Cloning and expression analysis of PCNA in Metapenaeus affinis
-
摘要: 增殖细胞核抗原 (Proliferating cell nuclear antigen, PCNA) 作为DNA聚合酶δ的辅助蛋白,在DNA复制过程中发挥重要作用。近缘新对虾 (Metapenaeus affinis) 卵巢发育过程中存在细胞增殖活动旺盛的阶段,但目前关于其卵巢发育的分子机制研究较少。利用RACE (Rapid amplification of cDNA ends) 技术获得近缘新对虾PCNA (MaPCNA) 基因的cDNA序列全长,并通过实时荧光定量PCR (qRT-PCR) 对其进行卵巢发育相关的表达分析。MaPCNA全长为1 144 bp,包含140 bp的5'非编码区,221 bp的3'非编码区,开放阅读框 (ORF) 为783 bp,编码260个氨基酸。MaPCNA蛋白的相对分子质量为28.82 kD,理论等电点为4.5。多重比对分析表明,PCNA氨基酸序列在甲壳动物中较为保守。组织表达结果显示,MaPCNA基因在检测的组织中均有表达,其中在卵巢中的表达量最为显著 (P<0.05)。在不同发育阶段的卵巢中,MaPCNA基因的表达出现变化 (P<0.05),从I期开始逐渐上升,到III期表达量最高,之后显著降低并趋于平稳。MaPCNA基因在幼体发育不同时期的表达呈现出一定的规律性趋势,在受精卵时期的表达量最高,之后呈下降趋势,从无节幼体VI期开始表达平稳。研究结果提示PCNA基因可能在近缘新对虾的卵巢发育过程中发挥重要作用。Abstract: As a coprotein of DNA polymerase δ, proliferating cell nuclear antigen (PCNA) plays an important role in the process of DNA replication. There is a stage of vigorous cell proliferation during the ovarian development in Metapenaeus affinis, but little attention has been paid to its molecular mechanism. In this study, we applied rapid amplification of cDNA ends (RACE) technique to obtain the full length cDNA sequences of PCNA in M. affinis (MaPCNA), and analyzed the expression of MaPCNA related to ovarian development by real-time fluorescent quantitative PCR (qRT-PCR). The total length of MaPCNA was 1 144 bp, including 140 bp of 5' untranslated region, 221 bp of 3' untranslated region and 783 bp ORF encoding 260 amino acids. The molecular mass of MaPCNA protein was 28.82 kD and the theoretical isoelectric point was 4.5. The protein homology analysis shows that MaPCNA had high homology with other crustaceans. The result of tissue expression shows that Ma-PCNA was expressed in all tested tissues, with the highest expression in ovary (P<0.05). The expression of MaPCNA in ovary at different developmental stages showed significant changes (P<0.05), increasing from stage I to stage III gradually, decreasing significantly and then tending to be stable. The expression of MaPCNA showed a regular change trend at different larval developmental stages. The expression level of MaPCNA was the highest in oosperm, then began to decline, and became stable from the Nauplius VI. The results suggest that PCNA may play an important role in the ovarian development of M. affinis.
