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1 2016 年 9 月, 23(5): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 斑节对虾增殖细胞核抗原基因克隆及表达分析 1, 王成扬 2 1,, 赵超 3 1,, 傅明骏 3 1, 3, 4, 邱丽华 1., ; 2., ; 3., ; 4., 摘要 : (proliferating cell nuclear antigen, PCNA) (Penaeus monodon), RACE PCNA (PmPCNA) cdna 978 bp, 135 bp 5 (5 UTR) 60 bp 3 UTR bp (ORF), PmPCNA, PmPCNA, ; PmPCNA, 2 ; (5-hydroxy tryptamine, 5-HT), PmPCNA, 48 h, 5 PCNA, Western Blot PCNA, PmPCNA, 关键词 : ; (PCNA); ; PCR; 5- (5-HT); 中图分类号 : S917 文献标志码 : A 文章编号 : (2016) (proliferating cell nuclear antigen, PCNA), DNA, DNA [1] Miyachi [2] 1978, PCNA, Tománek [3] PCNA (Sus scrofa) ; Motiwale [4] (Mus musculus), PCNA ; Witko-Sarsat [5], (Homo sapiens) PCNA PCNA, DNA DNA, [6 7], PCNA, PCNA, DNA [8 10], PCNA [11], PCNA, 收稿日期 : ; 修订日期 : 基金项目 : ( ); (ZDXM ); (A201300B03); (A201501A11); (948 ) (2015-Z20). 作者简介 : (1990 ),,,. wchengyangnhs@126.com 通信作者 : (1971 ),,,. qiu902@126.com

2 5 : 1053, PCNA [12 13] PCNA (Rattus norvegicus) [14] (Bos taurus) [15] (Papio hamadrayas) [16],, PCNA (Penaeus monodon), [17],, [18 19],,, PmPCNA, PCNA, PmPCNA (5-hydroxy tryptamine, 5-HT), [20], 5-HT, PCNA, 1 材料与方法 1.1 实验用虾 ( 83 ), 100~180 g, 50, 3 d, 24~25, 3 d,, [21], ~ HT 注射实验 5-HT PCNA, 5-HT 1 PBS 25 μg/μl ( 26~28, 20 ), 7 2, 18, 5-TH, 50 μg, 1 PBS, 0 h 12 h 24 h 48 h 72 h 96 h, 总 RNA 的提取与 cdna 的合成 Trizol (Invitrogen, USA) RNA, DEPC, 1.2% RNA, RNA (2 μg), Oligo-dT/adaptor [5 GGCCACGCGACTAGTAC (T) 16 3 ], M-MLV (Promega, USA) cdna, : min, min 1.4 斑节对虾 PmPCNA 基因的克隆与测序 PmPCNA EST, 3 RACE 3 PCNAF(5 GGAGGA- AGCAGTCGTTATTG 3 ) 3 PCNAR(5 CCAAG- ATTGAGGACGAAGACT 3 ) cdna SMARTTM RACE (Clontech, USA), PCR (touchdown PCR) PCR (semi-nest PCR) PmPCNA 3 [22] 1.5%, pmd18-t (TaKaRa, Japan), 16 4 h E. coli DH5α, 1.5 序列分析 Clustal W ( clustalw/) ; NCBI ORF Finder BLAST ( ncbi.nlm.nih.gov/blast.cgi) (open reading frame, ORF) ; NCBI BLAST ( SMART ( de/) Scan Prosite(

