2015 7, 22(4): 608 619 Journal of Fishery Sciences of China 研 究 论 文 DOI: 10.3724/SP.J.1118.2015.140383 柳 学 周 1, 史 宝 1 1,, 李 晓 晓 2, 徐 永 江 1, 王 珊 珊 1 1, 李 晓 妮 1. ;,, 266071; 2., 201306 摘 要 : (Cynoglossus semilaevis Günther), (mpr-like) (RACE), 2 002 bp cdna ;, 7 ; MEGA4.0 Clustal X mpr-like, mpr-like 鳉 (Oryzias latipes) (Gasterosteus aculeayus) mpr-like, 68% 72% PCR mpr-like mrna, mpr-like mrna,, ;, mpr-like mrna II V, VI (P<0.05);, mpr-like mrna II V, V (P<0.05), VI ;, mpr-like mrna, V (P<0.05), (P<0.05), IV V, ; : ; ; ; ; 中 图 分 类 号 : Q959; S917 文 献 标 志 码 : A 文 章 编 号 : 1005 8737 (2015)04 0608 12,, ;, [1],, [2] Masui [3],, Sadler [4] (Xenopus laevis),,, 收 稿 日 期 : 2014-09-14; 修 订 日 期 : 2014-11-05. 基 金 项 目 : (31201982); (CARS-50); (BS2013SW042);. 作 者 简 介 :,,. E-mail: liuxz@ysfri.ac.cn
4 : 609,, [5] α (mprα) β (mprβ) γ (mprγ), [6 8] (Paralichthys olivaceus), (mprl), ; mprl mrna ; IV mprl mrna V (P<0.05), V VI mprl [9], mprl (Cynoglossus semilaevis Günther),,, [10 11] mpr-like cdna,, mpr-like mrna,, mpr-like, 1 1.1,, F 1, 53~66 cm, 1 266.3~2 271.0 g :, 8~25, 27~31, ph 7.8~8.4, 5 mg/l, 4, 1 MS-222,,,,, 80 Davidesons AFA, 4 4~6 h, 12 000 r/min 10 min, 40 1.2 Davidesons AFA,,, LEICA RM 2235 ( ), 5 µm, HE,, LEICA DW4000B ( ), HE, [12 13] 1.3 RNA cdna 80, RNAiso Plus (TaKaRa ) RNA, 1% RNA Thermo Nanodrop 2000 RNA 1 g RNA, TaKaRa PrimerScript TM 1st Strand cdna Synthesis Kit, cdna, 20 1.4 mpr-like 1.4.1 mpr-like mprl/f( 1), cdna, mpr-like PCR peasy-t1, Trans1-T1 Phage Resistant, Amp LB 12 h, Amp, 37 8 h PCR 1.4.2 mpr-like RNA, SMARTer TM RACE
610 22 cdna Amplification Kit(Clontech ) 5 -RACE 3 -RACE cdna, cdna Advantage 2 PCR (Clontech ) PCR mprlgsp1/mprlngsp1( 1) 5, mprlgsp2/mprlngsp2( 1) 3, 表 1 半 滑 舌 鳎 mpr-like 基 因 克 隆 与 表 达 所 用 引 物 序 列 Tab. 1 Primer sequences for Cynoglossus semilaevis mrna cloning and expression analysis primer name mprlf mprlr mprlgsp1 mprlngsp1 mprlgsp2 mprlngsp2 RT-mPRLF RT-mPRLR RT-mPRLF2 RT-mPRLR2 18S F 18S R (5 3 ) sequence (5 3 ) TTCCAGCGCCACAAYGAGAC CACCTGGCACACCTTRCGCA CGTAACAGCAGCCGAGGCAGGACAGACA CTGGCATGAAGATCCCACGAACGTGTCG ACCGAGACCGTAGACTTTGTGGGTGA ACCATCGACACGTTCGTGGGATCTTCA CAGGGAGTGTAATGTGAAAACAGG AAAGAGGACAGAAGGAGAATCAACA TGTCCTCGTCCAGCGTCACT CCCTGTCCCAAGAAATCACACT GGTCTGTGATGCCCTTAGATGTC AGTGGGGTTCAGCGGGTTAC 1.5 NCBI BLAST, Dnastar ; SOSUI [14] ; I-TASSE(http: // zhanglab.ccmb.med.umich.edu/i-tasser/) ; Clustal X 2, mpr-like mpr- Like ; MEGA6.0 Neighbor-joining ( 1 000) 1.6 mpr-like mrna mpr-like mrna 4, 12 RNA TaKaRa PrimerScript TM RT reagent Kit with gdna Eraser cdna mpr-like mrna [15] : II III IV V VI RNA ; 4 mpr-like mrna : II III IV V VI 4, RNA (qrt-pcr) 20 µl: 1 µl cdna, 1.5 µl RT-mPRLF/R RT-mPRLF2/R2 (10 µmol/l), 10 µl SYBR Premix ExTaq TM II 6 µl dh 2 O PCR, 95 30 s, 95 5 s, 60 18 s 40 18S rrna, RNA PCR Mastercycler ep realplex PCR (Eppendorf ) (melting curve) 3, 3,, 1.7,, 4,, 7.2%, 8.9% 0.03 ng/ml 1.8 2 DDCT [16], SPSS16.0 (P<0.05) ± ( x ±SE), ± ( x ±SE), (P<0.05)
