三角帆蚌细胞周期蛋白基因筛选及其表达分析

doi:10.3969/j.issn.1004-1524.2021.11.06

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (11): 2041-2050.DOI: 10.3969/j.issn.1004-1524.2021.11.06

三角帆蚌细胞周期蛋白基因筛选及其表达分析

冯上乐^a(), 李雪男^a, 陈一格^a, 刘瑞琦^a, 白志毅^a^,^b^,^c, 李文娟^a^,^b^,^c^,^*()

a.上海海洋大学农业农村部淡水水产种质资源重点实验室,上海 201306
b.上海海洋大学水产种质资源发掘与利用教育部重点实验室,上海 201306
c.上海海洋大学水产科学国家级实验教学示范中心,上海 201306

收稿日期:2021-01-16 出版日期:2021-11-25 发布日期:2021-11-26
作者简介:*李文娟,E-mail: wjli@shou.edu.cn
冯上乐(1996—),男,河南信阳人,硕士研究生,研究方向为贝类生物学。E-mail: fengshangle@126.com
通讯作者: 李文娟
基金资助:
国家自然科学基金(31201991);国家重点研发计划(2018YFD0901406)

Screening and expression of cyclins gene in Hyriopsis cumingii

FENG Shangle^a(), LI Xuenan^a, CHEN Yige^a, LIU Ruiqi^a, BAI Zhiyi^a^,^b^,^c, LI Wenjuan^a^,^b^,^c^,^*()

a. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China
b. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
c. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China

Received:2021-01-16 Online:2021-11-25 Published:2021-11-26
Contact: LI Wenjuan

摘要/Abstract

摘要：

细胞周期蛋白(cyclin)对细胞生长繁殖起到关键的调节作用。该研究采用高通量测序技术,构建了三角帆蚌(Hyriopsis cumingii)转录组文库,进行了cyclins基因筛选与生物信息分析,通过qRT-PCR技术,探讨其在不同组织(闭壳肌、内脏团、外套膜、斧足、心脏、血液、鳃、性腺)与性别中的表达特征。转录组测序共得到257 457条unigene(N50为1 796 bp),注释率为86.7%;通过分析,cyclin A、cyclin D2、cyclin E被筛选出,且cyclin A、cyclin D2与cyclin E蛋白的氨基酸序列均与美洲牡蛎(Crassostrea virginica)、太平洋牡蛎(Crassostrea gigas)具有较高相似性。实时荧光定量分析结果表明,cyclin A、cyclin D2、cyclin E在三角帆蚌各组织中均有表达,且存在显著的组织差异:cyclin A、cyclin E基因在性腺中表达量显著高于其他组织(P<0.05),在斧足中表达量最低;cyclin D2基因在鳃中表达量最高(P<0.05),在血液中表达量最低。在同一组织的不同性别之间:cyclin A基因在性腺、闭壳肌、心脏、血液中表达存在显著性别差异(P<0.05);cyclin D2在外套膜、心脏、性腺中的表达具有显著(P<0.05)的性别差异;cyclin E在性腺、斧足、鳃、心脏中的表达具有显著(P<0.05)的性别差异;cyclin A、cyclin E基因在雌性心脏和性腺中表达量显著(P<0.05)高于雄性,而cyclin D2反之(P<0.05),cyclin D2基因在雌性外套膜表达量显著高于雄性(P<0.05),暗示cyclin A、cyclin E与性腺发育关系密切,cyclin D2可能与鳃损伤修复有关。本研究初步探究了三角帆蚌cyclin A、cyclin D2、cyclin E基因的功能,为建立三角帆蚌细胞系提供了分子基础。

关键词: 转录组, 三角帆蚌, 细胞周期蛋白, 组织表达谱

Abstract:

Cyclins plays an important role in cell cycle entry and progression, they can modulate the catalytic activities of cyclin-dependent kinases (CDKs), cyclins can binding the catalytic subunits of CDKs to regulate cell cycle.To select cyclin genes in Hyriopsis cumingii, explore the function of cyclins, total RNA from mixed sample of mantle, gonad, visceral mass and hemolymph were isolated and sequenced by Illumina HiSeq 2000.Based on the sequencing results, select the cyclin A, cyclin D2, cyclin E genes, through cloning their conserved sequence to verify cyclins genes in H. cumingii. Amino acid sequence similarity analysis showed that cyclin A, cyclin D2 and cyclin E of H. cumingii have high sequence identity with Crassostrea virginica and Crassostrea gigas. At the transcriptional level, quantitative real-time PCR (qRT-PCR) was used to detect expression of cyclin genes in different tissues. The result revealed that all cyclin genes were expressed in tested tissues (adductor, foot, visceral mass, gill, heart, mantle, gonad, blood) with variable levels, expression levels of cyclin A and cyclin E genes in gonad were significantly (P<0.05) higher than those in other tissues, expression level of cyclin D2 gene in gills was significantly(P<0.05) higher than those in other tissues.Between different genders in the same tissue, there were significant(P<0.05) gender differences in the relative expression of cyclin A gene in gonad, adductor, heart and blood.In gonads and heart,there were significant(P<0.05) gender differences in cyclin D2 and cyclin E expression.Cyclin A and cyclin E genes were significantly(P<0.05)higher expressed in female hearts and gonads than those in males,indicated that cyclin A and cyclin E genes play an important role in gonad development.The high expression of cyclin D2 in gill suggested that it might be associated with gill damage repair. In this study, cyclin A, cyclin D2 and cyclin E genes and their functions were preliminarily studied, which laid a molecular basis for further regulation of the cell growth in vitro.

Key words: transcriptome, Hyriopsis cumingii, cyclins, tissue expression profile

中图分类号:

S634.1

冯上乐, 李雪男, 陈一格, 刘瑞琦, 白志毅, 李文娟. 三角帆蚌细胞周期蛋白基因筛选及其表达分析[J]. 浙江农业学报, 2021, 33(11): 2041-2050.

FENG Shangle, LI Xuenan, CHEN Yige, LIU Ruiqi, BAI Zhiyi, LI Wenjuan. Screening and expression of cyclins gene in Hyriopsis cumingii[J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2041-2050.

图/表 11

表1 实验所用引物

Table 1 Primers used in the experiment

引物Primer	上游引物(5'-3')Forward primer(5'-3')	下游引物(5'-3')Reverse primer(5'-3')
Cyclin A	AGCAATGACACTTGGGATG	TTGTAGAGGTGGCAGAGG
Cyclin D2	TGTGTGTAGAGGCCGAATCA	TCCCTAGTGAAAGCCGACAC
Cyclin E	TATCCCCAATTCTGAGCATGGA	TATCACCTGGGCCTGAATGTC
Cyclin A-Q Cyclin D2-Q Cyclin E-Q EF1α	CACATCAGGGGGATAGAT GATAGCAGCAGGGAGTGT GTCCCGTCTGAGGATAGC GGAACTTCCCAGGCAGACTGTGC	AGAGGTGGCAGAGGAATA TTTGTGGATTCCGTTTCG CTCTGTGGAGGTGTAAGC TCAAAACGGGCCGCAGAGAAT

表2 三角帆蚌转录组文库筛选的cyclins相关unigene

Table 2 Screening of cyclin related genes in transcriptome library of Hyriopsis cumingii

基因ID Gene ID	基因 Gene	长度 Length/bp
comp121579_c0_seq1	cyclin A	1 929
comp136327_c0_seq1	cyclin A	3 881
MblContig8891	G2/mitotic-specific cyclin B	3 641
MblContig41239 MblContig65698 MblContig31421 MblContig39154 MblContig1030 comp77562_c0_seq1 MblContig9313	cyclin B3 cyclin B G1/S-specific cyclin D2 G1/S-specific cyclin-D2 G1/S-specific cyclin E cyclin G1 cyclin H	835 1 935 1 971 4 358 1 019 659 2 006

图1 三角帆蚌cyclin A基因预测的氨基酸序列黑色框内为周期蛋白框。

Fig.1 Predicted amino acid sequence of cyclin A gene in Hyriopsis cumingii Black frame was the cyclin box.