3 scanprosite/) ; SignalP ( NetNGlyc 2.0 ( cbs.dtu.dk/ services/netnglyc/); NetPhos 2.0 ( NetPhos/); Clustalx 2.0 [23] BioEdit ; Mega 5.0 [24] 1.6 PmPCNA 在转录水平上的时空表达特征 5-HT RNA, SYBR Premix Ex Taq TM Kit (TaKaRa, Japan) cdna PmPCNA QPCNAF(5 TGTGCC- GCTAACGATGAC 3 ) QPCNAR(5 GTTTCTGG AATGCCAAGATG 3 ) 1 (EF-1α), EF-F (5 AAGCC- AGGTATGGTTGTCAACTTT 3 ) EF-R (5 CGT- GGTGCATCTCCACAGACT 3 ) cdna, 3, LC480 (ROCHE Light Cycler 480/480II, Switzerland), :, s; PCR, 94 5 s, s, s, 40 ; min 2 ΔΔCt PmPCNA 1.7 斑节对虾 PCNA 的体外重组表达分析 PmPCNA : PEPCNAF (5 GGATCCATGTTCGAAGCCCGCCT- GGTGCAAGGG 3 ) PEPCNAR (5 GAATTCTG- AGTCTTCGTCCTCAATCTTGGGGGCC 3 ), 5 BamH I EcoR I, BamH I EcoR I PCNA pgex-4t-1 ;, T 4 DNA 16 E. coli DH5α,, PCR ; PCNApGEX-4T-1, E. coli BL21,, PCR 2 ml, 200 ml LB, 37, 220 r/min 3 h, OD 0.6~0.8, 2 ml, (IPTG) 0.6 mmol/l, 30, 2 h 2 ml, 2 h 4 h 6 h 8 h 4, 200 μl PBS, μl, 10 min, 10 μl (SDS-PAGE),, 1.8 免疫印迹 SDS-PAGE, GST Tag HRP ( ), 2000, PBST (1 PBS 3 ), 10 μl 20 ml PBST ; PBST, 3, 15 min; HRP-DAB ( ) 1.9 数据分析 SPSS 22.0, ± (x ±SD), P<0.05 Sigma Plot 结果与分析 2.1 PmPCNA 基因的 cdna 序列分析, SMART RACE PmPCNA cdna ( 1) PmPCNA cdna 978 bp, 135 bp 5 UTR 60 bp 3 UTR 783 bp ORF (GenBank : KU380336) ORF 260, PCNA kd BLAST SMART, PCNA PCNA/RFC (replication factor C), 208 aa 254 aa, PCNA SignalP 4.0, PmPCNA NetNGlyc 2.0 NetPhos 2.0 PmPCNA, PmPCNA 1 N- 12 ( 8

5 : 1055 1 PmPCNA cdna ; (ATG) (TAA); PCNA/RFC ; ; (AATAA). Fig.

4 5 : PmPCNA cdna ; (ATG) (TAA); PCNA/RFC ; ; (AATAA). Fig. 1 Nucleotide and deduced amino acid sequence of PmPCNA Numbers on the right and left of each row represent amino acid or nucleotide position. The boxed sequences represent the initiation code (ATG) and termination code (TAA), respectively. The PCNA/RFCL domain is shown in grey. The predicted N-glycosylation site is shown in the circle. The underlined sequence (AATAA) was polyadenylation signal. 1 2 ) NCBI PCNA, PCNA, PmPCNA (Litopenaeus vannamei) (99%), (Zootermopsis nevadensis) (86%) (Drosophila melanogaster) (81%) (Bos taurus) (75%) (Homo sapiens) (74%) (Xenopus laevis) (74%) ( 2), (Fenneropenaeus chinensis), (Litopenaeus vannamei) (Marsupenaeus japonicus), 溞 (Daphnia pulex) ( 3) 2.2 PmPCNA mrna 在组织中的表达差异 PCR PmPCNA, ( 4) PCR PmPCNA mrna, PCNA,,,, 4.8 4, 0.15 PmPCNA mrna

1056 23 2 PmPCNA PCNA,. GenBank : (AEJ89927.1), (ACA09718.1), (ABM66815.1), (KDR11193.1), (NP_002583.1), (NP_001029666.1), (NP_476905.1), (NP_001081011.1), 溞 (EFX70582.1), (NP_571479.2). Fig.