第4期 2 柳学周等: 半滑舌鳎新型膜孕激素受体基因分子特征及其在卵巢发育过程的作用 最高比例的卵细胞的对应时相来确定性腺的发育 结果与分析 2.1 611 时期 对实验样品进行分析, 将采集的卵巢组织 卵巢发育组织学分析 对半滑舌鳎卵巢组织切片的 HE 染色结果进 行分析, 采用性腺切面中平均面积超过50%或居 图1 样本分为 5 个期: II 期卵巢, III 期卵巢, IV 期卵巢, V 期卵巢和 VI 期卵巢(图 1) 半滑舌鳎各期卵巢组织切片 A: II 期卵巢, 比例尺=100 µm; B: III 期卵巢, 比例尺=100 µm; C: IV 期卵巢, 比例尺=200 µm; D 和 E: V 期卵巢, 比例尺=200 µm; F: VI 期卵巢, 比例尺=200 µm. 图中标注的 II III IV V 和 VI 分别指对应时相的卵母细胞. Fig. 1 The stained sections for each of ovarian stage of Cynoglossus semilaevis A: ovary of stage II, bar=100 µm; B: ovary of stage III, bar=100 µm; C: ovary of stage IV, bar=200 µm; D&E: ovary of stage V, bar=200 µm; F: ovary of stage VI, bar=200 µm. The symbols of II, III, IV, V, and VI were corresponding to the phases of oocytes. mpr-like 的序列分析和蛋白质结构分析 三刺鱼的 mpr-like 氨基酸序列进行比对, 发现 从半滑舌鳎卵巢内分离得到了 mpr-like 基因 半 滑 舌 鳎 mpr-like 氨 基 酸 序 列 与 其 他 鱼 类 的 全长 cdna, 该 cdna 全长为 2 002 bp, 5 非编码 mpr-like 氨基酸序列较为保守(图5) 对半滑舌 区为 163 bp, 3 非编码区 783 bp, 同时在 3 非编码 鳎 mpr-like 氨 基 酸 序 列 与 上 述 的 硬 骨 鱼 类 的 区发现有典型的加尾信号 AATAAA; 该基因的开 mpr-like 氨基酸序列同源性进行分析, 结果表 放阅读框为 1 059 bp, 编码 352 个氨基酸, 分子 明半滑舌鳎 mpr-like 与青 鳉 (Oryzias latipes)的 量为 40 kd (GenBank 登录号为 KF277065) (图 同源性为68%, 与三刺鱼(Gasterosteus aculeayus) 2) 采用 SOSUI 软件预测蛋白质二级结构, 发现 同源性为72% 2.2 半滑舌鳎 mpr-like 是具有 7 个跨膜区域的膜蛋白 为分析半滑舌鳎 mpr-like 的进化地位, 构建了 (图 3) 通 过 I-TASSER 软 件 预 测 了 半 滑 舌 鳎 14 个物种的 mpr 家族成员氨基酸序列系统进化树, mpr-like 蛋白质的三级结构, 发现在其蛋白质三 包括硬骨鱼类和哺乳类(图 6) 结果发现此进化树共 级结构中存在多个蛋白结合位点(图 4) 分为 3 大分支, 分别为 mprα 分支 mpr-like (缩写 2.3 mpr-like 氨基酸同源性比较及系统进化分析 为 mprl)分支 mprβ 分支; 半滑舌鳎 mpr-like 基 对半滑舌鳎 mpr-like 氨基酸序列与青 鳉 和 因属于硬骨鱼类 mpr 家族新基因分支
612 22 Fig. 2 2 mpr-like cdna The cdna sequence and its deduced amino acid sequence of mpr-like from Cynoglossus semilaevis
第4期 柳学周等: 半滑舌鳎新型膜孕激素受体基因分子特征及其在卵巢发育过程的作用 图3 Fig. 3 613 半滑舌鳎 mpr-like 蛋白跨膜结构预测 Proposed model for mpr-like transmembrane domains of Cynoglossus semilaevis NP 083105; 斑马鱼 mprβ, AAN78114.1; 斑点叉尾 鮰 mprβ, AAS45555.1; 红 鳍 东 方 鲀 mprβ, SINFRUP00000160848; 人类 mprβ, NP_588608.1 2.4 mpr-like mrna 在繁殖期的组织表达特征 qrt-pcr 的结果表明 mpr-like 基因在性成熟 半滑舌鳎雌鱼的脑 垂体 鳃 心脏 卵巢和肌 肉等 12 种组织中均有表达, 但其表达量存在差 异 分析表明, mpr-like 基因在卵巢组织中相对表 达量最高, 其次心脏 鳃 脾 胃和脑组织中的 表达量也较为丰富, 而在肝 肾和头肾组织中的 表达量很弱(图 7) 显著性检验和多重比较分析 半滑舌鳎 mpr-like 蛋白三维结构 发现在半滑舌鳎雌鱼的 12 个组织中, 该基因在 Tertiary structure of mpr-like protein of Cynoglossus semilaevis 卵 巢 和 其 他 11 种 组 织 的 表 达 水平均差异显著 图4 Fig. 4 Mega6.0 软件(Neighbor-Joining 法)进行聚类 分析所用不同蛋白序列 GenBank 登录号为: 半滑 (P<0.05) 2.5 mpr-like mrna 在不同发育阶段卵母细胞 表达 舌鳎 mprα, AFC90009; 大西洋绒须石首鱼 mprα, qrt-pcr 的分析结果表明 mpr-like 基因在半 ABU68407.1; 云纹犬牙石首鱼 mprα, AF262028_1; 滑舌鳎卵母细胞不同发育阶段均有表达, 但其表 牙鲆mPRα, AGE13751.1; 漠斑牙鲆mPRα, ACW83621; 达量有明显差异 mpr-like 基因的表达水平从 II 三刺鱼, BT026752; 青 鳉, BAC82706; 家鼠 mprβ, 时相卵母细胞到 V 时相卵母细胞持续升高, 而
614 22 5 mpr-like mpr-like C.S:, O.L: 鳉, G.A:. Fig. 5 Alignment amino acid of Cynoglossus semilaevis mpr-like with other teleosts C.S: Cynoglossus semilaevis; O.L: Oryzias latipes, G.A: Gasterosteus aculeayus. 6 MEGA 6.0 NJ mpr-like mpr Fig. 6 Phylogenetic tree of the mpr-like from Cynoglossus semilaevis and other vertebrates mpr family in MEGA 6.0 VI (P<0.05),, mpr-like ( 8) 2.6 mpr-like mrna qrt-pcr
