本發(fā)明屬于分子生物學(xué)領(lǐng)域，具體涉及一種基于新型灘涂模式魚(yú)類(lèi)-彈涂魚(yú)多型卵黃蛋白原(vitellogenin，vtg)基因全長(zhǎng)序列及其克隆方法。

背景技術(shù)：

魚(yú)類(lèi)的卵黃蛋白原(vitellogenin，Vtg)是在雌二醇(estradiol-17β，E2)作用下經(jīng)肝臟合成后分泌到血液中的卵黃前驅(qū)蛋白。通常情況下，Vtg是雌魚(yú)在卵黃形成時(shí)期血液中出現(xiàn)的雌特異性血清蛋白，但雄魚(yú)和稚魚(yú)在外源性雌激素作用下同樣能誘導(dǎo)體內(nèi)Vtg的生成。

以灘涂作為棲息地的魚(yú)類(lèi)中，彈涂魚(yú)Periophthalmus modestus是典型的代表種。彈涂魚(yú)隸屬于鱸形目、蝦虎魚(yú)亞目、彈涂魚(yú)科、彈涂魚(yú)屬，棲息于河口咸淡水水域、近岸灘涂處或底質(zhì)爛泥的低潮區(qū)，為沿岸暖水廣溫廣鹽性魚(yú)類(lèi)(倪勇，2008)。彈涂魚(yú)喜穴居，攝食底棲硅藻、藍(lán)綠藻、橈足類(lèi)及有機(jī)質(zhì)的特點(diǎn)，這些特征決定了它對(duì)底質(zhì)有很強(qiáng)的依存性。彈涂魚(yú)之用作海洋環(huán)境風(fēng)險(xiǎn)評(píng)估的指示生物，除了其體內(nèi)多項(xiàng)生理生化指標(biāo)對(duì)環(huán)境污染物敏感外(劉冰和周忠良，2007)，還具有其他一系列優(yōu)勢(shì)：分布區(qū)域廣，易捕獲，易運(yùn)輸，以底棲硅藻等為食，食物鏈較短，體內(nèi)各項(xiàng)指標(biāo)檢測(cè)能直觀地反映近岸環(huán)境的變化；較其他魚(yú)類(lèi)而言，彈涂魚(yú)具有更強(qiáng)的定居性，能更加準(zhǔn)確地反映棲息地的生態(tài)狀況；近年來(lái)彈涂魚(yú)作為養(yǎng)殖魚(yú)類(lèi)發(fā)展也很快，取得的研究成果可以直接用于近岸養(yǎng)殖和水體質(zhì)量評(píng)估。

隨著基因克隆和免疫生物化學(xué)分析在魚(yú)類(lèi)Vtg研究上的廣泛應(yīng)用，硬骨魚(yú)類(lèi)Vtg被證實(shí)是由若干基因編碼的相關(guān)蛋白組成的一個(gè)家族，即“多型”蛋白，且不同類(lèi)型Vtg對(duì)雌激素的感受性也存在差異(Matsubara等，2003；Ohkubo等，2004；Wang等，2005；Miracle等，2006)。針對(duì)不同類(lèi)型Vtg建立特異的檢測(cè)系，是保證雌激素暴露檢測(cè)準(zhǔn)確的第一步。然而傳統(tǒng)的針對(duì)單一型Vtg的檢測(cè)方法，未針對(duì)多型Vtg分離出相應(yīng)蛋白和基因，因此往往特異性不佳，甚至存在多型Vtg混雜的情況，檢測(cè)結(jié)果的準(zhǔn)確性亟待提高。

現(xiàn)在未見(jiàn)有關(guān)于彈涂魚(yú)多型vtg基因和其全長(zhǎng)cDNA序列的報(bào)道。

技術(shù)實(shí)現(xiàn)要素：

本發(fā)明的目的在于克服上述現(xiàn)有技術(shù)存在的不足，提供一種彈涂魚(yú)多型卵黃蛋白原(vitellogenin，vtg)基因全長(zhǎng)序列及其克隆方法。

本發(fā)明的目的是通過(guò)以下技術(shù)方案來(lái)實(shí)現(xiàn)的：

第一方面，本發(fā)明涉及一種彈涂魚(yú)卵黃蛋白原，其氨基酸序列如SEQ ID NO.2所示。

第二方面，本發(fā)明涉及一種編碼如前述的彈涂魚(yú)卵黃蛋白原的彈涂魚(yú)卵黃蛋白原基因，所述基因?yàn)榫幋a氨基酸序列如SEQ ID NO.2所示的彈涂魚(yú)卵黃蛋白原的彈涂魚(yú)VtgAa基因。

優(yōu)選的，彈涂魚(yú)VtgAa基因的核苷酸序列如SEQ ID NO.1中第3位～第5000位所示。

第三方面，本發(fā)明涉及一種彈涂魚(yú)卵黃蛋白原，其氨基酸序列如SEQ ID NO.4所示。

第四方面，本發(fā)明涉及一種編碼如前述的彈涂魚(yú)卵黃蛋白原的彈涂魚(yú)卵黃蛋白原基因，所述基因?yàn)榫幋a氨基酸序列如SEQ ID NO.4所示的彈涂魚(yú)卵黃蛋白原的彈涂魚(yú)VtgAb基因。

