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1 2016 年 9 月, 23(5): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 三疣梭子蟹细胞 Cdk7 基因克隆及其在卵巢发育中的表达 1, 贾复龙 2 1,, 孟宪亮 2 1,, 刘萍 2 1,, 李健 2 1, 2, 高保全 1.,, ; 2.,, 摘要 : RACE (Portunus trituberculatus) 7(Cdk7) cdna 5 3 (UTR) 23 bp 178 bp 1056 bp, 351, kd, / T-loop, CDK7 Cdk7, Cdk7 PCR, Cdk7 (P<0.05) Cdk7, I Ⅱ (P<0.05), 4, Cdk7, 关键词 : ; Cdk7; ; ; 中图分类号 : S917 文献标志码 : A 文章编号 : (2016) (cyclin-dependent kinase 7, Cdk7) Schachter [1] 1994 Cdk, Fisher [2] Cdk7 Cdk,, (Cyclins), [3] Cdk7 Cyclin H, (cyclin-activating kinase, CAK), G1 Cdk2 Cdk4 Cdk6, G1 S ; Cdk1 (maturation or M-phase promoting factor, MPF), G2 M, [3 6], Ⅱ H (TF Ⅱ H), Cdk7 TF Ⅱ H, [7 8], Cdk7 Fujii [9] Cdk7, Cdk7 [10 11],, [12 13] (Portunus trituberculatus) (Arthropoda) (Crustacea) (Decapoda) (Portunidae) 收稿日期 : ; 修订日期 : 基金项目 : ( , ); (LJNY ). 作者简介 : (1989 ),,, @qq.com 通信作者 :,,. liuping@ysfri.ac.cn

2 5 : Cdk7 1033,, [14 16],,, [17] SMART RACE, Cdk7 cdna, PCR, Cdk7 ; 1 材料与方法 1.1 实验材料, (560 mm 360 mm 280 mm) 7 d( 3 PVC, 6, ) (20±2) 33, ph 8.2,, 1/3 Trizol Reagen Invitrogen, SMART TM RACE Amplification Kit Advantage 2 PCR Kit Clontech, DNA ( ), SYBR Premix Ex Taq TM II PrimeScript RT reagent Kit PMD18-T DH5α TaKaRa 1.2 三疣梭子蟹卵巢不同发育时期卵巢组织的采集 , [18],, 5, HE, 1.3 去除眼柄实验 7 d 3, 40 3 s,, ,, RNA 1.4 实验方法 RACE 第一链合成 Trizol RNA,, RNA, RNA RNA, SMART TM RACE Amplification Kit 3 5 RACE cdna 三疣梭子蟹 Cdk7 基因 cdna 序列全长的克隆 Cdk7 EST, Primer Premier RACE ( 1) 3 5 Advantage 2 PCR Kit, SMART RACE, CDK CDK UPM, Cdk7 cdna 3 5 PCR : s, s, 72 2 min, 30, 1.0%, PMD18-T PMD18-T, DH5α,, AMP 6 h PCR 表 1 本实验用到的所有引物的序列 Tab. 1 The sequences of primers used in this study primer CDK-3-1 CDK-5-1 CDK7-F CDK7-R β-actin-f β-actin-r UPM (5 3 ) sequence(5 3 ) TCTTCACAGCACTTGGCACA CTCCTCACGGCTACCCAG CCTGGATGTGTTTGGCTACA GGGTGAGAATGATGTTGTCTG CGAAACCTTCAACACTCCCG GGGACAGTGTGTGAAACGCC CTAATACGACTCACTATAGGGC

3 生物信息学分析 VecScreen ( Contig Express Project Cdk7 EST Cdk7 cdna EditSeq (ORF), Cdk7 EditSeq BLAST ( ExPASy ( InterproScan ( html), NetPhos 2.0 Server ( GenBank Cdk7 Cdk7 DNAMAN, MEGA 5.0 Cdk7 NJ 组织表达分析 mrna, PrimeScript RT reagent Kit cdna Cdk7 β-actin cdna, 1 (β-actin-f β-actin-r CDK7-F CDK7-R) ( 1), SYBR Premix Ex Taq TM Ⅱ Cdk7 ( ) SYBR Premix Ex Taq TM II, 10 μl : 5 μl SYBR Premix Ex Taq TM II (2 ) 0.2 μl ROX Reference Dye II (50 ) 0.4 μl 10 μmol/l 3.0 μl DEPC 1.0 μl cdna : s; 95 5 s, s, 40 ; s, 60 1 min, s 2 ΔΔC t, SPSS19.0, P< 结果与分析 2.1 三疣梭子蟹 Cdk7 基因的获得及其序列分析 RNA, OD 260 nm /OD 280 nm 1.9~2.0; 1.0%, 28S 18S rrna, RNA, RACE SMART TM RACE Amplification Kit 3 5 RACE cdna, CDK CDK UPM 5 3 RACE, Cdk7 cdna, GenBank KT cdna 1257 bp, 1056 bp CDS( ) 23 bp 5 UTR 178 bp 3 UTR ( 1) 2.2 氨基酸序列分析 Cdk7 cdna (ORF) 1056 bp, ORF 351, kd, 8.69 Cdk (N-62, N-92, N-286, N-292) Cdk7 1 / (18~302 aa) 1 / (140~ 152 aa)( 1) 2.3 同源性及系统进化分析 BLAST, Cdk7 Cdk7, 93%; 72% 70% 70% 69%, / T-loop ( 2) MEGA5.0 Cdk7,, 10 Cdk7