-
Keywords:
- Metapenaeus affinis /
- PCNA /
- Cloning /
- Gene expression
-
鲷科鱼类(Sparidae)约有33属110种,主要分布于大西洋、印度洋和太平洋的暖热水域,许多种类为优质经济鱼类[1-2]。由于品种较多,而且许多品种外部形态接近,在野外利用形态学的标准如体型、牙齿形状和位置、鳍条或鳍棘长短或数目等对许多鲷科鱼类进行品种鉴定仍然存在较大的困难,因此,有必要在实验室进行进一步的鉴定。分子生物学技术由于具有要求样本量少、准确、快速等优点,为品种鉴定提供了新的手段。然而,目前采用分子技术对鲷科鱼类进行种类鉴定的研究仅有少量报道,如ALARCÓN和ALVAREZ[3]首先应用同工酶标记对8种鲷科鱼类进行了品种鉴定研究,但在mtDNA水平仍然缺乏科学有效的鉴定标准。
随着科技的发展,生物遗传资源保护和应用研究日益受到重视,mtDNA序列分析技术目前已经被广泛应用于许多物种的群体遗传学和生物资源的保护和管理等研究领域。应用mtDNA序列如控制区、细胞色素b(Cyt b)及16S rRNA等序列对鲷科种类的研究已经涉及到系统发育等研究领域,如ORRELL和CARPENTER[1]、HANEL和STURMBAUER[4]及刘红艳等[5]等。通过这些研究获得的鲷科鱼类的mtDNA序列数据可以从NCBI(http://www.ncbi.nlm.nih.gov)等分子生物学数据库得到。一般认为Cyt b序列较为保守,主要用于种以上水平遗传变异的分析[6]。为了了解鲷科鱼类种间及种内遗传关系为种类鉴定提供基础信息,本研究应用生物信息学的一些手段对部分已经登陆NCBI核苷酸数据库的鲷科鱼类mtDNA Cyt b序列数据进行研究,为鲷科鱼类的种类鉴定及进一步遗传育种研究工作提供参考。
1. 材料和方法
为了进行种内及种间遗传距离分析及获得尽可能多的序列信息,在NCBI分子生物学数据库中至少具有2条1 kb以上长度的Cyt b序列的种才被纳入本研究范围。最终,通过搜索NCBI核苷酸序列数据库,下载用于本研究的Cyt b序列共有鲷科鱼类10属12种的24条序列。这些序列的基本信息见表 1。
表 1 研究所采用的Cyt b序列信息Table 1 Cyt b sequences used in this study属名
genus种名
speciesNCBI序列号
accession number薄唇鲷属 Boops boops X81567、BBO319810 牙鲷属 Dentex dentex AF143197、DDE319814 重牙鲷属 Diplodus cervinus AF240723、DCE277367 兔牙鲷属 Oblada melanura AF240701、OME319813 玉颌鲷属 Lithognathus mormyrus AF240712、LMO277371 海鲷属 Pagellus bogaraveo PBO319818、PBO276880 acarne PAC319817、PAC276879 erythrinus PER319816、PER276881 真鲷属 Pagrus pagrus AF240729、PPA319815 黑海鲷属 Spondyliosoma cantharus AF240705、SCA319811 厚唇鲷属 Sarpa salpa AF240704、SSA319812 鲷属 Sparus aurata AF240735、SAU319809 注:中文种名没有提供
Note: Chinese name for each species was not provided.应用程序Clustal X[7]对所获得的Cyt b序列进行序列比对。采用程序Mega(Ver.2.1)[8]以Kimura 2-parameter模型进行系统发育及遗传距离分析,对未知数据采用成对删除法(pairwise deletion)进行处理,通过自展法(bootstrap method;重复次数500)计算自展值。采用Excel软件计算距离均值及标准偏差(SD),并以散点图表示种内种间的遗传距离分布情况。应用程序DnaSP(Ver.4.0)[9]计算序列GC含量、核苷酸多样性(Nucleotide diversity,Pi)、单倍型多样性(haplotype diversity,Hd)等其它相关数据。
2. 研究结果
2.1 序列分析
通过序列比对最终得到了1 070 bp的Cyt b共有序列。序列GC含量为0.461,其中核苷酸C含量最高,为30.3%,G最低,为15.8%,而T、A分别为29.7%、24.2%。序列中共检测到675个单态性位点,393个变异位点,2个未知位点(315,555),没有检测到插入缺失。变异位点中单一序列变异位点(singleton variable sites)有24个,简约性信息位点(parsimony informative sites)为369个。群体单倍型多样性(Hd)为0.986,而核苷酸多样性(Pi)为0.150。有关序列信息详见表 2。
表 2 研究鲷科鱼类Cyt b序列变异位点Table 2 Variable sites of Sparidae Cyt b sequences used in this study进一步对表 2中的序列数据进行分析发现在1 070 bp序列范围内各种类都有种特征性的变异位点(即种内所有个体都具有,而其它种没有的变异位点),如S. cantharus具有的种特征性位点最多,共24个,而O. melanura只有4个。所有种类具有4到24个不等的种特征性位点,平均约为12.42个(表 3)。
表 3 Cyt b序列种特征性位点信息Table 3 Species-specific site information of Cyt b sequences种名
species位点位置
site location小计