5 PmPCNA PCNA,. GenBank : (AEJ ), (ACA ), (ABM ), (KDR ), (NP_ ), (NP_ ), (NP_ ), (NP_ ), 溞 (EFX ), (NP_ ). Fig. 2 Multiple alignment of the deduced amino acid sequences of PCNA from Penaeus monodon and other species Residues in black and grey background indicate identical and similar sites, respectively. The GenBank access numbers are as follows: Litopenaeus vannamei(aej ), Marsupenaeus japonicas(aca ), Fenneropenaeus chinensis(abm ), Zootermopsis nevadensis(kdr ), Homo sapiens(np_ ), Bos taurus(np_ ), Drosophila melanogaster(np_ ), Xenopus laevis(np_ ), Daphnia pulex(efx ), Danio rerio(np_ ). : ( 5) 2.3 斑节对虾卵巢发育各时期 PmPCNA mrna 的相对表达 PCR PmPCNA mrna,, PmPCNA mrna, III (P>0.05), (P>0.05), III 2 ( 6)

5 : 1057 3 MEG A5.0 PCNA NJ 1000. Fig. 3 NJ phylogenetic tree based on amino acid sequence encoded by PCNA via MEGA 5.

6 5 : MEG A5.0 PCNA NJ Fig. 3 NJ phylogenetic tree based on amino acid sequence encoded by PCNA via MEGA 5.0 software The values at the forks indicate the percentage of trees in which this grouping occurred after bootstrapping the data (1000 replicates). 4 PCR EF-1α ; Marker, DNA ; Hea: ; Int: ; Hep: ; Sto: ; Ova: ; Gil: ; Mus: ; Bra:. Fig. 4 Electrophoresis results of PmPCNA and EF-1α expression in different tissues of Penaeus monodon Using EF-1α as the internal control gene. Marker, molecular weight. Hea: heart; Ins: intestines; Hep: hepatopancreas; Sto: stomach; Ova: ovary; Gil: gill; Mus: muscle; Bra: brain. 2.4 注射 5-HT 后斑节对虾卵巢中 PmPCNA mrna 的表达 PCR 5-HT PmPCNA,, 5-HT 12 h PmPCNA (P>0.05); 12 h, PmPCNA, 48 h (P<0.05), 5 ; 72 h,, 3 (P<0.05, 7) 2.5 斑节对虾 PCNA 蛋白体外重组表达结果, PCNA-pGEX-4T-1 55 kd, IPTG, SDS-PAGE, 2 h, ( 8A) 6 h 8 h, GST Western Blot, PCNA, ( 8B),, 3 讨论 PCNA, DNA, T 4 gene45 DNA III β, DNA, DNA [8, 25], PCNA, PCNA DNA [26], PCNA 2

1058 23 5 PmPCNA mrna (P<0.05). Hea: ; Int: ; Hep: ; Sto: ; Ova: ; Gil: ; Mus: ; Bra:. Fig.

7 PmPCNA mrna (P<0.05). Hea: ; Int: ; Hep: ; Sto: ; Ova: ; Gil: ; Mus: ; Bra:. Fig. 5 Relative mrna expression of PmPCNA in different tissues of Penaeus monodon Significant differences between various tissues were indicated with different letters above vertical bars (P<0.05). Hea: heart; Ins: intestines; Hep: hepatopancreas; Sto: stomach; Ova: ovary; Gil: gill; Mus: muscle; Bra: brain. 7 5-HT PmPCNA mrna PBS, 5-HT ; ** (P<0.01). Fig. 7 Mean relative expression levels of PmPCNA in ovaries of Peraeus monodon at different times after injection of 5-HT PBS was used in the control group. Extremely significant differences between trial and control were indicated with ** (P<0.01). 6 PmPCNA mrna (P ). I : ; II : ; III : ; IV : ; V :. Fig. 6 Relative expression levels of PmPCNA mrna in different development stages of ovary of Penaeus monodon Different letters above vertical bars indicate significant difference between different stages (P ). I: ovogonium stage; II: chromatin nucleolus stage; III: perinucleolus stage; IV: yolky stage; V: cortical rod stage. 8 PCNA-pGEX-4T-1 A. PCNA-pGEX-4T-1 SDS-PAGE ; B. PCNA-pGEX-4T-1. M: ; W: ; 2 h, 4 h, 6 h, 8 h. Fig. 8 Recombinant expression of PCNA-pGEX-4T-1 in vitro A. SDS-PAGE analysis of recombinant expression of PCNApGEX-4T-1; B. Western blot of PCNA-pGEX-4T-1. M: protein marker; W: non-induced; 2 h, 4 h, 6 h, 8 h represent the induced time. (cyclin-dependent kinases 2, CDK2) [27 28] PCNA,,,, PCNA

