军曹鱼nanos1的克隆及其在胚胎和性腺发育过程中的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1039

摘要/Abstract

摘要：

旨在探讨军曹鱼nanos1基因（Rcnanos1）的序列特征,及其在胚胎和性腺发育过程中的功能,利用cDNA末端快速扩增技术（RACE）克隆获得Rcnanos1基因的cDNA序列,全长1 290 bp,其中5'非编码区139 bp,3'非编码区470 bp,开放阅读框（ORF）681 bp,共编码226个氨基酸。序列多重比对结果显示,RcNanos1氨基酸序列包含两个连续的锌指功能结构域,与尖吻鲈（Lates calcarifer）Nanos1的一致性最高（99.6%）。系统进化分析表明军曹鱼Nanos1与尖吻鲈（Lates calcarifer）和斜带石斑鱼（Epinephelus coioides）的同源蛋白亲缘关系最近。实时荧光定量PCR（qRT-PCR）结果显示,Rcnanos1在150 dph（days post hatching）军曹鱼的13种组织中均有表达,但在性腺中的表达量显著高于其它组织。胚胎发育过程中,Rcnanos1在早期卵裂阶段的表达水平较低,16细胞期时表达量开始显著升高,并在1 dph仔鱼中的表达量上升至最大值。Rcnanos1在精巢和卵巢首周年发育过程中的表达模式相似,在精巢中（Ⅱ-Ⅴ期）,Rcnanos1的表达水平呈逐渐升高趋势,360 dph（Ⅴ期）时的表达量最高;在卵巢中（Ⅰ-Ⅲ期）,Rcnanos1的表达量同样在360 dph（Ⅲ期）时达到峰值。由此推测,Rcnanos1基因可能在胚胎和性腺发育过程发挥一定的调控作用,可为揭示军曹鱼生殖细胞发生发育的分子机理提供理论基础。

关键词: 军曹鱼, nanos1, 性腺发育, 胚胎发育, 表达分析

Abstract:

In order to explore the sequence characteristics of nanos1 gene and its function in embryonic and gonadal development of Rachycentron canadum,the full-length cDNA sequence of R.canadum nanos1（Rcnanos1）was cloned using the rapid amplification of cDNA ends（RACE）technology for the first time. In total,the sequence comprised of 1 290 bp,including a 5'-UTR of 139 bp,a 3'-UTR of 470 bp,and an open reading frame（ORF）of 681 bp which encoded a protein of 226 aa. The deduced amino acid sequence of RcNanos1 contained two continuous zinc finger functional domains. Comparisons of the deduced amino acid sequence with those of other teleosts revealed the highest percentage identity（99.6%）with Lates calcarifer. Phylogenetic analysis showed that the Nanos1 of R.canadum was the most closely related to the homologous proteins of Lates calcarifer and Epinephelus coioides. The results of real-time quantitative PCR（qRT-PCR）indicated that Rcnanos1gene was expressed in 13 tissues of 150 dph（days post hatching）R.canadum,and the expression in the gonad was significantly higher than that in other tissues.. During embryonic development of R.canadum,the expression of Rcnanos1 was low at early cleavage stage,and began to increase significantly at 16-cell stage,and reached the highest expression in 1dph larvae. The expression patterns of Rcnanos1 gene during annual development of testis and ovary were similar. The expression of Rcnanos1 in the testis（stage Ⅱ to Ⅴ）increased gradually and reached the maxmium at 360 dph（stage Ⅴ）. In ovary（stageⅠto Ⅲ）,the expression of Rcnanos1also reached the maxmium at 360 dph（stage Ⅲ）. These findings suggested that Rcnanos1 play a certain role in regulating embryonic and gonadal development and provide a theoretical basis for revealing the molecular mechanism of germ cell development in R.canadum.

Key words: Rachycentron canadum, nanos1, gonadal development, embryonic development, expression analysis

李豫, 陈刚, 马骞, 邝杰华, 陈有铭. 军曹鱼nanos1的克隆及其在胚胎和性腺发育过程中的表达分析[J]. 生物技术通报, 2022, 38(5): 175-182.

LI Yu, CHEN Gang, MA Qian, KUANG Jie-hua, CHEN You-ming. Cloning of nanos1 Gene of Rachycentron canadum and Its Expression Analysis in the Embryonic and Gonadal Development[J]. Biotechnology Bulletin, 2022, 38(5): 175-182.