sumB. boops 52a,372,429,624,625,685,717,774,837 9 D. dentex 21,27,102,133,147,171,180,240,267,348,429,534,574,645,666,687,705,728,876,945,1056,1062 22 D. cervinus 333,366,387,570,810,846,996 7 O. melanura 234,360,717,855 4 L. mormyrus 12,123,252,297,447,462,483,579,591,630,633,681,693,699,702,714,726,981 18 P. bogaraveo 18,543,555,572,702,864,996 7 P. acarne 42,297,738,843,846,864,969,1038 8 P. erythrinus 7,8,237,378,468,525,537,556,585,609,612,627,693,729,945,951,1035 17 P. pagrus 21,57,315,372,519,639,756,777,981,1017 10 S. cantharus 42,66,73,102,147,258,361,366,402,423,456,483,640,660,703,705,706,759,843,891,958,984,996,1047 24 S. salpa 240,264,448,612,759,873,1003 7 S. aurata 15,30,138,417,546,600,615,675,701,729,774,841,861,960,985,1065 16 注:“a”代表种特征性位点在序列中所处的位置(参考表 2中序列)
Note: “a” represents the site location in the Cyt b sequences (refer to the sequence in Tab. 2).2.2 遗传距离分析
12个鲷种间,L. mormyrus与S. cantharus之间具有最大的遗传距离[0.220±0.017(SE)],而P. bogaraveo与P. acarne间距离最小[0.105±0.010(SE)](表 4)。种间遗传距离平均值为0.180±0.0231(SD)。种内遗传距离最大的为P. pagrus [0.018±0.004(SE)],而其它各种的种内遗传距离都要小于这一数值。种内遗传距离平均值为0.007±0.006(SD)。因此,种间距离均值约为种内距离均值的26倍,种间遗传距离要远远大于种内遗传距离。图 1中各数据点的分布也反映了这一趋势。
表 4 鲷科鱼类种内种间遗传距离关系Table 4 The genetic distances among and within Sparid species种名
species1 2 3 4 5 6 7 8 9 10 11 Da 1 0.000 2 0.193a 0.000 3 0.158 0.179 0.013 4 0.165 0.187 0.117 0.007 5 0.172 0.196 0.152 0.166 0.009 6 0.178 0.196 0.152 0.158 0.168 0.000 7 0.160 0.198 0.140 0.156 0.175 0.105 0.005 8 0.208 0.175 0.198 0.205 0.200 0.202 0.216 0.000 9 0.191 0.157 0.188 0.186 0.196 0.207 0.198 0.141 0.018 10 0.174 0.216 0.161 0.195 0.220 0.187 0.175 0.219 0.197 0.011 11 0.154 0.194 0.174 0.167 0.180 0.169 0.171 0.188 0.180 0.171 0.014 12 0.172 0.217 0.167 0.166 0.182 0.191 0.170 0.202 0.210 0.207 0.182 0.007 注:表中数值1~12分别代表鲷种B. boops、D. dentex、D. cervinus、O. melanura、L. mormyrus、P. bogaraveo、P. acarne、P. erythrinus、P. pagrus、S. cantharus、S. salpa、S. aurata。‘a’显示种间遗传距离,‘Da’代表种内遗传距离,标准误(SE)没有提供
Note:Number 1~12 represent B. boops, D. dentex, D. cervinus, O. melanura, L. mormyrus, P. bogaraveo, P. acarne, P. erythrinus, P. pagrus, S. cantharus, S. salpa, S. aurata, respectively. ‘a’indicated the genetic distance among species, whereas ‘Da’indicated the genetic distance within species; Standard Error (SE) wasn′t provided.进一步通过序列数据集构建了所有序列的NJ系统发育树(文中系统树没有提供)。尽管有些种间的自展值相对较低(最低为20),如L. mormyrus与D. cervinus、O. melanura、P. bogaraveo及P. acar-ne,然而分析发现,同一种内的2条序列都被聚为一个类群,而且大多数都具有非常高的自展值(100)。因此,系统发育分析显示不同鲷科种能够通过Cyt b序列进行较好的区分。
3. 讨论
本研究通过对鲷科10属12个种的1 070 bp Cyt b序列进行分析,发现各鲷科种类在分析的序列范围内各种群都存在12个左右种群特征性的变异位点。刘红艳等[5]曾经报道鲷科同一种内不同个体的Cyt b基因序列具有一定的保守性。本研究也显示部分鲷科种内遗传距离为零,如B. boops、D. dentex、P. bogaraveo、P. erythrinus,而且12个鲷种种内遗传距离均值相对较低。因此,推测Cyt b基因种内保守性为鲷科种群的基本特征,在实际应用中可以应用这些保守的种特征性位点进行鲷科鱼类品种的鉴定。
