牦牛<i>ANXA5</i>基因的克隆、序列分析及表达研究

doi:10.7668/hbnxb.20193457

华北农学报 ›› 2023, Vol. 38 ›› Issue (2): 224-231. doi: 10.7668/hbnxb.20193457

所属专题： 畜牧；生物技术；

• 畜牧·水产·兽医 • 上一篇下一篇

牦牛ANXA5基因的克隆、序列分析及表达研究

范依琳¹^,²^,³, 赵丹¹^,²^,³, 马妍¹^,²^,³, 岳永起¹^,²^,³, 冯欣欣¹^,²^,³, 张姬越¹^,²^,³, 字向东¹^,²^,³, 熊显荣¹^,²^,³, 付伟¹^,²^,³, 熊燕¹^,²^,³

1.西南民族大学畜牧兽医学院,四川成都 610041
2.西南民族大学,青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都 610041
3.西南民族大学,青藏高原动物遗传资源保护与利用四川省重点实验室,四川成都 610041

收稿日期:2022-12-15 出版日期:2023-04-28
通讯作者:
熊燕(1987-),女,四川茂县人,副教授,博士,主要从事动物遗传育种与繁殖研究。
熊燕(1987-),女,四川茂县人,副教授,博士,主要从事动物遗传育种与繁殖研究。
作者简介:
范依琳(1996-),女,广东佛山人,在读硕士,主要从事动物遗传育种与繁殖研究。
基金资助:
四川省科技计划项目(2019YJ0258); 西南民族大学2022年研究生“创新型科研项目”资助重点项目(ZD2022966)

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

FAN Yilin¹^,²^,³, ZHAO Dan¹^,²^,³, MA Yan¹^,²^,³, YUE Yongqi¹^,²^,³, FENG Xinxin¹^,²^,³, ZHANG Jiyue¹^,²^,³, ZI Xiangdong¹^,²^,³, XIONG Xianrong¹^,²^,³, FU Wei¹^,²^,³, XIONG Yan¹^,²^,³

1. College of Animal & Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China
2. Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education,Southwest Minzu University,Chengdu 610041,China
3. Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province,Southwest Minzu University,Chengdu 610041,China

Received:2022-12-15 Published:2023-04-28

摘要/Abstract

摘要：

为了深入地讨论牦牛ANXA5基因序列的特点,阐述其组织及细胞表达特性,通过采集牦牛不同组织样品,如心、肝、脾、肺、肾、子宫、卵巢、输卵管等,提取总RNA。采用PCR、生物信息学软件、实时荧光定量PCR、免疫组化等技术对牦牛ANXA5基因进行克隆、序列分析及表达特性研究。结果显示,牦牛ANXA5基因CDS区长为966 bp,共编码321个氨基酸。与黄牛比较,发现共有6个碱基突变存在于牦牛ANXA5基因中。黄牛与牦牛的核苷酸和氨基酸同源性大于99%,与其他哺乳动物进行氨基酸同源性比较,结果也均在90%以上,说明ANXA5基因在长期进化中保守。在牦牛的肺组织中,ANXA5基因表达量最高,与其他组织差异极显著(P<0.01);在子宫和输卵管组织中表达量次之。ANXA5在牦牛不同时期卵巢颗粒细胞中均有表达,且表达量随时间增长而逐渐升高。结果显示,培养72 h颗粒细胞中ANXA5表达量极显著高于培养24,36 h颗粒细胞(P<0.01);培养48 h颗粒细胞中ANXA5表达量极显著高于培养24 h颗粒细胞(P<0.01),显著高于培养36 h颗粒细胞(P<0.05),表明ANXA5在黄体化的颗粒细胞中表达量更高。然而,免疫组化结果显示,ANXA5的亚细胞定位于细胞质,在黄体细胞中表达较高。

关键词: 牦牛, ANXA5, 基因克隆, 组织表达, 时序表达及定位

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

范依琳, 赵丹, 马妍, 岳永起, 冯欣欣, 张姬越, 字向东, 熊显荣, 付伟, 熊燕. 牦牛ANXA5基因的克隆、序列分析及表达研究[J]. 华北农学报, 2023, 38(2): 224-231. doi: 10.7668/hbnxb.20193457.

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.

http://www.hbnxb.net/CN/Y2023/V38/I2/224

图/表 8

表1 引物信息

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

图1 牦牛ANXA5基因克隆 A.牦牛ANXA5基因扩增结果:M.DL2000 DNA Marker,1-4.ANXA5目的条带;B.牦牛和黄牛ANXA5基因序列差异。

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.

表2 牦牛与10个物种ANXA5基因相似性比对

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

图2 基于ANXA5基因核苷酸构建的系统进化树

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

图3 牦牛ANXA5基因预测 A.牦牛ANXA5基因信号肽分析;B.牦牛ANXA5基因跨膜结构分析。

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

图4 牦牛ANXA5蛋白预测 A.牦牛ANXA5蛋白二级结构预测:e.延伸链,h.α-螺旋,c.自由卷曲,t.β-转角;B.牦牛ANXA5蛋白三级结构图;C.牦牛ANXA5蛋白质互作网络。

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.

图5 牦牛ANXA5基因组织表达与ANXA5蛋白定位 A.ANXA5在牦牛不同组织中的相对表达量(以肾表达水平作为对照,GAPDH作为内参基因);B.ANXA5在牦牛卵巢中的定位:a.阴性对照-次级卵泡,b.阴性对照-黄体,c.阴性对照-闭锁卵泡,d.ANXA5阳性染色-次级卵泡,e.ANXA5阳性染色-黄体,f.ANXA5阳性染色-闭锁卵泡;C.ANXA5在颗粒细胞及黄体中的平均染色强度。不同小写字母表示差异显著(P<0.05);不同大写字母表示差异极显著(P<0.01)。图6同。

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.

图6 ANXA5在牦牛不同时期卵巢颗粒细胞中的相对表达量

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

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牦牛ANXA5基因的克隆、序列分析及表达研究

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

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