图/表 7

表1 本实验所用引物序列

Table 1 Primer sequences used in this study

引物名称 Primer	序列Sequence（5'-3'）	用途 Usage
Rcnanos1-F	ATGGATTTTCTCAACCACAACTATT	CDS序列克隆 CDS sequence cloning
Rcnanos1-R	TTAGAATATTTTCATCCTCTTACCACC	CDS序列克隆 CDS sequence cloning
Rcnanos1-3' F1	GCCCAAAATCTGCGTCTTCTGC	3' 末端序列克隆 3' RACE
Rcnanos1-3' F2	TCAAAAGACCAGCCATCCCAGC	3' 末端序列克隆 3' RACE
Rcnanos1-5' R1	GCTCCTGGAAGCGGGTTTGC	5' 末端序列克隆 5' RACE
Rcnanos1-5' R2	CGATTACGCACGGACACGCC	5' 末端序列克隆 5' RACE
Rcnanos1-F1	AGAACCCCAACTCCATCACC	Rcnanos1的qRT-PCR检测 Expression ofRcnanos1
Rcnanos1-R1	TCTGGAAGGGGCTCAGTATC	Rcnanos1的qRT-PCR检测 Expression ofRcnanos1
β-actin-F	AGGGAAATTGTGCGTGAC	内参基因 Internal control gene
β-actin-R	AGGCAGCTCGTAGCTCTT	内参基因 Internal control gene

图1 Rcnanos1 cDNA全长序列和氨基酸序列分析粗体表示起始密码子和终止密码子;粗体斜体表示加尾信号;灰色阴影表示锌指结构

Fig.1 Complete sequence of Rcnanos1 cDNA and analysis of deduced amino acid sequence The initiation codon ATG and stop codon TAA are in bold. The polyadenylation signal ATTAAA are in bold and italic. The zinc finger motif are highlighted in grey shadow

图2 军曹鱼nanos1氨基酸序列多重序列比对分析方框表示保守的锌指结构域

Fig.2 Multiple alignment of R. canadum nanos1 deduced amino acid sequence The frame regions indicate the zinc finger motif

图3 基于nanos1氨基酸序列构建的系统进化树（NJ树）节点上的数字为各分支的置信度,标尺0.05为进化距离

Fig.3 Phylogenetic tree of nanos1 amino acid sequences based on Neighbor-Joining（NJ）method Numbers indicates the confidence level of each branch,the scale bar 0.05 in terms of genetic distance is indicated below the tree

图4 qRT-PCR分析Rcnanos1在军曹鱼不同组织中的表达 LI：肝脏;SP：脾;KI：体肾;BR：脑;HE：心脏;GI：鳃;TE：精巢;OV：卵巢;ST：胃;IN：肠;MU：肌肉;SK：皮肤;EY：眼睛;上标不同字母表示差异显著（P<0.05）

Fig.4 Expressions of Rcnanos1 in the tissues of R. canadum by qRT-PCR LI：Liver. SP：Spleen. KI：Kidney. BR：Brain. HE：Heart. GI：Gill. TE：Testis. OV：Ovary. ST：Stomach. IN：Intestines. MU：Muscle. SK：Skin. EY：Eye. Different superscripts indicate significant difference（P<0.05）

图5 Rcnanos1在胚胎发育过程中的表达分析 1：1细胞期;2：2细胞期;3：4细胞期;4：8细胞期;5：16细胞期;6：多细胞期;7：囊胚期;8：原肠胚期;9：神经胚期;10：器官形成期;11：孵化期;12：孵化后1 d;上标不同字母表示差异显著（P<0.05）

Fig.5 Expressions of Rcnanos1 during the embryonic deve-lopment stages 1：1-cell stage;2：2-cell stage;3：4-cell stage;4：8-cell stage;5：16-cell stage;6：poly-cell stage;7：blastocyst stage;8：gastrulation stage;9：neuroblast stage;10：organ formation stage;11：hatching stage;12：1 day after hatching. Different superscripts indicate significant difference（P<0.05）

图6 军曹鱼性腺周年发育过程中Rcnanos1 mRNA的表达水平 A：Rcnanos1 mRNA在精巢中的表达水平;B：Rcnanos1 mRNA在卵巢中的表达水平;上标不同字母表示差异显著（P<0.05）

Fig.6 Rcnanos1 mRNA expression in annual gonadal deve-lopment of R. Canadum A：Rcnanos1 mRNA expression in testis;B：Rcnanos1 mRNA expression in ovary. Different superscripts indicate significant difference（P<0.05）

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