比较分析显示,不同鲷科鱼类种间与种内的遗传距离差异较大,种间遗传距离[0.105±0.010(SE)~0.220±0.017(SE)]要远远大于种内遗传距离[0~0.018±0.004(SE)]。另外,系统发育分析显示,来源于同一种的2条序列都被清晰的聚为同一类群,而来源于不同种的序列分别处于不同的类群。因此,不同鲷种能够通过Cyt b序列进行较好的区分。基于以上鲷科鱼类种间与种内遗传距离数据,提出以下假设,当2个鲷科鱼类个体通过以上Cyt b序列计算得到的遗传距离如小于0.018时,可以初步判断来源于同一种,如大于0.105,则为不同种,如处于2个数据之间,则有待于进一步结合其它的数据如形态学特征、种特征性位点等进行分析以确定种群,保证鉴定的准确性。
ALARCÓN和ALVAREZ[3]首先应用同工酶标记对8种鲷科鱼类进行了品种鉴定研究。由于同工酶标记技术相对于mtDNA序列分析而言,具有制样更严格,且受个体发育时期限制等缺点,因此,本研究结果相对于前人的研究而言具有更大的实际应用价值。然而,值得注意的是,尽管以上研究对于鲷科鱼类新种的鉴定、个体或者小组织样本的品种鉴定和杂交育种的选育具有重要意义,但是,由于本研究所采用的同一种内的Cyt b序列数目相对较少,而且相对于整个鲷科鱼类而言,包含的品种数目也较少,因此,采用以上方法对鲷科鱼类中最近形成的种进行鉴定时可能由于其序列变异相对较少,从而可能出现错误的品种鉴定。另外,由于mtDNA是母性遗传的,鱼类杂种也可能被不正确的鉴定。因此,利用以上标准对鲷科鱼类进行品种鉴定时仍然要谨慎,最好与其它标准同时使用。为了准确地对鲷科鱼类进行分子鉴定,建立一个所有鲷科鱼类的Cyt b序列数据库非常必要。
-
图 5 MaPCNA 基因在不同组织中的相对表达量
注:图中数值为“平均值±标准差”(n=3),小写字母不同表示各组之间具有显著差异 (P<0.05);后图同此。
Figure 5. Relative expression of MaPCNA gene in different tissues
Note: The values are "$ {\rm{Mean}} \pm {\rm{SD}}$" (n=3). Different lowercase letters indicate significant difference among groups (P<0.05); the same case in the following figures.
表 1 实验所使用的引物序列
Table 1 Oligonucleotide primers used in experiment
引物
Primer引物序列 (5'—3')
Primer sequence (5'−3')用途
FunctionMaPCNA-F GGAAGTCTGTTGAAGAAGGTGTTGGA 验证序列 MaPCNA-R GTATTCTGCCACAAAGCCATAGTAAGC MaPCNA-5GSP1 TTTGGACATGCTGGTGAGGTTCATGCCC 5'RACE MaPCNA-5GSP2 CATGCCCATGATGAGGTTTCGGTCGC MaPCNA-3GSP1 AAAGCAACACCCCTTTCCCCACAGG 3'RACE MaPCNA-3GSP2 TCCCTGTCCATGTCTCCTGATGTACCCC UPM-long CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT RACE UPM-short CTAATACGACTCACTATAGGGC NUP AAGCAGTGGTATCAACGCAGAGT MaPCNA-qF TCATCGAGATGCAGGAGCCAGTTA qPCR MaPCNA-qR ATCAGGAGACATGGACAGGGAGAC EF-1α-qF AAGCCAGGTATGGTTGTCAACTTT 内参 EF-1α-qR CGTGGTGCATCTCCACAGACT -
[1] MIYACHI K, FRITZLER M J, TAN E M. Autoantibody to a nuclear antigen in proliferating cells[J]. J Immunol, 1978, 121(6): 2228-2234. doi: 10.4049/jimmunol.121.6.2228
[2] BRAVO R, CELIS J E. A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells[J]. J Cell Biol, 1980, 84(3): 795-802. doi: 10.1083/jcb.84.3.795
[3] SCHRECKER M, CASTABEDA J C, DEVBHANDARI S, et al. Multistep loading of a DNA sliding clamp onto DNA by replication factor C[J]. ELIFE, 2022, 11: e78253. doi: 10.7554/eLife.78253
[4] GONZALEZ-MAGANA A, BLANCO F J. Human PCNA structure, function, and interactions[J]. Biomolecules, 2020, 10(4): 570. doi: 10.3390/biom10040570
[5] YOUNG P S, JEONG M S, CHANG W H, et al. Structural and functional insight into proliferating cell nuclear antigen[J]. J Microbiol Biotechnol, 2016, 26(4): 637-647. doi: 10.4014/jmb.1509.09051