優(yōu)選的，彈涂魚(yú)VtgAb基因的核苷酸序列如SEQ ID NO.3中第3位～第5048位所示。

第五方面，本發(fā)明涉及一種彈涂魚(yú)卵黃蛋白原，其氨基酸序列如SEQ ID NO.6所示。

第六方面，本發(fā)明涉及一種編碼如前述的彈涂魚(yú)卵黃蛋白原的彈涂魚(yú)卵黃蛋白原基因，所述基因?yàn)榫幋a氨基酸序列如SEQ ID NO.6所示的彈涂魚(yú)卵黃蛋白原的彈涂魚(yú)VtgC基因。

優(yōu)選的，彈涂魚(yú)VtgC基因的核苷酸序列如SEQ ID NO.5中第7位～第3732位所示。

第七方面，本發(fā)明涉及一種前述的彈涂魚(yú)卵黃蛋白原在作為判斷性別和雌魚(yú)成熟度的指示蛋白中的用途。

第八方面，一種彈涂魚(yú)三型Vtg基因全長(zhǎng)CDS序列，所述CDS序列包括如SEQ ID NO.1中第3位～第5000位，如SEQ ID NO.3中第3位～第5048位和如SEQ ID NO.5中第7位～第3732位。

第九方面，本發(fā)明還涉及一種克隆彈涂魚(yú)Vtg三型基因全長(zhǎng)序列的方法，所述Vtg三型基因包括VtgAa基因、VtgAb基因和VtgC基因；所述方法包括如下步驟：

S1、從性成熟期彈涂魚(yú)雌魚(yú)的肝臟組織中提取總RNA，進(jìn)行反轉(zhuǎn)錄擴(kuò)增；

S2、分別設(shè)計(jì)Vtg的簡(jiǎn)并引物進(jìn)行擴(kuò)增反應(yīng)，獲得VtgAa基因、VtgAb基因和VtgC基因片段；

S3、以彈涂魚(yú)總RNA為模板根據(jù)RACE法分別制備VtgAa基因、VtgAb基因和VtgC基因3’端和5’端cDNA序列；

S4、分別設(shè)計(jì)特異性全長(zhǎng)引物，擴(kuò)增獲得Vtg三型基因全長(zhǎng)序列。

優(yōu)選的，步驟S2中，VtgAa基因?qū)?yīng)的簡(jiǎn)并引物為如SEQ ID NO.7所示的vtgAa-degenerate-F1和如SEQ ID NO.8所示的vtgAa-degenerate-R1；VtgAb基因?qū)?yīng)的簡(jiǎn)并引物為如SEQ ID NO.9所示的vtgAb-degenerate-F1和如SEQ ID NO.10所示的vtgAb-degenerate-R1；VtgC基因?qū)?yīng)的簡(jiǎn)并引物為如SEQ ID NO.11所示的vtgC-degenerate-F1和如SEQ ID NO.12所示的vtgC-degenerate-R1。

優(yōu)選的，步驟S3中，VtgAa基因?qū)?yīng)的RACE引物為如SEQ ID NO.13所示的vtgAa-5′RACE-GSP1和如SEQ ID NO.14所示的vtgAa-3′RACE-GSP2；VtgAb基因?qū)?yīng)的RACE引物為如SEQ ID NO.15所示的vtgAb-5′RACE-GSP1和如SEQ ID NO.16所示的vtgAb-3′RACE-GSP2；VtgC基因?qū)?yīng)的RACE引物為如SEQ ID NO.17所示的vtgC-5′RACE-GSP1和如SEQ ID NO.18所示的vtgC-3′RACE-GSP2。

優(yōu)選的，步驟S4中，VtgAa基因?qū)?yīng)的特異性全長(zhǎng)引物為如SEQ ID NO.19所示的vtgAa-full primer-F1和如SEQ ID NO.20所示的vtgAa-full primer-R1；VtgAb基因?qū)?yīng)的特異性全長(zhǎng)引物為如SEQ ID NO.21所示的vtgAb-full primer-F1和如SEQ ID NO.22所示的vtgAb-full primer-R1；VtgC基因?qū)?yīng)的特異性全長(zhǎng)引物為如SEQ ID NO.23所示的vtgC-full primer-F1和如SEQ ID NO.24所示的vtgC-full primer-R1。

