3, ∗ P < 0.05; ∗∗ P < 0.01 vs. buffy coat). Error bars showed the SD. All tests were performed in triplicate
Tissue distribution of OaCdc42 mRNA from healthy sheep. The relative value of OaCdc42 mRNA was calculated using 2-ΔΔCt method and β-actin as the reference gene. Data were presented as mean ±SD (n = 3, ∗ P < 0.05; ∗∗ P < 0.01 vs. buffy coat). Error bars showed the SD. All tests were performed in triplicate

Fig. 5

The differential expression of OaCdc42 mRNA between Bm-infected sheep and S2-vaccinated sheep. Relative value of OaCdc42 mRNA was calculated using 2-ΔΔCt method and β-actin as the reference gene. Data were presented as mean ±SD (n = 9, ∗P < 0.05; ∗∗P < 0.01). Error bars showed the SD. All tests were performed in triplicate
The differential expression of OaCdc42 mRNA between Bm-infected sheep and S2-vaccinated sheep. Relative value of OaCdc42 mRNA was calculated using 2-ΔΔCt method and β-actin as the reference gene. Data were presented as mean ±SD (n = 9, ∗P < 0.05; ∗∗P < 0.01). Error bars showed the SD. All tests were performed in triplicate

Primers used for PCR amplification

Primer Nucleotide sequence (5’-3’) Method
Cdc42 F1 GTGTGAGACAAGGCCCGTAGGTATG 3’- RACE; outer
Cdc42 F2 TGGCCCCTTCCCCTCTCAATACTAG 3’-RACE; inner
Cdc42 R1 GCCATACCTACGGGCCTTGTCTCAC 5’-RACE; outer
Cdc42 R2 AGGTGCAGGGCATTTGTCATTATTG 5’-RACE; inner
Cdc42 F’ GTTGTTGTGGGTGATGGTGCTGTTG Real-time PCR
Cdc42 R’ CACTGAGAGGCAGACCAGAAACACG Real-time PCR
β-actin F’ CCCAAGGCCAACCGTGAGAAGATGA Real-time PCR
β-actin R’ CGAAGTCCAGGGCCACGTAGCAGAG Real-time PCR
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Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
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