Cloning and expression analysis of Runx2b in Culter alburnus

doi:10.3969/j.issn.1004-1524.20230755

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (5): 1024-1031.DOI: 10.3969/j.issn.1004-1524.20230755

• Animal Science • Previous Articles Next Articles

Cloning and expression analysis of Runx2b in Culter alburnus

HE Changxi¹^,²(), ZHENG Jianbo², MA Jianbo³, JIA Yongyi¹, LIU Shili², JIANG Wenping², CHI Meili², CHENG Shun², LI Fei²^,^*()

1. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
2. Key Laboratory of Freshwater Aquaculture Genetic and Breeding, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, Zhejiang, China
3. Yuyao Aquaculture Technology Extension Center, Yuyao 315400, Zhejiang, China

Received:2023-06-13 Online:2024-05-25 Published:2024-05-29

Abstract

Abstract:

In order to explore the preliminary function of Runx2b gene of Culter alburnus during the ossification process of intermuscular bones (IBs), we obtained the full-length coding sequence of Runx2b gene by transcriptome data and PCR verification. In this study, we staged the different periods of IBs formation by alizarin red staining, and spatial and temporal expression level of Runx2b were investigated by qRT-PCR. The results showed that the open reading frame (ORF) of Runx2b was 1 401 bp, which encoded 466 amino acids with a predicted molecular weight of 50.676 ku and a theoretical isoelectric point of 9.35. The results of amino acid multi-alignment showed that the topmouth culter Runx2b protein displayed a higher identity of 95% with other cyprinidae fish specie, and shared highly conserved Runt and RunxI domains. Phylogenetic tree analysis showed that the topmouth culter Runx2b was most closely related to that of Megalobrama amblycephala. Alizarin red staining showed that the fins and axial skeleton had developed completely before IBs formation. A few IBs of small length emerged in the tail at 15 d, and had a mature morphology and length at 30 d. In addition, mRNA expression abundance was detected in all tested tissues, with the highest expression level in the gill, followed by muscle. Moreover, the expression pattern of Runx2b gene at different growth stages of juveniles was highly consistent with the timing of IBs ossification, indicating its regulation role in IBs development. Our data will contribute to generating IBs-free new varieties and revealing the mechanism of IBs formation.

Key words: Culter alburnus, intermuscular bone, Runx2b, gene expression, ossification

CLC Number:

S917.4

HE Changxi, ZHENG Jianbo, MA Jianbo, JIA Yongyi, LIU Shili, JIANG Wenping, CHI Meili, CHENG Shun, LI Fei. Cloning and expression analysis of Runx2b in Culter alburnus[J]. Acta Agriculturae Zhejiangensis, 2024, 36(5): 1024-1031.

Figures/Tables 7

References 27

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引物名称 Primer name	引物序列 Primer sequences (5'→3')	目的 Application
Runx2b-F	ATGGCATCTAACAGCCTCTTTAGTT	全长扩增
		Full length amplification
Runx2b-R	TCAATACGGCCGCCACACACCTTCG	全长扩增
		Full length amplification
RT-Runx2b-F	AGCAGCCCCAACTCCTTCAC	qRT-PCR
RT-Runx2b-R	AGCAGCCCCAACTCCTTCAC	qRT-PCR
β-actin-F	ACATGAGGCAGACCGTTGCT	qRT-PCR
β-actin-R	GGCAGCCTTCTGTGCAGATT	qRT-PCR

引物名称 Primer name	引物序列 Primer sequences (5'→3')	目的 Application
Runx2b-F	ATGGCATCTAACAGCCTCTTTAGTT	全长扩增
		Full length amplification
Runx2b-R	TCAATACGGCCGCCACACACCTTCG	全长扩增
		Full length amplification
RT-Runx2b-F	AGCAGCCCCAACTCCTTCAC	qRT-PCR
RT-Runx2b-R	AGCAGCCCCAACTCCTTCAC	qRT-PCR
β-actin-F	ACATGAGGCAGACCGTTGCT	qRT-PCR
β-actin-R	GGCAGCCTTCTGTGCAGATT	qRT-PCR

氨基酸类型 Type of amino acid	数量 Numbers	百分比 Percentage/%	氨基酸类型 Type of amino acid	数量 Numbers	百分比 Percentage/%
丙氨酸Ala (A)	27	5.8	精氨酸Arg (R)	33	7.1
天冬氨酸Asn (N)	18	3.9	天冬氨酸Asp (D)	20	4.3
半胱氨酸Cys (C)	4	0.9	谷氨酰胺Gln (Q)	27	5.8
谷氨酸Glu (E)	13	2.8	甘氨酸Gly (G)	33	7.1
组氨酸His (H)	9	1.9	异亮氨酸Ile (I)	7	1.5
亮氨酸Leu (L)	28	6.0	赖氨酸Lys (K)	8	1.7
甲硫氨酸Met (M)	13	2.8	苯丙氨酸Phe (P)	20	4.3
脯氨酸Pro (P)	53	11.4	丝氨酸Ser (S)	64	13.7
苏氨酸Thr (T)	38	8.2	色氨酸Trp (W)	4	0.9
酪氨酸Tyr (Y)	15	3.2	缬氨酸Val (V)	32	6.9

氨基酸类型 Type of amino acid	数量 Numbers	百分比 Percentage/%	氨基酸类型 Type of amino acid	数量 Numbers	百分比 Percentage/%
丙氨酸Ala (A)	27	5.8	精氨酸Arg (R)	33	7.1
天冬氨酸Asn (N)	18	3.9	天冬氨酸Asp (D)	20	4.3
半胱氨酸Cys (C)	4	0.9	谷氨酰胺Gln (Q)	27	5.8
谷氨酸Glu (E)	13	2.8	甘氨酸Gly (G)	33	7.1
组氨酸His (H)	9	1.9	异亮氨酸Ile (I)	7	1.5
亮氨酸Leu (L)	28	6.0	赖氨酸Lys (K)	8	1.7
甲硫氨酸Met (M)	13	2.8	苯丙氨酸Phe (P)	20	4.3
脯氨酸Pro (P)	53	11.4	丝氨酸Ser (S)	64	13.7
苏氨酸Thr (T)	38	8.2	色氨酸Trp (W)	4	0.9
酪氨酸Tyr (Y)	15	3.2	缬氨酸Val (V)	32	6.9

Cloning and expression analysis of Runx2b in Culter alburnus

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