图2 三角帆蚌与其他物种cyclin A氨基酸相似性序列比对 cyclin A氨基酸比对所用物种登录号:斑马贻贝,AAC35953.1;美洲牡蛎,XP_022320685.1;太平洋牡蛎,XP_034299932.1;虾夷扇贝,XP_021364053.1;斑节对虾,ACH72071.1;非洲爪蟾,NP_001081579.1;斑马鱼,AAH68323.2;长棘海星,XP_022085019.1。

Fig.2 Amino acid similarity alignment of cyclin A among Hyriopsis cumingii and other species GenBank accession numbers of cyclin A: Dreissena polymorpha, AAC35953.1;Crassostrea virginica, XP_022320685.1; Crassostrea gigas, XP_034299932.1;Mizuhopecten yessoensis, XP_021364053.1;Penaeus monodon, ACH72071.1;Xenopus laevis, NP_001081579.1;Danio rerio, AAH68323.2;Acanthaster planci, XP_022085019.1.

图3 三角帆蚌cyclin D2基因预测的氨基酸序列黑色框内为cyclin box。

Fig.3 Predicted amino acid sequence of cyclin D2 gene in Hyriopsis cumingii Black frame was the cyclin box.

图4 三角帆蚌与其他物种cyclin D2氨基酸相似性序列比对 cyclin D2氨基酸比对所用物种登录号:斑马贻贝,AAM44813.1;美洲牡蛎,XP_022292709.1;太平洋牡蛎,XP_011430967.2;虾夷扇贝,XP_021360132.1;非洲爪蟾,CAA61665.1;斑马鱼,NP_001082914.1;长棘海星,XP_022112210.1。

Fig.4 Amino acid sequence similarity alignment of cyclin D2 among Hyriopsis cumingii and other species GenBank accession numbers of cyclin D2: Dreissena polymorpha, AAM44813.1;Crassostrea virginica, XP_022292709.1;Crassostrea gigas, XP_011430967.2;Mizuhopecten yessoensis, XP_021360132.1;Xenopus laevis, CAA61665.1;Danio rerio, NP_001082914.1;Acanthaster planci, XP_022112210.1.

图5 三角帆蚌cyclin E基因预测的氨基酸序列

Fig.5 Predicted amino acid sequence of cyclin E gene in Hyriopsis cumingii

图6 三角帆蚌与其他物种cyclin E氨基酸相似性序列比对 cyclin E氨基酸比对所用物种登录号:美洲牡蛎,XP_022336414.1;太平洋牡蛎,XP_011433150.1;虾夷扇贝,OWF54564.1;斑节对虾,AGW23550.1;非洲爪蟾,CAA78370.1;斑马鱼,CAA58574.1;长棘海星,XP_022092193.1。

Fig.6 Amino acid sequence similarity alignment of cyclin E among Hyriopsis cumingii and other species GenBank accession numbers of cyclin E: Crassostrea virginica, XP_022336414.1;Crassostrea gigas, XP_011433150.1;Mizuhopecten yessoensis, OWF54564.1;Penaeus monodon, AGW23550.1;Xenopus laevis, CAA78370.1;Danio rerio, CAA58574.1; Acanthaster planci, XP_022092193.1.

图7 cyclin A基因的相对表达量组间无相同字母表示差异显著(P<0.05);*表示差异显著(P<0.05),**表示差异极显著(P<0.01)。下同。

Fig.7 Relative expression level of cyclin A Values without the same letters were significantly different (P<0.05);* indicated P<0.05, ** indicated P<0.01. The same as below.

图8 cyclin D2基因的相对表达量

Fig.8 Relative expression level ofcyclin D2

图9 cyclin E基因的相对表达量

Fig.9 Relative expression level of cyclin E

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三角帆蚌细胞周期蛋白基因筛选及其表达分析

Screening and expression of cyclins gene in Hyriopsis cumingii

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