[6] NAIR A G, RABAS N, LEJON S, et al. Unorthodox PCNA binding by chromatin assembly factor 1[J]. Int J Mol Sci, 2022, 23(19): 11099. doi: 10.3390/ijms231911099
[7] 李忠邦. LYZL4, LYZL6和PCNA在牦牛睾丸中的表达定位及组织学分析[D]. 兰州: 西北民族大学, 2019: 22-39. [8] XU B, HUA J, ZHANG Y W, et al. Proliferating Cell Nuclear Antigen (PCNA) regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries[J]. PLoS One, 2017, 6(1): e16046.
[9] MUHAMMAD F. Ontogenesis of main organs and cloning and expression of two immune-related genes in Litopenaeus vannamei[D]. 青岛: 中国海洋大学, 2012: 78-88. [10] XIE Y S, WANG B, LI F H, et al. Molecular cloning and characteri-zation of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis[J]. Comp Biochem Physiol B, 2008, 151: 225-229. doi: 10.1016/j.cbpb.2008.07.006
[11] ZHANG Z P, SHEN B L, WANG Y L, et al. Molecular cloning of proliferating cell nuclear antigen and its differential expression analysis in the developing ovary and testis of penaeid shrimp Marsupenaeus japonicus[J]. DNA Cell Biol, 2010, 29: 163-170. doi: 10.1089/dna.2009.0958
[12] PENG L, JIE Z, YI K, et al. Identification, mRNA expression and characterization of proliferating cell nuclear antigen gene from Chinese mitten crab Eriocheir sinensis[J]. Comp Biochem Physiol A, 2010, 157(2): 170-176. doi: 10.1016/j.cbpa.2010.06.163
[13] 王成扬, 赵超, 傅明骏, 等. 斑节对虾增殖细胞核抗原基因克隆及表达分析[J]. 中国水产科学, 2016, 23(5): 1052-1062. [14] 姜虎成. 克氏原螯虾转录组测序数据发掘和性腺发育相关基因功能初步研究[D]. 上海: 上海海洋大学, 2015: 43-63. [15] 杨亚男, 黄辉洋, 尚丽丽, 等. 近缘新对虾Cyclin B基因的克隆与原核表达[J]. 水产学报, 2013, 37(2): 184-191. [16] DINCER T, AYDIN İ. Proximate composition and mineral and fatty acid profiles of male and female jinga shrimps (Metapenaeus affinis, H. Milne Edwards, 1837)[J]. Turk J Vet Anim Sci, 2014, 38: 445-451. doi: 10.3906/vet-1301-15
[17] KESHAVARZIFARD M, VAZIRZADEH A, SHARIFINIA M. Occurrence and characterization of microplastics in white shrimp, Metapenaeus affinis, living in a habitat highly affected by anthropogenic pressures, northwest Persian Gulf[J]. Mar Pollut Bull, 2021, 169: 112581. doi: 10.1016/j.marpolbul.2021.112581
[18] KESHAVARZIFARD M, VAZIRZADEH A, SHARIFINIA M. Implications of anthropogenic effects on the coastal environment of Northern Persian Gulf, using Jinga shrimp (Metapenaeus affinis) as indicator[J]. Mar Pollut Bull, 2020, 159: 111463. doi: 10.1016/j.marpolbul.2020.111463
[19] GERAMI M H, GHORBANI R, PAIGHMABARI S Y, et al. Reproductive season, maturation size (LM50) and sex ratio of Metapenaeus affinis (Decapoda: Penaeidae) in Hormozgan shrimp fishing grounds, south of Iran[J]. Int J Aquat Biol, 2013, 1(2): 48-54.