4 5 : Cdk Cdk7 /, T-loop,. Fig. 1 Nucleotide sequence and deduced amino acid sequence of Portunus trituberculatus s Cdk7 gene The catalytic domain of the serine/threonine kinase, T-loop, initiation codon and stop codon were marked with shadow, underline and black box, respectively., Cdk7 Cdk7,, 鮰 ( 3) 2.4 组织差异表达分析 Cdk7, (P<0.05), ( 4) 2.5 三疣梭子蟹 Cdk7 基因在不同发育时期卵巢组织中的差异表达分析 Cdk7 (P<0.05), II, I Ⅲ VI IV, V ( 5) 2.6 去眼柄后三疣梭子蟹卵巢组织中 Cdk7 基因差异表达分析 PCR, 1 4 7, Cdk7, 4, 10 Cdk7 ; 7, 4 (P<0.05, 6)

1036 23 2 Cdk7 Cdk7 Cdk7 GeneBank : (KT804694), (AIX11620.1), (XP_003396102.1), (XP_012528779.1), (XP_011694124.1). Fig.

5 Cdk7 Cdk7 Cdk7 GeneBank : (KT804694), (AIX ), (XP_ ), (XP_ ), (XP_ ). Fig. 2 Multiple alignments of the amino acid sequerces deduced by Cdk7 gene of Portunus trituberculatu with other species The GenBank accession numbers of Cdk7 gene were as follows: Portunus trituberculatus (KT804694), Penaeus monodon (AIX ), Bombus terrestris (XP_ ), Monomorium pharaonis (XP_ ), Wasmannia auropunctata (XP_ ). 3 讨论 Cdk7 cdna, CAK, Cdk7 Cdks, [19], Cdk7 Cdk / (17~315 aa), T-loop (162~ 189 aa, DFGLARFFGSPNRQYSHQVVTRWYRSPE) T-loop Cdks [20], Cdk7 T-loop Martinez [21] Larochelle [22] (Xenopus laevis) Cdk7 T-loop Ser-170

5 : Cdk7 1037 3 Cdk7 NJ Ck7 GenBank : (EHJ67061), (NP_001166283), (KOC69523), (NP_511044), (KT804694), (AIX11620), (NP_001253586), (NP_001790), 鮰 (ADO28014), (NP_998126). Fig.

6 5 : Cdk Cdk7 NJ Ck7 GenBank : (EHJ67061), (NP_ ), (KOC69523), (NP_511044), (KT804694), (AIX11620), (NP_ ), (NP_001790), 鮰 (ADO28014), (NP_998126). Fig. 3 NJ tree based on Cdk7 amino acids The GenBank accession numbers of different species were as follows: Danaus plexippus (EHJ67061), Bombyx mori (NP_ ), Habropoda laboriosa (KOC69523), Drosophila melanogaster (NP_511044), P. trituberculatus (KT804694), Penaeus monodon (AIX11620), Macaca mulatta (NP_ ), Homo sapiens (NP_001790), Ictalurus furcatus (ADO28014), Danio rerio (NP_998126). CAK, Cdk7 CAK,, [3] Cdk7,, 4 Cdk7 (P<0.05). Fig. 4 Distribution of Cdk7 in different tissues of Portunus trituberculatus The different letters up the error bars indicate significant difference between different tissue (P<0.05). Thr-176 Ser-164 Thr-170, Cdk7 T-loop 4 (Ser-171, Ser-177, Thr-182, Ser-187), 5 Cdk7 O: ; (P<0.05). Fig. 5 The expression of Cdk7 gene in ovary of Portunus trituberculatus in different gonadal stages O: ovary. Different letters up the error bars indicated significant difference between different overy stages (P<0.05).