與現(xiàn)有技術(shù)相比，本發(fā)明具有如下有益效果：

1、本發(fā)明獲得了彈涂魚(yú)Vtg家族中三型Vtg基因全長(zhǎng)CDS序列，填補(bǔ)了彈涂魚(yú)Vtg基因家族的空白。

2、彈涂魚(yú)是我國(guó)東南沿海廣泛分布的常見(jiàn)河口性魚(yú)類(lèi)，Vtg是了解河口咸淡水水域、近岸灘涂處或底質(zhì)爛泥的低潮區(qū)環(huán)境雌激素污染的重要生物標(biāo)志物，獲得多型Vtg基因全長(zhǎng)，對(duì)于更準(zhǔn)確指示相應(yīng)水域污染狀況，以及更深入探究水生生物在污染水域的蛋白表達(dá)等問(wèn)題具有重要意義。

3、本發(fā)明通過(guò)設(shè)計(jì)特異性引物，建立了彈涂魚(yú)三型Vtg和ef1-α(內(nèi)參)基因的實(shí)時(shí)熒光定量PCR方法。運(yùn)用熒光定量PCR法檢測(cè)了Vtg三型基因在性成熟不同時(shí)期的表達(dá)變化，結(jié)果表明：未成熟的幼魚(yú)體內(nèi)沒(méi)有該基因的表達(dá)，只有在雌魚(yú)中才有表達(dá)，且隨著卵巢的成熟，表達(dá)量呈現(xiàn)上升趨勢(shì)。

附圖說(shuō)明

圖1為彈涂魚(yú)三型卵黃蛋白原VtgAa、VtgAb、VtgC基因的cDNA全長(zhǎng)電泳圖；

其中，M：Marker，1：VtgAa，2：VtgAb，3：VtgC；

圖2為彈涂魚(yú)三型卵黃蛋白原VtgAa、VtgAb、VtgC基因在不同發(fā)育階段表達(dá)水平的差異。

具體實(shí)施方式

下面結(jié)合實(shí)施例對(duì)本發(fā)明進(jìn)行詳細(xì)說(shuō)明。以下實(shí)施例將有助于本領(lǐng)域的技術(shù)人員進(jìn)一步理解本發(fā)明，但不以任何形式限制本發(fā)明。應(yīng)當(dāng)指出的是，對(duì)本領(lǐng)域的普通技術(shù)人員來(lái)說(shuō)，在不脫離本發(fā)明構(gòu)思的前提下，還可以做出若干調(diào)整和改進(jìn)。這些都屬于本發(fā)明的保護(hù)范圍。

實(shí)施例

本實(shí)施例涉及一種彈涂魚(yú)多型卵黃蛋白原(vitellogenin，vtg)基因、編碼蛋白及其應(yīng)用、克隆cDNA全長(zhǎng)序列的方法。

VtgAa基因：本發(fā)明的彈涂魚(yú)多型VtgAa基因，其特征在于VtgAa序列全長(zhǎng)5223bp，包含起始密碼子ATG、終止密碼子TAG及polyA。該基因的開(kāi)放閱讀框(ORF)位于3～5000bp，長(zhǎng)4998bp，共編碼1665個(gè)氨基酸，其中信號(hào)肽為15個(gè)氨基酸。根據(jù)推算，其編碼蛋白分子量為185kDa。該編碼氨基酸序列具有典型的完全型卵黃蛋白原結(jié)構(gòu)：1個(gè)vitellogenin結(jié)構(gòu)域(位于編碼氨基酸肽鏈的24～594號(hào)氨基酸)、1個(gè)VWFD結(jié)構(gòu)域(位于編碼氨基酸肽鏈的1423～1555號(hào)氨基酸)，1個(gè)多聚絲氨酸區(qū)域(位于編碼氨基酸肽鏈的1064～1195號(hào)氨基酸)，另外還含有1個(gè)蛋白水解酶切位點(diǎn)(RSRR)。將該推導(dǎo)氨基酸序列與其他魚(yú)類(lèi)Vtg氨基酸序列比對(duì)后發(fā)現(xiàn)，該氨基酸序列包含LvH、LvL、Pv和β組分4個(gè)部分。

本發(fā)明所述彈涂魚(yú)VtgAa基因的核苷酸序列如SEQ ID NO.1所示。

所獲得的全長(zhǎng)VtgAa cDNA序列的編碼區(qū)為從第3個(gè)核苷酸到5000個(gè)核苷酸。

本發(fā)明所述彈涂魚(yú)VtgAa基因的氨基酸序列如SEQ ID NO.2所示。

VtgAb基因：本發(fā)明的彈涂魚(yú)Vtg Ab基因，其特征在于序列全長(zhǎng)5198bp，包含起始密碼子ATG、終止密碼子TGA及polyA。開(kāi)放閱讀框(ORF)位于3～5048bp，長(zhǎng)5046bp，共編碼1681個(gè)氨基酸，其中信號(hào)肽為15個(gè)氨基酸。根據(jù)推算，其編碼蛋白分子量為187kDa。將該推導(dǎo)氨基酸序列與其他魚(yú)類(lèi)Vtg氨基酸序列比對(duì)后發(fā)現(xiàn)，該氨基酸序列同樣包含LvH、LvL、Pv和β組分4個(gè)部分。

