Cloning and expression analysis of PCNA in Metapenaeus affinis
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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中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.
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Key words:
- Metapenaeus affinis /
- PCNA /
- Cloning /
- Gene expression
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图 2 MaPCNA与其他物种的PCNA氨基酸序列基因之间的多重序列比对图
Figure 2. Multiple sequence alignment between MaPCNA and PCNA of other species
图 4 基于氨基酸序列比较的PCNA在不同物种中的系统进化树
Figure 4. Phylogenetic tree of PCNA in different species based on amino acid sequence
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 "$ \overline { X} \pm {\rm{SD}}$" (N=3). Different lowercase letters indicate significant difference among groups (P<0.05); the same case in the following figures.
图 6 MaPCNA基因在卵巢发育不同阶段的相对表达量
Figure 6. Relative expression of MaPCNA gene at different ovarian developmental stages
图 7 MaPCNA基因在幼体发育各阶段的相对表达量
Figure 7. Relative expression of MaPCNA gene at different larval developmental stages
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Function MaPCNA-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 
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[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 -
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