Cloning of Dmrta1 Gene and Its Temporal and Spatial Expression in Tissue of Silurus lanzhouensis

doi:10.7668/hbnxb.20193954

Abstract

Abstract:

In order to understand the biological characteristics of Dmrta1 gene and its physiological role in the growth and development of Silurus lanzhouensis,and to provide theoretical basis and technical support for the realization of full male S.lanzhouensis population production.Homologous cloning and cDNA terminal rapid cloning(RACE)techniques were used to obtain the full-length cDNA sequence of S.lanzhouensis Dmrta1.qRT-PCR was used to detect the expression characteristics of this gene in different tissues of S.lanzhouensis such as embryo development,larvae and juveniles,third instar hermaphroditic and third instar hermaphroditic.The results showed that the cDNA length of Dmrta1 gene was 1 415 bp and that of ORF was 1 158 bp,encoding 385 amino acids.SMART prediction showed that the gene belonged to a typical Dmrta subfamily protein with conserved DM and DMA domains.The sequence alignment and phylogenetic tree analysis showed that S.lanzhouensis and S.meridionalis had the highest similarity(95.47%) and were closely related.The results of tissue expression showed that Dmrta1 mRNA was expressed in all stages of unfertilized and embryonic development,and the highest expression was in unfertilized egg(P<0.05).In different developmental stages of S.lanzhouensis within 40 days after incubation,the expression of Dmrta1 mRNA in XX individuals was significantly higher than that in XY individuals,and rapidly reached the peak value on 35 days(P<0.05).The expression of Dmrta1 mRNA in the ovary and gills of female catfish of the third age was significantly higher than that in other tissues(P<0.05),and the expression in the ovary was significantly higher than that in the gills of male and female catfish.Except that the expression in the ovary was 47.07 times that in the sperms,there was no difference between male and female in other tissues.The expression characteristics of the third instar hermaphrodite and the third instar hermaphrodite were the same,and the egg expression was significantly higher than that of the gill and germ nest(P<0.05).These results indicated that Dmrta1 gene had parental genetic characteristics and belongs to the mother gene.This gene may play an important role in the process of sex differentiation,ovum formation and gill arch formation of S.lanzhouensis,and it has the same role in the gonads of both hermaphroditic and hermaphroditic S.lanzhouensis.

Key words: S.lanzhouensis, Dmrta1 gene, Gene cloning, Early life stages, Hermaphroditism, mRNA expression

LIU Ting, LI Minmin, LAI Zhanglong, LIU Yanbin, LIU Kai, XIAO Wei, SAI Qingyun, TIAN Yonghua, YANG Liqiang, RUAN Chaoling, YANG Ruilan, LIU Zhe, LIAN Zongqiang. Cloning of Dmrta1 Gene and Its Temporal and Spatial Expression in Tissue of Silurus lanzhouensis[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 228-238. doi: 10.7668/hbnxb.20193954.

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Figures/Tables 14

References 39

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引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Usage
Dmrta1-F	GTCTCCAGTGGCAAAGAT	中间序列克隆
Dmrta1-R	TCCAAGTTGAGGGTTTATT
Dmrta1-qF	ACCTCAAAGTCTGCCTTCTCCCCA	荧光定量PCR
Dmrta1-qR	CAGGTACGGAGAGATAAAACTCGG
β-actin-F	AAGATCATTGCCCCACCTGA
β-actin-R	CCTGCTTGCTGATCCACATC
Dmrta1-5gsp1	CCCTCTTTAGAAGTGAGCAGCAG	5'RACE-PCR
Dmrta1-5gsp2	CAGCTCGATGGTCTTTACAATGTC
Dmrta1-3gsp1	CTTTAGGAAATGATGGCTTCTACAG	3'RACE-PCR
Dmrta1-3gsp2	GCGTGTTGCTTATTCCGGTTC
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE通用引物
UPM short primer	CTAATACGACTCACTATAGGGC
Y-F	AACCAGGTTTGAAATGAAAAGAG	性别鉴定
Y-R	TGATTGCATACATTGGAGATAGA
X-F	CATTGAAACGTGCAATGATACG
X-R	CCATCAACTGTTATACTCTCATATTC
XY-F	CTGCGTCTGCTCTGTCCAGCTACT
XY-R	TCTGCACTGCATTCATGAATGCTG