本發(fā)明所述彈涂魚(yú)VtgAb基因的核苷酸序列如SEQ ID NO.3所示。

所獲得的全長(zhǎng)VtgAb cDNA序列的編碼區(qū)從第3個(gè)核苷酸到5048個(gè)核苷酸。

本發(fā)明所述彈涂魚(yú)VtgAb基因的氨基酸序列如SEQ ID NO.4所示。

VtgC基因：本發(fā)明彈涂魚(yú)VtgC基因，其特征在于序列全長(zhǎng)3770bp，包含起始密碼子ATG、終止密碼子TAA。開(kāi)放閱讀框(ORF)位于7～3732bp，長(zhǎng)3726bp，共編碼1241個(gè)氨基酸，其中信號(hào)肽為15個(gè)氨基酸。根據(jù)推算，其編碼蛋白分子量為141kDa。將該推導(dǎo)氨基酸序列與其他魚(yú)類(lèi)Vtg氨基酸序列比對(duì)后發(fā)現(xiàn)，該氨基酸序列僅包含LvH和LvL兩個(gè)部分。

本發(fā)明所述彈涂魚(yú)VtgC基因的核苷酸序列如SEQ ID NO.5所示。

所獲得的全長(zhǎng)VtgC cDNA序列的編碼區(qū)從第7個(gè)核苷酸到3732個(gè)核苷酸。

本發(fā)明所述彈涂魚(yú)VtgC基因的氨基酸序列如SEQ ID NO.6所示。

本發(fā)明通過(guò)設(shè)計(jì)特異性引物，引物為如SEQ ID NO.25～32所示，建立了彈涂魚(yú)三型Vtg和ef1-α(內(nèi)參)基因的實(shí)時(shí)熒光定量PCR方法。運(yùn)用熒光定量PCR法檢測(cè)了Vtg三型基因在性成熟不同時(shí)期的表達(dá)變化，結(jié)果表明：未成熟的幼魚(yú)體內(nèi)沒(méi)有該基因的表達(dá)，只有在雌魚(yú)中才有表達(dá)，且隨著卵巢的成熟，表達(dá)量呈現(xiàn)上升趨勢(shì)。

克隆cDNA全長(zhǎng)序列的方法：從性成熟期彈涂魚(yú)雌魚(yú)的肝臟組織中提取總RNA，進(jìn)行反轉(zhuǎn)錄擴(kuò)增，設(shè)計(jì)不同類(lèi)型Vtg的簡(jiǎn)并引物，獲得相應(yīng)基因片段；在此基礎(chǔ)上，采用SMART RACE技術(shù)獲得5’和3’端未知序列；最后設(shè)計(jì)全長(zhǎng)上下游引物，克隆獲得相應(yīng)的全長(zhǎng)cDNA序列。用Macvector軟件確認(rèn)完整開(kāi)放閱讀框(ORF)并分析其各功能域。通過(guò)完整氨基酸序列比對(duì)建立系統(tǒng)分析圖，確定其Vtg類(lèi)型。

在實(shí)施過(guò)程中，克隆cDNA全長(zhǎng)序列的技術(shù)方案可采用如下具體步驟：

1、動(dòng)物組織器官樣品采集：

采集新鮮的懷卵彈涂魚(yú)雌魚(yú)的肝臟，剪碎后置于RNAlater中保存用于總RNA提取。

2、彈涂魚(yú)總RNA的提取及反轉(zhuǎn)錄擴(kuò)增

按照ISOGEN II試劑盒操作說(shuō)明提取總RNA，具體方法如下：

(1)將肝臟組織(約100mg)放入1.5ml離心管，加入1ml ISOGEN II，電動(dòng)勻漿棒勻漿后，加入0.4ml RNase free水，15s劇烈振蕩后，室溫靜置10min；在4℃下，12K×g，離心10min。

(2)取上清1ml，加入5μl對(duì)溴苯甲醚，15s劇烈振蕩后，室溫靜置3min；在4℃下，12K×g，離心10min。

(3)取上清1ml，加入1ml(等體積)異丙醇，顛倒混勻后室溫靜置10min；在4℃下，12K×g，離心10min，管底及管壁的白色沉淀即為RNA沉淀。

(4)棄上清，在RNA沉淀中加入0.5ml 75％乙醇洗滌沉淀，在4℃下，7K×g，離心3min，棄上清；重復(fù)操作此步驟一次。

(5)室溫遮光干燥沉淀2min。

(6)加入50μl(或適量)RNase free水溶解沉淀，此溶液即為總RNA溶液。

(7)用0.8％瓊脂糖TAE凝膠電泳檢測(cè)總RNA完整性；用Thermo Fisher NanoDrop2000分光光度計(jì)測(cè)定總RNA濃度及純度。