8 5 : 1059 PmPCNA cdna ( 1), 783 bp ORF, 135 bp 5 UTR 60 bp 3 UTR, 978 bp, PCNA 260, 4.59 kd kd, 1 N- 12 PCNA/RFC (replication factor C), PCNA DNA, DNA [29] Thompson [30], RFC PCNA, RFC PCNA PCNA, PCNA DNA [31] [32], PmPCNA PCNA, PCNA PmPCNA mrna ( 5), PmPCNA,,,, de-la-re-vega [7] PCNA, ; Xie [31], PCNA, PCNA, PCNA PmPCNA,, PmPCNA, III PmPCNA 4, 4, III 2 ( 5) [21], I II, ; III IV,, ; V III PmPCNA ( 5), PmPCNA ;, PmPCNA PmPCNA, PCNA, 5-HT,,, [33] 5-HT, Tinikul [34], 5-HT (Macrobrachium rosenbergii) ; Wongprasert [35], 5-HT ; [36] (Procambrus clarkii) 5-HT, 5-HT PmPCNA, 5-HT, 5-HT PmPCNA,, 5-HT 48 h (P<0.01), 5, 72 h, 3 ( 7) PCNA PCNA-pGEX-4T-1,,, PCNA-pGEX-4T-1 2 h, ( 7), 55 kd, Western Blot, GST ( 8B),

