Cloning and Expression Analysis of Rana dybowskii LEPR Gene During Infection

doi:10.7668/hbnxb.20193754

Abstract

Abstract:

In order to explore the biological function of LEPR gene in amphibians,we infected Rana dybowskii with Aeromonas hydrophila(Ah)to establish an inflammatory model,and analyzed expression profile of LEPR gene after Ah infection.Firstly,the LEPR gene was cloned by RT-PCR technology and bioinformatics analysis was performed,and then the inflammation model of Rana dybowskii was constructed.The histopathological changes during infection were observed by hematoxylin eosin staining(HE staining),and the differences of tissue expression of LEPR gene in physiological and infectious states were analyzed by qRT-PCR.Finally,the changes of LEPR protein expression in kidney,skin and muscle tissues were verified by immunohistochemistry staining.The results showed that the length of LEPR gene sequence of Rana dybowskii was 3 604 bp,and its open reading frame was 3 405 bp,encoding 1 134 amino acids.Subcellular localization showed that the LEPR protein was a membrane protein with a primary transmembrane domain.The homology analysis confirmed that the homology between Rana dybowskii and amphibians was more than 52.6%,indicating that LEPR was less conservative.Based on qRT-PCR results,LEPR mRNA was expressed in the heart,liver,spleen,lung,kidney,skin,muscle and stomach of the healthy Rana dybowskii,and its relative expression in skin tissue was significantly higher than that in other tissues.After Ah infection,the LEPR gene was significantly up-regulated in different tissues,but the response time and level were different.Immunohistochemical results showed that the change trend of LEPR protein expression in kidney,skin and muscle was basically consistent with that of qRT-PCR.The strong response of the LEPR gene in the skin tissue also indicates that it may participate in the infection process,which lays a foundation for further expanding the immunological function of the amphibian LEPR gene.

Key words: Rana dybowskii, Aeromonas hydrophila, LEPR gene, Tissue expression

XU Dongmei, LIU Tingting, LIU Yiming, LIU Yufen, LIU Peng, ZHAO Wenge. Cloning and Expression Analysis of Rana dybowskii LEPR Gene During Infection[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(3): 227-238. doi: 10.7668/hbnxb.20193754.

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Figures/Tables 12

Tab.1 Primers used in this study

基因 Gene	引物序列(5'-3') Primer sequences (5'-3')	退火温度/℃ Tm	产物长度/bp Product size	目的 Purpose
LEPR-A	F:ACACTCGCCAAACACTCA	47.3	969	RT-PCR
	R:AACGCACTTCAAACACCAG
LEPR-B	F:CGCTGGTGTTTGAAGTGC	53.8	683	RT-PCR
	R:GGGAATGACGCAGACAGG
LEPR-C	F:GAGGACGGATTTTACAGATG	54.5	1 252	RT-PCR
	R:AGGATTAAGGGGAAACTGGG
LEPR-D	F:ATGAGATGCAGAATAATG	51.5	1 099	RT-PCR
	R:ACTATCTTTGCAGCAAAC
LEPR-q	F:GGGAATGACGCAGACAGG	52.0	121	qRT-PCR
	R:GGAGGACGGATTTTACAGAT
β-actin	F:AAGAATGAGGGCTGGAACA	52.0	176	qRT-PCR
	R:GTGCGTGACATCAAGGAGAAGC

Fig.1 Cloning of LEPR gene in Rana dybowskii

Fig.2 Physicochemical properties of LEPR protein in Rana dybowskii A.The transmembrane domain of LEPR protein;B.Prediction of LEPR protein phosphorylation sites;C.Hydrophilic/hydrophobic analysis of LEPR protein;D.Prediction of LEPR protein functional domain.

Fig.3 Protein secondary and tertiary structures and protein interaction networks A.LEPR protein secondary structure;B.LEPR protein tertiary structure;C.LEPR protein interaction network.

Fig.4 Homology analysis from LEPR and construction of amino acid phylogenetic tree of Rana dybowskii A.LEPR gene homology;B.LEPR phylogenetic tree.

Fig.5 Histopathological section of Rana dybowskii challenged with Ah Observation by HE staining in different tissues of Rana dybowskii infected by Ah.A.Liver;B.Skin;1,2,3.0,36,72 h.

Fig.6 Expression level of LEPR gene in different tissues of Rana dybowskii Different lowercase letters indicate significant differences in relative expression among tissues(P<0.05).

Fig.7 Changes of relative expression of LEPR gene in Rana dybowskii infected by Ah A.Heart;B.Liver;C.Spleen;D.Lung;E.Kidney;F.Skin;G.Muscle;H.Stomach.Different lowercase letters indicate significantly different at 5% level(P<0.05).

Fig.8 Immunohistochemical staining of kidney at different time points after Ah infection A-G respectively represent the kidney tissue in the control group,8,16,24,36,48,72 h after infection.

Fig.9 Immunohistochemical staining of skin at different time points after Ah infection A-G respectively represent the skin tissue in the control group,8,16,24,36,48,72 h after infection.

Fig.10 Immunohistochemical staining of muscle at different time points after Ah infection A-G respectively represent the muscle tissue in the control group,8,16,24,36,48,72 h after infection.

Fig.11 Differential expression of LEPR in kidney,skin and muscle of Rana dybowskii infected with Ah Different capital letters are significantly at 1% level(P<0.01).

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