(8)將上述總RNA按照VILO^TM cDNA Synthesis Kit(Invitrogen)反轉(zhuǎn)錄成cDNA模板。

注：以上所述離心管，勻漿棒等均為RNase free處理。

2、簡(jiǎn)并引物擴(kuò)增各型Vtg基因片段

Vtg基因?qū)僭谛蛄猩暇哂斜Ｊ匦?，根?jù)庸鰈、真鯛、鯔魚(yú)、食蚊魚(yú)、矛尾刺蝦虎魚(yú)三種vtg基因即VtgAa/VtgAb/VtgC基因CDS序列，用Blast進(jìn)行序列比對(duì)，根據(jù)Vtg基因的保守序列分別設(shè)計(jì)三對(duì)簡(jiǎn)并引物，序列如下：

PCR擴(kuò)增反應(yīng)體系為10μl體系：F和R端引物(10μM)各1μl，cDNA模板0.5μl，ddH₂O2.5μl，Master Mix 5μl，反應(yīng)條件為95℃預(yù)變性5min，35個(gè)循環(huán)(95℃，30s；54℃，30s；72℃，1min)，72℃延伸10min。

4、PCR產(chǎn)物的回收和純化

取5μl PCR反應(yīng)物進(jìn)行1.5％的瓊脂糖凝膠電泳，用于確認(rèn)中間序列擴(kuò)增產(chǎn)物。將目標(biāo)條帶產(chǎn)物用GENECLEAN Turbo Kit(MP Biomedicals Europe，F(xiàn)rance)割膠純化回收。

5、PCR擴(kuò)增目的片段的連接轉(zhuǎn)化

(1)連接：在0.2ml PCR管中加入PCR產(chǎn)物3μl，pGEM-T EasyVector(Promega，USA)1μl，T4ligase 1μl，Buffer 5μl，輕輕混勻后，4℃靜置過(guò)夜。

(2)轉(zhuǎn)化：加連接產(chǎn)物5μl于大腸桿菌DH5d感受態(tài)細(xì)胞中，輕彈混勻，冰浴30min；42℃熱激45s，立刻置于冰上2min；涂平板，將培養(yǎng)皿倒置放入37℃培養(yǎng)箱過(guò)夜。

6、陽(yáng)性克隆的鑒定、測(cè)序及序列分析

在0.2ml PCR管中加入10ul如下混合液：AmpliTaqMaster Mix 5μl，T7和SP6引物(10μM)各0.5μl，無(wú)菌水4ul，用無(wú)菌牙簽取單一克隆后放入上述PCR管中，使之充分接觸后進(jìn)行菌落PCR驗(yàn)證。PCR反應(yīng)條件如下：95℃預(yù)變性5min；35個(gè)循環(huán)(95℃變性30s，50℃退火30s，72℃延伸1min)；72℃延伸10min。確認(rèn)包含目的片段的克隆，擴(kuò)大培養(yǎng)，用T7和SP6引物測(cè)序。結(jié)果分別獲得579bp、745bp和694bp的三型Vtg基因家族(分別是VtgAa，VtgAb和VtgC)片段。

7、5’RACE和3’RACE擴(kuò)增彈涂魚(yú)Vtg5’端和3’端基因序列

利用Macvector設(shè)計(jì)RACE引物如下：

按照RACE 5’/3’Kit(Clontech)說(shuō)明書(shū)進(jìn)行，以彈涂魚(yú)總RNA為模板制備其3’端和5’端cDNA，與特異性引物(GSP)配制成總體積為25μl的PCR反應(yīng)液：包括2.5μl 10×Advantage 2PCR buffer，0.5μl dNTPs mix(10mM)，0.5μl GSP(10μM)，2.5μl 10×UPM，1.25μl模板和0.5μl 10×Advantage 2polymerase mix(Clontech)。PCR反應(yīng)條件如下：5個(gè)循環(huán)(94℃，30s；72℃，3min)，5個(gè)循環(huán)(94℃，30s；70℃，30s；72℃，3min)，25個(gè)循環(huán)(94℃，30s；68℃，30s；72℃，3min)。

5’RACE和3’RACE進(jìn)行PCR反應(yīng)后連接轉(zhuǎn)化步驟同5。

8、擴(kuò)增Vtg三型基因全長(zhǎng)序列

根據(jù)RACE法獲得的3’端和5’端cDNA序列，分別設(shè)計(jì)特異性全長(zhǎng)引物如下：

配置如下PCR反應(yīng)液：包括10×Advantage 2PCR buffer(5μl)，dNTPs mix(10mM，1μl)，上下游引物GSP(10μM，各1 μl)，模板(1μl)和10×Advantage 2polymerase mix(1μl)，加N.F.W.至總反應(yīng)體積為50μl。PCR反應(yīng)條件如下：95℃預(yù)變性1min；5個(gè)循環(huán)(95℃，30s；68℃，3min)，25個(gè)循環(huán)(94℃，30s；68℃，30s；72℃，3min)，68℃延伸3min。