[20] GULNER M, İLKER A. Some reproductive characteristics of Meta-penaeus affinis (H. Milne Edwards, 1837) in Izmir Bay (Eastern Aegean Sea, Turkey)[J]. Pakistan J Zool, 2017, 49(5): 1913-1916.
[21] DASH G, MOHAMMED KOYA K, SEN S, et al. Fishery, population dynamics and stock status of Jinga shrimp, Metapenaeus affinis (H. Milne Edwards, 1837) from Gujarat waters of India[J]. Indian J Geo-Mar Sci, 2018, 47(1): 2267-2277.
[22] ABDEL RAZEK F A, RAGHEB E, EI-DEEB R S, et al. Growth pattern and stock assessment of Jinga shrimp Metapenaeus affinis (H. Milne Edwards, 1837) (Decapoda, Penaeidae) from the southeastern Mediterranean of the Egyptian coasts[J]. Egy J Aquat Res, 2022, 48(1): 83-90. doi: 10.1016/j.ejar.2021.11.008
[23] SAPUTRA S W, SOLICHIN A, TAUFANI W T. Stock Analysis of Metapenaeus affinis (H. Milne Edwards, 1837) on the north coast of central Java, Indonesia[J]. IOP Conf Ser: Earth Environ Sci, 2018, 116(1): 012004.
[24] ZHANG Y, LI Y D, YANG Q B, et al. Transcriptome analysis of Metapenaeus affinis reveals genes involved in gonadal development[J]. Isr J Aquacult-Bamid, 2022, 74: 1722195.
[25] HABIB-UI-HASSAN. Early developmental stages of Metapenaeus affinis (Decapoda, Penaeidae) reared in a laboratory[J]. ICES J Mar Sci, 1980, 39(1): 30-43. doi: 10.1093/icesjms/39.1.30
[26] 江世贵, 杨丛海, 周发林. 等. 斑节对虾种虾繁育技术[M]. 北京: 海洋出版社, 2013: 80-81. [27] ALMAGRO AEMENTEROS J J, TSIRIGOS K D, SØNDERBY C K, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks[J]. Nat Biotechnol, 2019, 37: 420-423. doi: 10.1038/s41587-019-0036-z
[28] CHOU K C, SHEN H B. Cell-PLoc: a package of web servers for predicting subcellular localization of proteins in various organi-sms[J]. Nat Protoc, 2008, 3: 153-162. doi: 10.1038/nprot.2007.494
[29] GUPTA R, BRUNAK S. Prediction of glycosylation across the human proteome and the correlation to protein function[J]. Pac Symp Biocomput. 2002, 7: 310-322.
[30] BLOM N, SICHERITZ-PONTEN T, GUPTA R, et al. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence[J]. Proteomics, 2004, 4(6): 1633-1649. doi: 10.1002/pmic.200300771
[31] WILKINS M R, GASTEIGER E, BAIROCH A, et al. Protein identification and analysis tools in the ExPASy server[J]. Methods Mol Biol, 1999, 112: 531-552.
[32] KROGH A, LARSSON B, HEIJINE G V, et al. Sonnhammer: predicting transmembrane protein topology with a hidden markov model: application to complete genomes[J]. J Mol Biol, 2001, 305: 567-580. doi: 10.1006/jmbi.2000.4315
[33] FINN R D, BATEMAN A, CLEMENTS J, et al. Pfam: the protein families database[J]. Nucleic Acids Res, 2014, 42(D1): D222-D230. doi: 10.1093/nar/gkt1223
[34] GEOURJON C, DELEAGE G. SOPM: a self-optimized method for protein secondary structure prediction[J]. Protein Eng, 1994, 7(2): 157-164. doi: 10.1093/protein/7.2.157
[35] WATERHOUSE A, BERTONI M, BIENERT S, et al. SWISS-MODEL: homology modelling of protein structures and complexes[J]. Nucleic Acids Res, 2018, 46(W1): W296-W303. doi: 10.1093/nar/gky427
[36] LARKIN M A, BLACKSHIELDS G, BROWN N P, et al. Clustal W and Clustal X version 2.0[J]. Bioinformatics, 2007, 21(23): 2947-2948.