9 , PmPCNA PmPCNA, PmPCNA, 5-HT PmPCNA PmPCNA, 5-HT PmPCNA, PCNA PmPCNA, 参考文献 : [1] Carrasco-Miranda J S, Lopez-Zavala A A, Arvizu-Flores A A, et al. Crystal structure of the shrimp proliferating cell nuclear antigen: structural complementarity with WSSV DNA polymerase PIP-box[J]. PLoS ONE, 2014, 9(4): e [2] Miyachi K, Fritzler M J, Tan E M. Autoantibody to a nuclear antigen in proliferating cells[j]. Immunology, 1978, 121(6): [3] Tománek M, Chronowska E. Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary[j]. Folia Histochem Cytobiol, 2006, 44(4): [4] Motiwale L, Ingle A D, Rao K V K. Mouse skin tumor promotion by sodium arsenate is associated with enhanced PCNA expression[j]. Cancer Lett, 2005, 223(1): [5] Witko-Sarsat V, Mocek J, Bouayad D, et al. Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival[j]. J Exp Med, 2010, 207(12): [6] Bravo R, Frank R, Blundell P, et al. Cyclin/PCNA is the auxiliary protein of DNA polymerase-δ[j]. Nature, 1987, 326 (6112): [7] de-la-re-vega E, Muhlia-Almazan A, Arvizu-Flores A A, et al. Molecular modeling and expression of the Litopenaeus vannamei proliferating cell nuclear antigen (PCNA) after white spot syndrome virus shrimp infection[j]. Results Immunol, 2011, 1(1): [8] Wang K, Li R G, Cheng D L. Development of function and the crystal structure of proliferating cell nucleus antigen (PCNA)[J]. Journal of Shandong Polytechnic University, 2013, 25(3): [,,. (PCNA) [J]. :, 2013, 25(3): ] [9] Strzalka W, Ziemienowicz A. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation[j]. Ann Bot, 2011, 107(7): [10] Moldovan G L, Pfander B, Jentsch S. PCNA, the maestro of the replication fork[j]. Cell, 2007, 129(4): [11] Zhao H, Ho P C, Lo Y H, et al. Interaction of proliferation cell nuclear antigen (PCNA) with c-abl in cell proliferation and response to DNA damages in breast cancer[j]. PLoS ONE, 2012, 7(1): e2941. [12] Atilgan R, Kuloğlu T, Boztosun A, et al. Investigation of the effects of unilateral total salpingectomy on ovarian proliferating cell nuclear antigen and follicular reserve: experimental study[j]. Eur J Obstet Gynecol Reprod Biol, 2015, 188: [13] Langerak P, Nygren A O, Krijger P H, et al. A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNAK164 modification[j]. J Exp Med, 2007, 204(8): [14] Oktay K, Schenken R S, Nelson J F. Proliferating cell nuclear antigen marks the initiation of follicular growth in the rat[j]. Biol Reprod, 1995, 53(2): [15] Wandji S, Srsen V, Voss A, et al. Initiation in vitro of growth of bovine primordial follicles[j]. Biol Reprod, 1996, 55(5): [16] Wandji S, Srsen V, Nathanielsz P, et al. Initiation of growth of baboon primordial follicles in vitro[j]. Hum Reprod, 1997, 12(9): [17] Yang Q B, Jiang S, Huang J H, et al. The compensatory growth of Penaeus monodon after starvation[j]. South China Fisheries Science, 2013, 9(5): [,,,. [J]., 2013, 9(5): ] [18] Zhou J. Reseach of some hormones and genes being correlation with ovary development from Penaeus monodon[d]. Shanghai: Shanghai Ocean University, [. [D]. :, 2012.] [19] Okumura T, Sakiyama K. Hemolymph levels of vertebratetype steroid hormones in female kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae) during natural reproductive cycle and induced ovarian development by eyestalk ablation[j]. Fish Sci, 2004, 70(3): [20] Chen Y N, Fan H F, Hsieh S L, et al. Physiological involvement of DA in ovarian development of the freshwater giant prawn, Macrobrachium rosenbergii[j]. Aquaculture, 2003, 228(1 4): [21] Huang J H, Zhou F L, Ma Z M, et al. Morphological and histological observation on ovary development of Penaeus monodon from northern South China Sea[J]. Journal of