PCR產(chǎn)物用1％瓊脂糖凝膠電泳檢測(cè)擴(kuò)增效果如圖1所示，由圖1可知，以cDNA為模板，分別以SEQ ID19和SEQID20引物擴(kuò)增獲得了預(yù)期的全長(zhǎng)5000bp左右的VtgAa條帶；以SEQ ID21和SEQID22引物擴(kuò)增獲得了預(yù)期的全長(zhǎng)5000bp左右的VtgAb條帶；以SEQ ID23和SEQID24引物擴(kuò)增獲得了預(yù)期的全長(zhǎng)4000bp左右的VtgC條帶。其中，M：Marker，1：VtgAa，2：VtgAb，3：VtgC。

9、實(shí)時(shí)熒光定量PCR分析各型Vtg基因表達(dá)差異

分別從大腸桿菌中提取含有各型Vtg基因片段的重組質(zhì)粒，在分光光度計(jì)上測(cè)量其DNA濃度和純度。以制備的質(zhì)粒cDNA(目標(biāo)基因和內(nèi)參基因)為模板，稀釋成10¹至10⁶拷貝數(shù)6個(gè)梯度，每個(gè)梯度2個(gè)重復(fù)，制作目的基因Vtg和ef1-α(內(nèi)參)基因的標(biāo)準(zhǔn)曲線。根據(jù)標(biāo)準(zhǔn)曲線計(jì)算出目的基因的實(shí)際絕對(duì)定量。

實(shí)時(shí)熒光定量PCR反應(yīng)采用Power SYBR Green PCR Master Mix(Applied Biosystems，USA)配置20μl反應(yīng)體系：2μl cDNA，F(xiàn) and R primer(終濃度200nM)，SYBR Green Mix(10μl)，N.F.W。同時(shí)進(jìn)行無(wú)反轉(zhuǎn)錄樣本對(duì)照和組間對(duì)照。PCR反應(yīng)條件為：1個(gè)循環(huán)(50℃，2min；95℃，10min)，40個(gè)循環(huán)(95℃，15s；60℃，1min)。

根據(jù)已得到的3型卵黃蛋白原全長(zhǎng)cDNA序列，利用Primer3Plus(http：//www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/)設(shè)計(jì)如下引物進(jìn)行實(shí)時(shí)熒光定量PCR反應(yīng)。

隨著卵巢的發(fā)育，Vtg濃度呈現(xiàn)出遞增的趨勢(shì)(圖2)，特別在卵黃形成中期至完全成熟期，表現(xiàn)出急速增長(zhǎng)趨勢(shì)。從表達(dá)量來(lái)看，vtgAa＞vtgAb＞vtgC。

SEQUENCE LISTING

<110> 上海海洋大學(xué)

