兰州鲇<i>Dmrta1</i>基因克隆及其组织时空表达规律

doi:10.7668/hbnxb.20193954

华北农学报 ›› 2023, Vol. 38 ›› Issue (6): 228-238. doi: 10.7668/hbnxb.20193954

• 畜牧·水产·兽医 • 上一篇

兰州鲇Dmrta1基因克隆及其组织时空表达规律

柳婷¹^,², 李敏敏¹^,², 赖章龙⁵, 刘彦斌²^,³^,⁴, 刘凯²^,³^,⁴, 肖伟²^,³^,⁴, 赛清云²^,³^,⁴, 田永华²^,³^,⁴, 杨立强¹^,², 阮超岭⁵, 杨瑞兰¹^,², 刘哲¹, 连总强¹^,²^,³^,⁴^,⁵

¹ 甘肃农业大学动物科学技术学院,甘肃兰州 730070
² 宁夏回族自治区水产研究所,宁夏银川 750001
³ 宁夏渔业工程技术研究中心,宁夏银川 750001
⁴ 宁夏渔业科技院士工作站,宁夏银川 750001
⁵ 喀什大学生命与地理科学学院,新疆喀什 844000

收稿日期:2023-07-22 出版日期:2023-12-28
通讯作者:
刘哲(1970—),男,甘肃镇原人,教授,博士,博士生导师,主要从事鱼类学、水产养殖相关研究。
连总强(1980—),男,甘肃靖远人,高级工程师,博士,主要从事鱼类遗传育种与繁殖研究。
作者简介:
柳婷(1997—),女,甘肃庄浪人,在读硕士,主要从事鱼类遗传育种与繁殖研究。
基金资助:
国家重点研发计划项目(2018YFD0901202); 宁夏重点研发计划项目(2017BN06)

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

LIU Ting¹^,², LI Minmin¹^,², LAI Zhanglong⁵, LIU Yanbin²^,³^,⁴, LIU Kai²^,³^,⁴, XIAO Wei²^,³^,⁴, SAI Qingyun²^,³^,⁴, TIAN Yonghua²^,³^,⁴, YANG Liqiang¹^,², RUAN Chaoling⁵, YANG Ruilan¹^,², LIU Zhe¹, LIAN Zongqiang¹^,²^,³^,⁴^,⁵

¹ College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China
² Ningxia Fisheries Research Institute,Yinchuan 750001,China
³ Ningxia Fishery Engineering Technology Research Center,Yinchuan 750001,China
⁴ Academician Work Station of Ningxia Fishery Science and Technology,Yinchuan 750001,China
⁵ The College of Life and Geographic Sciences,Kashi University,Kashi 844000,China

Received:2023-07-22 Published:2023-12-28

摘要/Abstract

摘要：

为了解Dmrta1基因的生物学特性及其在兰州鲇生长发育中的生理作用,以及为加快实现全雄兰州鲇群体生产提供理论依据和技术支撑。采用同源克隆和cDNA末端快速克隆(RACE)技术获得兰州鲇Dmrta1全长cDNA序列,通过qRT-PCR技术检测该基因在兰州鲇胚胎发育、仔稚幼鱼以及3龄雌雄异体和3龄雌雄同体不同组织中的表达特征。结果表明,兰州鲇Dmrta1基因cDNA全长1 415 bp,ORF全长1 158 bp,共编码385个氨基酸,包含20种氨基酸;经SMART预测显示,该基因属于典型的Dmrta亚家族蛋白,具有保守的DM和DMA结构域。同源性比对和系统进化树分析结果表明,兰州鲇与南方鲇的相似性最高(95.47%),且亲缘关系最近。组织表达结果表明,Dmrta1 mRNA在未受精卵粒及胚胎发育各个时期均有表达,而在未受精卵粒中表达最高(P<0.05);在兰州鲇孵化后40 d内不同发育时期中,Dmrta1 mRNA在同期XX个体组织中表达均显著高于XY个体,且在35 d迅速达到峰值(P<0.05);Dmrta1 mRNA在3龄雌雄异体兰州鲇雌性卵巢、鳃和雄性鳃组织中表达显著高于其他组织(P<0.05),且卵巢表达显著高于雌性和雄性鳃,除卵巢表达是精巢的47.07倍外,其他组织中不存在雌雄性别差异;3龄雌雄同体和3龄雌雄异体表达特征一致,都是卵巢表达显著高于鳃和精巢(P<0.05)。以上结果说明,Dmrta1基因属于母源基因,该基因可能在兰州鲇性别分化、卵子形成和鳃弓形成过程中发挥重要作用,在雌雄同体和雌雄异体兰州鲇性腺中的作用一致。