[37] ROBERT X, GOUET P. Deciphering key features in protein structures with the new ENDscript server[J]. Nucleic Acids Res, 2014, 42(W1): W320-W324. doi: 10.1093/nar/gku316
[38] KUMAR S, STECHER G, TAMURA K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Mol Biol Evol, 2016, 7(33): 1870-1874.
[39] HILBE J M. A review of current SPSS products: SPSS 12, SigmaPlot 8.02, SigmaStat 3.0, Part 1[J]. Am Stat, 2003, 57(4): 310-315.
[40] EDWARDS P M. Origin 7.0: Scientific graphing and data analysis software[J]. J Chem Inf Comput Sci, 2002, 42(5): 1270-1271. doi: 10.1021/ci0255432
[41] QIAN J, CHEN Y Y, XU Y X, et al. Interactional similarities and differences in the protein complex of PCNA and DNA replication factor C between rice and arabidopsis[J]. BMC Plant Biol, 2019, 19: 257. doi: 10.1186/s12870-018-1600-2
[42] BHARDWAJ V K, PUROHIT R. A new insight into protein-protein interactions and the effect of conformational alterations in PCNA[J]. Int J Biol Macromol, 2020, 148: 999-1009. doi: 10.1016/j.ijbiomac.2020.01.212
[43] YU S M, QIAO X S, YANG Y, et al. The proliferating cell nuclear antigen (PCNA) is a potential proliferative marker in oyster Crassostrea gigas[J]. Fish Shellfish Immunol, 2022, 122: 306-315. doi: 10.1016/j.fsi.2022.02.018
[44] MANSILLA S F, de la VEGA M B, CALZETTA N L, et al. CDK-independent and PCNA-dependent functions of p21 in DNA replication[J]. Genes, 2020, 11(6): 593. doi: 10.3390/genes11060593
[45] FRANCO A, JOUAUX A, MATHIEU M, et al. Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues[J]. Cell Tissue Res, 2010, 340: 201-210. doi: 10.1007/s00441-009-0923-6
[46] 李治龙. 羽衣甘蓝PCNA基因克隆、亚细胞定位及蛋白表达分析[D]. 哈尔滨: 东北林业大学, 2017: 32-48. [47] MELO R M C, MARTINS Y S, LUZ R K, et al. PCNA and apoptosis during post-spawning ovarian remodeling in the teleost Oreochromis niloticus[J]. Tissue Cell, 2015, 47(6): 541-549. doi: 10.1016/j.tice.2015.10.002
[48] GUZMAN J M, LUCKENBACH J A, YAMAMOTO Y, et al. Expression profiles of fsh-regulated ovarian genes during oogenesis in coho salmon[J]. PLoS One, 2014, 9(12): e114176. doi: 10.1371/journal.pone.0114176
[49] KORFSMEIER K H. PCNA in the ovary of zebrafish (Brachydanio rerio, Ham.- Buch. )[J]. Acta Histochem, 2002, 104: 73-76. doi: 10.1078/0065-1281-00632
[50] LEUNG A Y, LEUNG J C, CHAN L Y, et al. Proliferating cell nuclear antigen (PCNA) as a prolifera-tive marker during embryonic and adult zebrafish hematopoiesis[J]. Histochem Cell Biol, 2005, 124(2): 105-111. doi: 10.1007/s00418-005-0003-2
[51] BALLA M, ANGELOPOULOU R, LAVRANOS G, et al. Follicular cells versus oocytes: cell population dynamics in the developing ovary[J]. Tissue Cell, 2008, 40(5): 373-381. doi: 10.1016/j.tice.2008.03.006
[52] ROA S, AVDIEVICH E, PELED J U, et al. Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombina-tion and is required for meiotic progression[J]. Proc Natl Aca Sci, 2008, 105(42): 16248-16253. doi: 10.1073/pnas.0808182105