<120> 彈涂魚(yú)多型卵黃蛋白原Vtg基因全長(zhǎng)序列及其克隆方法

<130> 2017

<160> 32

<170> PatentIn version 3.5

<210> 1

<211> 5223

<212> DNA

<213> Periophthalmus modestus（VtgAa）

<400> 1

acatggggac aacccactca gccatgagag tcgttgtact agccttggct ctagcccttg 60

tggctggcca gcacgataac ttggctccta gttttgctcc cggactaacc tatgtgtaca 120

agtacaatgc ccagagcctg ggaggtctgt ctgaacagca cctagctaaa gccggactca 180

acttcaccag caatgtcaag atcagtgttg cccaagaaaa cgtcctcatg cttcagcttg 240

agaatcctcg gatctatgag ttcagtggtg tttggccaaa ggattcctac gtacaaactc 300

acctccgtgc cgacctggaa gctcatctca aaaccgccat caagttcaaa tatgaccatg 360

ggattgtgag agagatcctg gccccagaga gtgtccccat actgctgctc aacatcttta 420

gaggcatcct caacttcttc cagctgaaca tcaagaagtc acagaatgtt tatgaactgc 480

aggaggaggg agcccagggc gtgtgcaaga cccagtatgc catcacagag aacgataagg 540

ctgagcgcat ccttttgacc aagagcagga acttgaatca ctgccaggag aaggtcatga 600

gggatatcgg gttggcatac actaagacat ggcataagtg ccgggagatt tccaaaaacc 660

tgaggggaac cacaggatac ttctacaagc tgaaggcagc cccaggcggc ttgatcattg 720

agaaggcctc cggaaaggaa gtcattcagt tcacaccttt taatgaccag aatggtgctg 780

cttctatgca gacaatccaa accctggatt tcattgctgc catcaaggct cctattgttc 840

ccatcagtgc tccgtaccat ccacgtggct ccctgaaata tcagttttcc actgagcttc 900

tgcagagccc cctcaggatc cttaagatgt cagacgtgaa ggaacaggtt gctgatgttc 960

tgaacaacct ggttgtcaat aacagagaca aagtacatga ggatgctcct ctcaagtttt 1020

ttgagctgat cctgctgctg cgtgcttctg atttgactga actgcgcaac ctgctgacta 1080

cctacaaaag cagacctctt gagaggcggt ggttgatgga cgccatcagc aacactggaa 1140

caaaggctgc tttggagatt gtcatggctg agatccagaa gagagagcta tctgttcctg 1200

aagccgctca agttttgatt ggaactctgc acatgttgaa gcccactgac gagatcatcc 1260

agaaggtttg gagctacatt gagcagctct cacaggaaca aggacgctac gaacaagttg 1320

tgcgcaaagc tctgttcctg ggctatggta gcatcatcca cagacagagt gttcagaggg 1380

ctgagtggaa tgatcgtgac attcagcgca tccagtcaga ttttgagagg gcctttgctg 1440

agaaaaacac acaggagctt gttctgttgg ctaaagttat ggctaatgct gctcagcctt 1500

ggggctacaa acctattaca aagctcctac caatccatgg cacagctggt gagcaactgt 1560

cccaatcagt tcacattgaa gccatcctgg ccctgagagg cattgccaaa cagaagccca 1620

aagaggttca gaacttggct ctgcagctgt ttatggacac aactctgcag cctgagcagc 1680

gtatgcttgc cgtcatgaca ctctttgaga ccaacccttc aatggccgtc atgactaatg 1740

ttatcaacgt tgtcaagtct gacaacagcc agccagtgat cagctttact tactctctca 1800

tcaagtctca gtccagaagc acagctaacc cctcagtggc acctgaggct aatattgctc 1860

tcagactctt gggccaaagg agacagagca tgaagctgag caaggctttc aaagcggact 1920

tctacagcca tcctctgatg cttggtgctg ctgcaagtat ttattacatc aatgaggctg 1980

ctaccatcct ccccaaagcg gttatagcta agacgagtgc ctatgtcgct ggagctgctg 2040

ctgatgtttt tgagattgga gtcagaagtg agggattcca ggagtacttc ctgaaaaagg 2100

agagctctga tgtctctgat agaaccacca agatgcagcg catcattaaa gctttcacca 2160

actggaagtc tttgccaatc agcaaattgc tgggctctgt gaatgtcaag gttctgggac 2220

aggaaatcgc ttttgttgac attgacgagc agctcattga ggaggcaatg aggattcgct 2280

ctgaaattga catcaaggag tatggcttaa acttactccg tcacttgttc caaaatggtg 2340

tctccgcaca cttggtcaaa gcagtgatgc ccgctgagat cagacgtgtc atgcctactg 2400

ccgcaggcct gccaatggag cttgccttct acactgttgc tgtcactgca gcaaatgtcc 2460

aggccagatt ccaggccaac cttccacaga acttccatgt ttctgacctt ctgaagcaga 2520

agaccaacat tgaggctgac ataaagccaa gcatggccct caacacattt gctgtgatgg 2580

gaatgaacac tgacatcgtt caggctgcca tagtctcaag agccaaggtc caggtcaacg 2640

tgcccgccaa gatagctgct tctttggact tagctgagaa caactttaag atcagtgctc 2700

ttccggttcg cctctctgaa aatgttgcag ttgctgttga cgttgatact ctggccattg 2760

caagacaggc caaacgggta acccctctga ttcccgaaga tgcttctccc caagcatctt 2820

ccgaaacatc ttcatctgct agcgcctcca attctaggga gatactgggt aacatgcagc 2880

aagttcagga caggcccctt cccacgacaa gagttcccag atctgacaag aagttctgca 2940

ctgttactgc tggagtgaag atctgcatca acatcagctc cagcaatgcc aagttcatca 3000

cagattccgc tctctccaga ctggctggaa agcacgctgt tctcatgtct gtcgaacaat 3060

ctgaaagtga caatgtcgag aagtgggaaa tggagcttca gcttggagcc aaggccgcaa 3120