7 (P<0.05),, Cdk7 Cdk7, (I II ),, Cdk7 Cdk7 Cdc2 ( MPF ) [23], Cdk7 Cdc2, MPF Preechaphol [29], 1 p23,, MPF, [29 30] Cdk7 CAK, MPF Uawisetwathana [31] Cdk7 Cyclin B Cdc2, Cdk7,, Cdk7 MPF RACE Cdk7 cdna,,, 参考文献 : 6 Cdk7 (P<0.05). Fig. 6 The expression of Cdk7 gene in ovary of Portunus trituberculatus after eyestalk ablation The different letters up the error bars indicated significant difference between groups at the same time (P<0.05). X-, (GIH),,, [24 25], [26 28] [1] Schachter M M, Fisher R P. The Cdk-activating kinase Cdk7: taking yes for an answer[j]. Cell Cycle, 2013, 12(20): [2] Fisher R P. Secrets of a double agent: Cdk7 in cell-cycle control and transcription[j]. J Cell Sci, 2005, 118(22): [3] Chu L, Qian M, Yan Y C. Advances in the studies of cyclin development kinase-activating kinase (CAK)[J]. Chinese Bulletin of Life Sciences, 2006, 18(2): [, 旻,. (CAK) [J]., 2006, 18(2): ] [4] Fesquet D, Labbe J C, Derancourt J, et al. The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues[j]. EMBO J, 1993, 12(8): [5] Kaldis P. The cdk-activating kinase (CAK): from yeast to mammals[j]. Cell Mol Life Sci, 1999, 55(2): [6] Phinyo M, Nounurai P, Hiransuchalert R, et al. Characterization and expression analysis of Cyclin-dependent kinase 7

8 5 : Cdk gene and protein in ovaries of the giant tiger shrimp Penaeus monodon[j]. Aquaculture, 2014, 432: [7] Roy R, Adamczewski J P, Seroz T, et al. The MO15 cell cycle kinase is associated with the TF II H transcription- DNA repair factor[j]. Cell, 1994, 79(6): [8] Shiekhattar R, Mermelstein F, Fisher R P, et al. Cdk-activating kinase complex is a component of human transcription factor TF II H[J]. Nature, 1995, 374(6519): [9] Fujii W, Nishimura T, Kano K, et al. CDK7 and Cyclin H are components of CDK-activating kinase and are required for meiotic progression of pig oocytes[j]. Biol Reprod, 2011, 85(6): [10] Kelso T W, Baumgart K, Eickhoff J, et al. Cyclin-dependent kinase 7 controls mrna synthesis by affecting stability of preinitiation complexes, leading to altered gene expression, cell cycle progression, and survival of tumor cells[j]. Mol Cell Biol, 2014, 34(19): [11] Ji Q B, Xu X J, Zhang Q, et al. Construction and identification of plasmid vector of Cdk7[J]. Military Medical Sciences, 2014, 38(12): [,,,. CDK7 [J]., 2014, 38(12): ] [12] Darbon J M, Devault A, Taviaux S, et al. Cloning, expression and subcellular localization of the human homolog of p40mo15 catalytic subunit of cdk-activating kinase[j]. Oncogene, 1994, 9(11): [13] Ganuza M, Santamaria D. Cdk7: open questions beyond the prevailing model[j]. Cell Cycle, 2012, 11(19): [14] Tune S. Crabs of Japan and Adjacent Seas[M]. Tokyo: Kodansha, 1976: [15] Sun Y M, Yan Y, Sun J J. Larval development of the swimming crab, Portunus trituberculatus[j]. J Fish China, 1984, 8: [16] Meng X L, Liu P, Jia F L, et al. De novo transcriptome analysis of Portunus trituberculatus ovary and testis by RNA-Seq: Identification of genes involved in gonadal development[j]. PLoS One, 2015, 10(6): e [17] Jia L. Preliminary study on the ovary development of the crab Portunus trituberculatus[d]. Qingdao: Ocean University of China, [. (Portunus trituberculatus) [D]. :, 2008.] [18] Wu X G, Yao G G, Yang X Z, et al. A study on the ovarian development of Portunus trituberculatus in East China Sea during the first reproductive cycle[j]. Acta Oceanologica Sinica, 2007, 29(4): [,,,. [J]. ( ), 2007, 29(4): ] [19] Devos M, Mommaerts E, Migeot V, et al. Fission yeast Cdk7 controls gene expression through both its CAK and C-terminal domain kinase activities[j]. Mol Cell Biol, 2015, 35(9): [20] 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.] [21] Martinez A M, Afshar M, Martin F, et al. Dual phosphorylation of the T-loop in cdk7: its role in controlling cyclin H binding and CAK activity[j]. EMBO J, 1997, 16(2): [22] Larochelle S, Chen J, Knights R, et al. T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity[j]. EMBO J, 2001, 20(14): [23] Wang L, Zuo D, Lv W W, et al. Characterization of Cdc2 kinase in the red claw crayfish (Cherax quadricarinatus): Evidence for its role in regulating oogenesis[j]. Gene, 2013, 515(2): [24] Guan Z B, Shui Y, Zhou X, et al. Participation of calmodulin in ovarian maturation induced by eyestalk ablation in red swamp crayfish Procambarus clarkia[j]. Aquac Res, 2013, 44(10): [25] Shui Y, Shi Y H, Xu Z H, et al. Advances of growth and development in crustaceans induced by eyestalk ablation[j]. Guangdong Agricultural Sciences, 2013, 40(21): [,,,. [J]., 2013, 40(21): ] [26] Lee S O, Jeon J M, Oh C W, et al. Two juvenile hormone esterase-like carboxylesterase cdnas from a Pandalus shrimp (Pandalopsis japonica): cloning, tissue expression, and effects of eyestalk ablation[j]. Comp Biochem Physiol B Biochem Mol Biol, 2011, 159(3): [27] Bai H K, Qiao H, Li F J, et al. Molecular characterization and developmental expression of vitellogenin in the oriental river prawn Macrobrachium nipponense and the effects of RNA interference and eyestalk ablation on ovarian maturation[j]. Gene, 2015, 562(1): [28] Kang B J, Okutsu T, Tsutsui N, et al. Dynamics of vitellogenin and vitellogenesis-inhibiting hormone levels in adult and subadult whiteleg shrimp, Litopenaeus vannamei: relation to molting and eyestalk ablation[j]. Biol Reprod, 2014, 90(1): 12. [29] Preechaphol R, Klinbunga S, Yamano K, et al. Molecular cloning and expression of progestin membrane receptor component 1 (Pgmrc1) of the giant tiger shrimp Penaeus monodon[j]. Gen Comp Endocrinol, 2010, 168(3):

