Cloning of nanos1 Gene of Rachycentron canadum and Its Expression Analysis in the Embryonic and Gonadal Development

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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）

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）

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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