10 5 : 1061 Tropical Ocean Ography, 2006, 25(3): [,,,. [J]., 2006, 25(3): ] [22] Dai W T, Fu M J, Zhao C, et al. Molecular cloning and expression analysis of CDK2 gene from black tiger shrimps (Penaeus monodon)[j]. South China Fisheries Science, 2015, 11(2): [,,,. CDK2 cdna [J]., 2015, 11(2): 1 11.] [23] Larkin M A, Blackshields G, Brown N P, et al. Clustal W and Clustal X version 2.0[J]. Bioinformatics, 2007, 23(21): [24] Mukhopadhyay N, Almasy L, M, Mulvihill W, et al. Mega 2: data-handling for facilitating genetic linkage and association analyses[j]. Bioinformatics, 2005, 21(10): [25] Giovanni M, Ulrich H. Proliferating cell nuclear antigen (PCNA): a dancer with many partners[j]. J Cell Sci, 2003, 116(15): [26] Song N M, Sang J L, Xu H. Development of molecular structure and the biological function of proliferating cell nucleus antigen (PCNA)[J]. Progress in Natural Science, 2006, 16(10): [,,. (PCNA) [J]., 2006, 16(10): ] [27] Koundrioukoff S, Jónsson Z O, Hasan S, et al. A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation[j]. J Biol Chem, 2000, 275(30): [28] Zhu X N, Kim D H, Lin H R, et al. Proteolysis of Xenopus Cip-type CDK inhibitor, p16xic2, is regulated by PCNA binding and CDK2 phosphorylation[j]. Cell Div, 2013(8): 5. [29] Matsumiya S, Ishino S, Ishino Y, et al. Physical interaction between proliferating cell nuclear antigen and replication factor C from Pyrococcus furiosus[j]. Genes Cells, 2002, 7(9): [30] Thompson J A, Marzahn M R, O donnell M, et al. Replication factor C is a more effective proliferating cell nuclear antigen (PCNA) opener than the checkpoint clamp loader, Rad24-RFC[J]. J Biol Chem, 2012, 287(3): [31] Xie Y S, Wang B, Li F H, et al. Molecular cloning and characterization of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis[j]. Comp Biochem Physiol B: Biochem Mol Biol, 2008, 151(2): [32] 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(4): [33] Dong Y N. Study on collaborative maturation-promoting effect of 5-hydroxytryptamine and eyestalk ablation on the ovary of female Marsupenaeus japonicas[d]. Xiamen: Xiamen University, [. 5- [D]. :, 2014.] [34] Tinikul Y, Mercier A J, Sobhon P. Distribution of dopamine and octopamine in the central nervous system and ovary during the ovarian maturation cycle of the giant freshwater prawn, Macrobrachium rosenbergii[j]. Tissue Cell, 2009, 41(6): [35] Wongprasert K, Asuvapongpatana S, Poltana P, et al. Serotonin stimulates ovarian maturation and spawning in the black tiger shrimp Penaeus monodon[j]. Aquaculture, 2006, 261(4): [36] Wei H, Sha X J, Chen A Q, et al. Distribution of 5-HT and DA in the ovary and hepatopancreas during the ovarian development in Procambrus clarkii[j]. Freshwater Fisheries, 2013, 42(6): [,,,. 5-HT DA [J]., 2013, 42(6): ]

11 Molecular cloning and expression analysis of PCNA from black tiger shrimp, Penaeus monodon WANG Chengyang 1, 2, ZHAO Chao 1, 3, FU Mingjun 1, 3 1, 3, 4, QIU Lihua 1. South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou , China; 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai , China; 3. Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, Guangzhou , China; 4. Tropical Aquaculture Research and Development Center of South China Sea Fisheries Research Institute, Sanya , China Abstract: Proliferating cell nuclear antigen (PCNA) is a member of the sliding clamp protein family that plays important roles in DNA replication, and is strongly expressed in proliferating tissues. Several studies have discovered other biological roles of PCNA, for example, in DNA repair, cell cycle regulation, and chromosome recombination. When culturing shrimp, the first problem is how to induce maturation of the ovary; therefore, it is important to understand ovarian development. We focused on PCNA because it plays an important role in cell proliferation. The PCNA gene (PmPCNA) was cloned from Penaeus monodon using the rapid amplification of cdna ends technique. The sequence encoded a protein of 260 amino acids that showed high homology to PCNA proteins from other species. The full-length PmPCNA cdna was 978 bp long and contained a 5 -untranslated region (UTR) of 135 bp, a 3 -UTR of 60 bp, and an open reading frame of 783 bp. The protein was predicted to have a molecular weight of kda and an isoelectric point of Analyses of PmPCNA transcript levels by quantitative real-time PCR indicated that it was transcribed in all tissues, with high transcript levels in the ovary and brain. During ovarian maturation, there were higher transcript levels of PmPCNA in the stage III ovary than in ovaries at other stages (P<0.05). Previous research has shown that 5-hydroxytryptamine (5-HT) can stimulate ovary growth and development. Injection of 5-HT resulted in increased transcript levels of PmPCNA in the ovaries, with the peak transcript levels at 48 h post-injection. The PmPCNA protein was successfully expressed in a prokaryote, providing a platform for further research. These results provide important information on the role of PmPCNA in the development of the shrimp ovary. Key words: Penaeus monodon; ovarian development; PmPCNA; 5-HT; qrt-pcr; prokaryotic expression Corresponding author: QIU Lihua. qiu902@126.com

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吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８