9 [30] Paul S, Banerjee N, Chatterjee A, et al. Arsenic-induced promoter hypomethylation and over-expression of ERCC2 reduces DNA repair capacity in humans by non-disjunction of the ERCC2-Cdk7 complex[j]. Metallomics, 2014, 6(4): [31] Uawisetwathana U, Leelatanawit R, Klanchui A, et al. Insights into eyestalk ablation mechanism to induce ovarian maturation in the black tiger shrimp[j]. PLoS One, 2011, 6(9): e Cloning and expression analysis of Cdk7, a gene involved in ovarian development, from swimming crab (Portunus trituberculatus) JIA Fulong 1, 2, MENG Xianliang 1, 2, LIU Ping 1, 2, LI Jian 1, 2, GAO Baoquan 1, 2 1. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China; 2. Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao , China Abstract: The full-length cdna encoding Cdk7 was cloned from swimming crab (Portunus trituberculatus) using the rapid amplification of cdna ends technique (RACE). The complete cdna was 1257 bp long, and contained an open reading frame (ORF) of 1056 bp, a 23-bp 3ʹ untranslated region, and a 178-bp 5ʹ untranslated region. The ORF encoded a polypeptide of 351 amino acids containing a serine/threonine kinase domain and a T-loop. An homology analysis revealed that the Cdk7 of P. trituberculatus had high identity with those from other species. Quantitative real-time PCR analyses showed that Cdk7 transcripts were present in all of the tissues examined, with the highest transcript levels in the ovary. The high transcript level of Cdk7 in the ovary was consistent with its role in regulating cell division. The Cdk7 mrna levels in oogonia were higher in the first and the second stages than in the other stages of overy development, indicating that Cdk7 may play an important role in oogonial mitosis. After eyestalk ablation, the transcript levels of Cdk7 significantly increased in the ovary to peak on day 4, and then decreased. Together, these results suggested that Cdk7 may play important roles in ovarian development, especially oogenesis, in P. trituberculatus. These data provide useful information for further research on the regulation of gonad development in this species and other crustaceans. Key words: Portunus trituberculatus; Cdk7; gene cloning; eyestalk ablation; gene expression Corresponding author: LIU Ping. liuping@ysfri.ac.cn

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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