4 : 615 7 mpr-like mrna B:, P:, G:, H:, HK:, K:, L:, S:, St:, I:, O:, M:. (P<0.05). Fig. 7 Expression level of mpr-like mrna in different tissues of Cynoglossus semilaevis B: Brain, P: Pituitary, G: Gill, H: Heart, HK: Heard kidney, K: Kindney, L: Liver, S: Spleen, St: Stomach, I: Intestine, O: Ovary, M: Muscle. Columns marked with different letter do differ significantly from each other (P<0.05). 8 mpr-like mrna (P<0.05). Fig. 8 Expression level of mpr-like mrna at various stages of oogenensis Columns marked with different letters do differ significantly from each other (P<0.05)., mpr-like mrna II V (P<0.05), V, VI, mpr-like mrna, II V, IV, V (P<0.05), mpr-like mrna II III, IV (P<0.05), V, VI ( 9) 2.7 (progesterone, P) (P<0.05), II III, IV V, VI ( 10), 3 mpr-like cdna, 352, 40 kd mpr-like (PAQRs) 7,, mpr-like
616 22 Fig. 9 9 mpr-like mrna (P<0.05). Expression level of mpr-like mrna in brain, pituitary and ovary of reproductive cycle of Cynoglossus semilaevis Columns marked with different letters do differ significantly from each other (P<0.05). 10 (P<0.05). Fig. 10 Progesterone levels of Cynoglossus semilaevis in the reproductive cycle Columnars marked by the different letter do differ significantly from each other (P<0.05)., mpr-like, mpr-like mpr-like 68%~72%, mpr-like, mpr-like, mpr-like mpr-like,,, mpr-like qrt-pcr mpr-like, mpr-like mrna, mpr-like Kazeto [17] qrt-pcr 鮰 (Ictalurus punctatus), mprα mprβ mrna ; mprγ mrna mpr-like mrna, (Micropogonias undulatus), ;,, [18] qrt-pcr mpr-like, mpr-like mrna, mprα mprβ [7, 19], mpr-like
4 : 617 ; mpr-like mpr-like mrna, 3, mpr-like, mpr-like mpr-like mrna II V V (P<0.05), mpr-like mrna, mpr-like Kazeto [20] qrt-pcr 鮰, mprα mrna, ; mprβ mprγ mrna 鮰 mprα mpr-like mrna, mpr-like, mpr-like,, mpr-like 参 考 文 献 : [1] Yamashita M. Molecular meachanisms of meiotic maturation and arrest in fish and amphibian oocytes[j]. Semin Cell Dev Biol, 1998, 9(5): 569 579. [2] Nagahama Y, Yamashita M. Regulation of oocyte maturation in fish[j]. Dev Growth Differ, 2008, 50(s1): S195 S219. [3] Masui Y, Markert C L. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes[j]. J Exp Zool, 1971, 177 (2) : 129 145. [4] Sadler S E, Maller J L. Progesterone inhibits adenylate cyclase in Xenopus oocytes. Action on the guanine nucleotide regulatory protein[j]. J Biol Chem, 1981, 256(2): 6368 6373. [5] Rahman M A, Ohta K, Yoshikuni M, et al. Characterization of ovarian membrane receptor for 17, 20β-dihydroxy- 4-pregnen-3-one, a maturation-inducing hormone in yellowtail, Seriola quinqueradiata[j]. Gen Comp Endocrinol, 2001, 127(1): 71 79. [6] ZhuY, Rice C D, Pang Y, et al. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocyte[j]. Proc Natl Acad Sci USA, 2003, 100: 2231 2236. [7] Josefsberg Ben-Yehoshua L, Lewellyn A, Thomas P, et al. The role of Xenopus membrane progesterone receptor β in mediating the effect of progesterone on oocyte maturation[j]. Mol Endocrinol, 2007, 21(3): 664 673. [8] Karteris E, Zervou S, Pang Y, et al. Progesterone signaling in human myometrium through two novel membrane G protein coupled receptors: potential role in functional progesterone withdrawal at term[j]. Mol Endocrinol, 2006, 20 (7): 1519 1534. [9] Shi B. Study on the molecular mechanisms of reproductive endocrinology in Paralichthys olivaceus and Paralichthys lethostigma[d]. Qingdao: Ocean University of China, 2010: 1 176. [. [D]. :, 2010: 1 176. ] [10] Liu X Z, Sun Z Z, Ma A J, et al. Study on the technology of spawner culture and eggs collection of Cynoglossus semilaevis Günther[J]. Marine Fisheries Research, 2006, 27(2): 25 32. [,,,. [J]., 2006, 27(2): 25 32.] [11] Liu X Z, Xu Y J, Liu N Z, et al. Study on histological and morphometric characters of gonad development of Cynoglossus semilaevis Günther[J]. Progress in Fishery Sciences, 2009, 30(6): 25 35. [,,,. [J]., 2009, 30(6): 25 35.] [12] Liu Yun. The reproductive physiology of cultured fish in China[M]. Beijing: China Aguriculture Press, 1993: 42 46. [. [M]. :, 1993: 42 46.] [13] Chen C F, Wen H S, Chen X Y, et al. Studies on ovarian development and spawn type of cultured half-smooth tongue sole, Cynoglossus semilaevis[j]. Marine Sciences, 2010,
618 22 34(8): 29 34. [,,,. [J]., 2010, 34(8): 29 34.] [14] Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and secondary structure prediction system for membrane proteins[j]. Bioinformatics, 1998, 14: 378 379. [15] Li X X. Study on the physiological function of membrane progestin receptor in the reproductive cycle of flatfish [D]. Shanghai: Shanghai Ocean University, 2013: 1 73. [. [D]. :, 2013: 1 73.] [16] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 method[j]. Methods, 2001, 25(4): 402 408. DDCT [17] Kazeto Y, Goto-kazeto R, Thomas P, et al. Molecular characterization of three forms of putative membrane-bound progestin receptors and their tissue-distribution in channel catfish, Ictalurus punctatu[j]. Mol Endocrinol, 2005, 34: 781 791. [18] Thomas P, Pang Y, Zhu Y, et al. Multiple rapid progestin actions and progestin membrane receptor subtypes in fish[j]. Steroids, 2004, 69: 567 573. [19] Tokumoto T, Tokumoto M, Oshima T, et al. Characterization of multiple membrane progestin receptor (mpr) subtypes from the goldfish ovary and their roles in the induction of oocyte maturation[j]. Gen Comp Endocrinol, 2012, 177: 168 176. [20] Kazeto Y, Goro-Kazeto R, Trant J M. Membrane-bound progestin receptors in channel catfish and zebrafish ovary: Changes in gene expression associated with the reproductive cycles and hormonal reagents[j]. Gen Comp Endocrinol, 2005, 142: 204 211.