关键词: 兰州鲇, Dmrta1基因, 基因克隆, 早期生活史, 雌雄同体, mRNA表达

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

柳婷, 李敏敏, 赖章龙, 刘彦斌, 刘凯, 肖伟, 赛清云, 田永华, 杨立强, 阮超岭, 杨瑞兰, 刘哲, 连总强. 兰州鲇Dmrta1基因克隆及其组织时空表达规律[J]. 华北农学报, 2023, 38(6): 228-238. doi: 10.7668/hbnxb.20193954.

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.

http://www.hbnxb.net/CN/Y2023/V38/I6/228

图/表 14

表1 本研究所用引物序列

Tab.1 Primer sequences were used in this study

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

表2 生物信息学分析软件

Tab.2 Bioinformatics analysis software

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

图1 兰州鲇Dmrta1蛋白二级结构预测紫色.无规则卷曲;蓝色.α-螺旋;绿色.β-折叠;红色.延伸。

Fig.1 The predicted secondary structure of Dmrta1 protein in S.lanzhouensis Purple.Random coil;Blue.Alpha-Helix;Green.Beta-Sheet;Red.Extended strand.

图2 兰州鲇和人Dmrta1基因共线性

Fig.2 Syntenic analysis for Dmrta1 genes in S.lanzhouensis and H.sapiens

图3 兰州鲇和其他硬骨鱼类Dmrta1基因共线性

Fig.3 Syntenic analysis for Dmrta1 genes in S.lanzhouensis and other teleost species

表3 兰州鲇Dmrta1氨基酸序列与其他物种同源性比对

Tab.3 Sequence alignment of Dmrta1 amino acid sequence in other species and S.lanzhouensis

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

图4 不同物种Dmrta1氨基酸部分序列比对分析

Fig.4 Sequence alignment of Dmrta1 amino acid sequences in different species

图5 不同物种Dmrta1氨基酸序列系统进化树

Fig.5 Phylogenetic tree of Dmrta1 amino acid sequences of different species

图6 兰州鲇胚胎发育过程中Dmrta1 mRNA表达特性 1.未受精;2.胚盘;3.卵裂;4.囊胚期;5.原肠;6.神经胚;7.器官形成期;8.出膜。内参基因.β-actin。不同字母表示差异显著(P<0.05),相同字母表示差异不显著(P>0.05)。图8—11同。

Fig.6 The temporal expression pattern of Dmrta1 mRNA during embryonic development of S.lanzhouensis 1.Unfertilized;2.Blastodisc;3.Cleavage;4.Blastula;5.Gastrula;6.Neurula;7.Organogenetic period;8.Hatching.Internal reference gene.β-actin.Different letters indicated significant differences(P<0.05),while the same letter indicated no significant difference(P>0.05).The same as Fig.8—11.

图7 兰州鲇遗传性别鉴定

Fig.7 Identification of genetic sex of S.lanzhouensis

图8 兰州鲇仔稚幼鱼生长发育过程中Dmrta1 mRNA表达水平变化

Fig.8 Temporal expression pattern of Dmrta1 mRNA in larvae and juveniles of S.lanzhouensis

图9 3龄雌雄异体兰州鲇不同组织中Dmrta1基因表达

Fig.9 Expression of Dmrta1 gene in different tissues of 3-year-old male and female of S.lanzhouensis

图10 3龄雌雄同体兰州鲇不同组织中Dmrta1基因表达

Fig.10 Expression of Dmrta1 gene in different tissues of 3-year-old hermaphroditic S.lanzhouensis

图11 3龄雌雄同体和雌雄异体兰州鲇性腺中Dmrta1基因表达

Fig.11 Expression of Dmrta1 gene in the gonads of 3-year-old hermaphroditic and dioecious S.lanzhouensis

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兰州鲇Dmrta1基因克隆及其组织时空表达规律

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