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Usage
Dmrta1-F	GTCTCCAGTGGCAAAGAT	中间序列克隆
Dmrta1-R	TCCAAGTTGAGGGTTTATT
Dmrta1-qF	ACCTCAAAGTCTGCCTTCTCCCCA	荧光定量PCR
Dmrta1-qR	CAGGTACGGAGAGATAAAACTCGG
β-actin-F	AAGATCATTGCCCCACCTGA
β-actin-R	CCTGCTTGCTGATCCACATC
Dmrta1-5gsp1	CCCTCTTTAGAAGTGAGCAGCAG	5'RACE-PCR
Dmrta1-5gsp2	CAGCTCGATGGTCTTTACAATGTC
Dmrta1-3gsp1	CTTTAGGAAATGATGGCTTCTACAG	3'RACE-PCR
Dmrta1-3gsp2	GCGTGTTGCTTATTCCGGTTC
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE通用引物
UPM short primer	CTAATACGACTCACTATAGGGC
Y-F	AACCAGGTTTGAAATGAAAAGAG	性别鉴定
Y-R	TGATTGCATACATTGGAGATAGA
X-F	CATTGAAACGTGCAATGATACG
X-R	CCATCAACTGTTATACTCTCATATTC
XY-F	CTGCGTCTGCTCTGTCCAGCTACT
XY-R	TCTGCACTGCATTCATGAATGCTG

软件名称 Software name	网址 Website	用途 Function
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	开放阅读框分析
BLAST	https://blast.ncbi.nlm.nih.gov/Blast.cgi	同源性比对分析
ProtParam	https://web.expasy.org/prot-param/	蛋白质理化性质分析
ProtScale	https://web.Expasy.org/protscale/	蛋白质亲疏水性分析
SignalP 4.1	http://www.cbs.dtu.dk/services/SignalP-4.1/	蛋白质信号肽分析
TMHMM	http://www.cbs.dtu.dk/services/TMHMM/	蛋白质跨膜结构预测
SMART	http://smart.emblheidelberg.de/	蛋白质结构域预测
PSORT	http://psort.hgc.jp/form2.html	蛋白亚细胞定位预测
SOPMA	https://npsa-prabi.ibcp.fr/	蛋白质二级结构预测
Clustal Omega	http://www.ebi. ac.uk/Tools/msa/clustalo/	氨基酸序列比对

软件名称 Software name	网址 Website	用途 Function
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	开放阅读框分析
BLAST	https://blast.ncbi.nlm.nih.gov/Blast.cgi	同源性比对分析
ProtParam	https://web.expasy.org/prot-param/	蛋白质理化性质分析
ProtScale	https://web.Expasy.org/protscale/	蛋白质亲疏水性分析
SignalP 4.1	http://www.cbs.dtu.dk/services/SignalP-4.1/	蛋白质信号肽分析
TMHMM	http://www.cbs.dtu.dk/services/TMHMM/	蛋白质跨膜结构预测
SMART	http://smart.emblheidelberg.de/	蛋白质结构域预测
PSORT	http://psort.hgc.jp/form2.html	蛋白亚细胞定位预测
SOPMA	https://npsa-prabi.ibcp.fr/	蛋白质二级结构预测
Clustal Omega	http://www.ebi. ac.uk/Tools/msa/clustalo/	氨基酸序列比对

物种 Species	基因登录号 GenBank No.	相似性/% Similarity
斑点叉尾鮰Ictalurus punctatus	XP_017316285.1	85.40
虎头鲨Pangasianodon hypophthalmus	XP_026800957.1	84.25
黄颡鱼Tachysurus fulvidraco	XP_027013753.1	80.11
南方鲇Silurus meridionalis	XP_046698647.1	95.47
大西洋鳕Gadus morhua	XP_030194020.1	43.34
大黄鱼Larimichthys crocea	XP_010735509.1	48.04
白斑狗鱼Esox lucius	XP_010886381.2	49.87
褐鳟Salmo trutta	XP_029615604.1	51.57
虹鳟Oncorhynchus mykiss	XP_021433981.1	50.52
金钱鱼Scatophagus argus	AYN77807.1	47.81
团头鲂Megalobrama amblycephala	AHB63700.1	47.91
西方爪蟾Xenopus tropicalis	NP_001072447.1	44.80
人类Homo sapiens	NP_071443.2	41.36
绵羊Ovis aries	XP_004004458.3	43.06
中华鳖Pelodiscus sinensis	XP_025043468.1	36.02

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