Cloning,Sequence Analysis and Expression of the ANXA5 Gene in Yak

doi:10.7668/hbnxb.20193457

Abstract

Abstract:

In order to obtain the molecular characterization of the yak ANXA5 gene,discuss its expression characteristics in tissues and cells,and larify its medium localization in granulosa cells.Samples of yak heart,liver,spleen,lung,kidney,uterus,ovaries and oviducts were collected.After extraction of total RNA,we used PCR,bioinformatics software,qPCR techniques and immunocytochemistry to clone and analyze sequence of the yak ANXA5 gene and investigated its expression and localization.The results showed that the CDS region of the yak ANXA5 gene was 966 bp in length,encoding 321 amino acids.Compared to the cattle ANXA5 genes,there were 6 base mutations in the yak ANXA5 gene.Both the nucleotide homology and amino acid homology of yak and cattle were more than 99%,and the amino acid homology were more than 90% with other mammals,indicating that the ANXA5 gene was more conservative in evolution.ANXA5 expression level was highest in lung,which was extremely significant higher than the other tissues(P<0.01),followed by uterus and oviduct.ANXA5 was expressed in ovarian granulosa cells of yak at different stages,and the expression level increased gradually with time.The results showed that the expression level of ANXA5 in cultured 72 h granulosa cells were extremely significant higher than that in cultured 24,36 h granulosa cells(P<0.01).The expression level of ANXA5 in cultured 48 h granulosa cells were extremely significant higher than that in cultured 24 h granulosa cells(P<0.01)and significantly higher than that in cultured 36 h granulosa cells(P<0.05),indicating that the expression level of ANXA5 in luteinized granulosa cells was higher.However,the immunohistochemical results showed that the subcellular localization of ANXA5 was in the cytoplasm,and corpus luteum cells were highly expressed.

Key words: Yak, ANXA5, Gene cloning, Tissue expression, Temporal expression and location

FAN Yilin, ZHAO Dan, MA Yan, YUE Yongqi, FENG Xinxin, ZHANG Jiyue, ZI Xiangdong, XIONG Xianrong, FU Wei, XIONG Yan. Cloning,Sequence Analysis and Expression of the ANXA5 Gene in Yak[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 224-231. doi: 10.7668/hbnxb.20193457.

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Figures/Tables 8

Tab.1 Primers information

引物 Primer	引物序列(5'-3') Primer sequences(5'-3')	退火温度/℃ Tm	产物长度/bp Product size	用途 Utilization	登录号 Accession number
ANXA5	F1:GTCGCTCACCTTCTCCTGAC	57	1 168	ANXA5 cDNA扩增	NM_001040477
	R1:CAGAAAGGAGCACCGCTACA
ANXA5	F2:GGATGTTGGTGGTCCTCCTT	60	241	实时荧光定量PCR	NM_001040477
	R2:CCAGAAGTCTCCCGGTCAAT
GAPDH	F:TGTTGTGGATCTGACCTGCC	60	135	实时荧光定量PCR	NM_001034034.2
	R:AAGTCGCAGGAGACAACCTG

Fig.1 Cloning of yak ANXA5 gene A.Amplification of yak ANXA5 gene:M.DL2000 DNA Marker,1-4.ANXA5 target band;B.Sequence difference of ANXA5 gene between yak and cattle.

Tab.2 Gene similarity between yak ANXA5 gene and other 10 species%

物种 Species	氨基酸相似性 Amino acid similarity	核苷酸相似性 Nucleotide similarity
黄牛Bos taurus	99.69	99.38
瘤牛Bos indicus	99.38	99.17
山羊Capra hircus	99.07	98.24
绵羊Ovis aries	98.75	97.68
猫Felis catus	97.20	91.72
人Homo sapiens	96.26	91.92
兔Oryctolagus cuniculus	93.89	87.89
大鼠Rattus norvegicus	92.43	85.83
小鼠Mus musculus	92.52	86.66
原鸡Gallus gallus	77.57

Fig.2 Phylogenetic tree based on nucleotide sequence of yak ANXA5 gene

Fig.3 Prediction of yak ANXA5 gene A.Signal peptide analysis of yak ANXA5 gene;B.Transmembrane analysis of yak ANXA5 gene.

Fig.4 Prediction of yak ANXA5 protein A.Secondary structure prediction of yak ANXA5 protein:e.Extended strand,h.α-helix,c.Random coil, t.β-turn;B.Tertiary structure of yak ANXA5 protein;C.Protein interaction network of yak ANXA5 protein.

Fig.5 Tissue expression of ANXA5 gene and mapping of ANXA5 protein in yak A.The relative expression level of yak ANXA5 gene in different organizations(The kidney expression level was used as the control,GAPDH as the internal reference gene);B.Localization of ANXA5 in yak ovary:a.Negative control-Secondary follicle,b.Negative control-Corpus luteum,c.Negative control-Atresia follicle,d.ANXA5 positive staining-Secondary follicle,e.ANXA5 positive staining-Corpus luteum,f.ANXA5 positive staining-Atresia follicle;C.Average staining intensity of yak ANXA5 in granular cells and corpus luteum.Different small letter superscripts indicate significant difference(P<0.05);different capital letter superscripts indicate extremely significant difference(P<0.01).The same as Fig.6.

Fig.6 The relative expression of yak ANXA5 gene in ovarian granular cells at different stages

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