4 : 619 Molecular characterization of the novel membrane progestin receptor gene and its role during ovarian development in the half smooth tongue sole Cynoglossus semilaevis Günther LIU Xuezhou 1, SHI Bao 1, LI Xiaoxiao 1, 2, XU Yongjiang 1, WANG Shanshan 1, LI Xiaoni 1 1. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China Abstract: To improve techniques of artificial breeding, this research focused on the novel membrane progestin receptor (mprl) of half smooth tongue sole Cynoglossus semilaevis Günther. The full-length cdna encoding mprl was cloned from the half smooth tongue sole by means of homology cloning and RACE PCR analyses. The complete cdna sequence of mprl (GenBank accession number: KF277065) was 2 002 bp in length, consisting of a 5 untranslated region (UTR) of 163 bp, a coding sequence of 1 056 bp, and a 3 UTR of 783 bp. The molecular weight of mprl was 17.62 kd. Structural analysis of the translated cdna suggested that it encoded membrane protein with seven transmembrane domains. Tertiary structure of the mprl protein showed that it had several binding sites. The rooted phylogenetic tree was constructed using the neighbor-joining method on MEGA4.0 for comparative analysis of half smooth tongue sole mprl and selected vertebrate sequences. The similarity (%, identity) of half smooth tongue sole mprl in comparison with other representative sequences was analyzed using Clustal X. The results indicated that the half smooth tongue sole mprl was clustered with mprl of other fish. Half smooth tongue sole had 68% identity with Japanese medaka (Oryzias latipes) and 72% with three-spined stickleback (Gasterosteus aculeatus). The mprl mrna expression in half smooth tongue sole was detected by quantitative real-time PCR. And found to be widely, although not homogeneously expressed. mprl transcripts were highly abundant in the brain, ovary, heart, gill, spleen, and stomach. In addition, positive signals were obtained for mprl mrna in the pituitary gland, muscle, and intestine, although the resulting transcript levels were lower than in the ovary. Representative sections of ovaries showed the morphological characteristics during development, which was divided into five stages. The expression level of mprl mrna at various stages of oogenensis in the sole showed that the level of mprl mrna in the brain and ovary significantly increased from stage II to stage V (P<0.05), climaxing at stage V, then sharply decreasing in stage IV. The transcript level of mprl mrna in the pituitary peaked at stagev, although itwas not significant (P>0.05). Serum progesterone levels were measured using radioimmunoassay to evaluate the relationship between levels of mprl transcript and serum progesterone. Changes in the serum progesterone were dramatic at stage II (P<0.05), from which progesterone values increased rapidly, reached a maximum at stage V. Expression in multiple tissue simplies that mprl plays various biological roles in half smooth tongue sole. We can infer from the mprl mrna expression pattern at various stages of oogenensis and the reproductive cycle that mprl also contributes to the process of oocyte maturation of the half smooth tongue sole. Key words: Cynoglossus semilaevis Günther; mpr; gene cloning; expression; progesterone Corresponding author: LIU Xuezhou. E-mail: liuxz@ysfri.ac.cn