acaagctgat caaaaacatc aacttggaca tggatgaggt cctagagggc acacctattc 3180

tgtccaaact caagagaatc ctgaatccaa gcatgaaaaa caacacctcc tctagctcct 3240

ccagcagctc caggtccaga gttcgtagca gccatccttc ctcctcatcc tcctcctcct 3300

cctcatcgtc caagtctcac atggccggta aagttatcag caccatgggc aaaatcatcg 3360

gggttaacca aaagaggagc agcagcagca gcagcagcag taggagtcag caaaaccgca 3420

agaggcaaag gtccactgtg tccagcctga gctctctgtt cagtgctagc tccagctcct 3480

cacagttttt ccccaagtct cagcgccaga gctctcgttc caaattccag ccaaaccacc 3540

agaagacgac atccaagcgg cactcaggat ctgcctcctc tgcaagaacc tttgaggaca 3600

tcaggaaaca gaacaaattc cttggcaata ccgttgagcc agtttttgca ctgatcctcc 3660

gtgctgtcag agctaacaac aacaatccac tgggctacca gatcgctgcc tataaggatg 3720

gagacagagt tcagatgatc atggctgccc tggcttctca tgacaactgg aggctttgtg 3780

ctgatgccat taaacttagc aagaacaaag ctgctgctaa aattgcatgg ggagagaagt 3840

gccagaaata tgagaccatg attacagctg agtctggccg tgttcagaac aagggacaat 3900

ctcaagatgc agctcgtgtg agagtggcct ggaaaagact gcccactgct gttgtcaaag 3960

atgtcaaaat gatctacaac tccatcattg ccccttactt gtctagtggc tatctgcaga 4020

agagatcaga tgcgaccagg cagatttcct tcactgtggt tgttgagcct aagaaacagc 4080

ttggctttat ttggaaatca ccagcatttg tctacaggag taatgtgcct cttcccatca 4140

ctctgccaat cgatgagctg aaggaagtgc tgccatttga tgaaatgctt gacaacgctc 4200

actatctgtt ggcaaagact actggaattc agtgcagatt taatgaagga cagcttacca 4260

cttccagcaa aagacaacac aagaactaca tgccaaattc ttgctaccag ctgctggctc 4320

aggattgcac cgatgagctt aaattcattg ttttgctgaa gaaggatagc gcaggccgtt 4380

acatggtcaa tgtgaagatt ggtacaaggg atattgacat gttctttaat ggagaaagac 4440

cagctgtcat gatcaatgga aaggaaattg ccagagagcg cttgccatat gacagagatt 4500

cagtgacgat tctgctgatg aacggcaagc tgtttctccg tgctctggac tttggcattg 4560

ctgaactcca attcagtgca tcagaagtga cgatcaatgt tccagagtat ttgaggaaca 4620

aagtctgcgg tctctgcggc caaggcaatg gagacaggag aaacgattac cgcatgccca 4680

atggacgtat cactgataac cccatcagct tcgcccattc ctggactctg ccctctcaga 4740

gttgcagcga tgagactgaa tgtcgtttga ctcatgagtc cattgaactg gagagagaaa 4800

ttaacgatca cggtgtgccg tctaagtgct tctctgtgga ctctgtgctg cgctgtcgcc 4860

ctggctgcac tcccactaag accaccatgt ccagtgtgag cttccactgc agacccctca 4920

atgacaacag ccaagtgtca gatatccgta accgcagcat tgacatgact gagtccgttg 4980

aagcccatct tgactgcagt tgcacttctc agtgtgctta gatcttgtcg tcttatattg 5040

tgtctatgtg taattttaac taaataaatg aaggcatctc aaaagacaaa aaaaaaaaaa 5100

aaaaaaaaaa aaaaaaaaaa tataaagaaa aaaaaaaaaa aaaaaaggta ctctgcgctg 5160

ataccactgc ttgccctata gtgagtcgta ttagaatcga attcccgcgg ccgcatggcg 5220

gcc 5223

<210> 2

<211> 1665

<212> PRT

<213> Periophthalmus modestus（VtgAa）

<400> 2

Met Arg Val Val Val Leu Ala Leu Ala Leu Ala Leu Val Ala Gly Gln

1 5 10 15

His Asp Asn Leu Ala Pro Ser Phe Ala Pro Gly Leu Thr Tyr Val Tyr

20 25 30

Lys Tyr Asn Ala Gln Ser Leu Gly Gly Leu Ser Glu Gln His Leu Ala

35 40 45

Lys Ala Gly Leu Asn Phe Thr Ser Asn Val Lys Ile Ser Val Ala Gln

50 55 60

Glu Asn Val Leu Met Leu Gln Leu Glu Asn Pro Arg Ile Tyr Glu Phe

65 70 75 80

Ser Gly Val Trp Pro Lys Asp Ser Tyr Val Gln Thr His Leu Arg Ala

85 90 95

Asp Leu Glu Ala His Leu Lys Thr Ala Ile Lys Phe Lys Tyr Asp His

100 105 110

Gly Ile Val Arg Glu Ile Leu Ala Pro Glu Ser Val Pro Ile Leu Leu

115 120 125

Leu Asn Ile Phe Arg Gly Ile Leu Asn Phe Phe Gln Leu Asn Ile Lys

130 135 140

Lys Ser Gln Asn Val Tyr Glu Leu Gln Glu Glu Gly Ala Gln Gly Val

145 150 155 160

Cys Lys Thr Gln Tyr Ala Ile Thr Glu Asn Asp Lys Ala Glu Arg Ile

165 170 175

Leu Leu Thr Lys Ser Arg Asn Leu Asn His Cys Gln Glu Lys Val Met

180 185 190

Arg Asp Ile Gly Leu Ala Tyr Thr Lys Thr Trp His Lys Cys Arg Glu

195 200 205

Ile Ser Lys Asn Leu Arg Gly Thr Thr Gly Tyr Phe Tyr Lys Leu Lys

210 215 220

Ala Ala Pro Gly Gly Leu Ile Ile Glu Lys Ala Ser Gly Lys Glu Val

225 230 235 240

Ile Gln Phe Thr Pro Phe Asn Asp Gln Asn Gly Ala Ala Ser Met Gln

245 250 255

Thr Ile Gln Thr Leu Asp Phe Ile Ala Ala Ile Lys Ala Pro Ile Val

260 265 270

Pro Ile Ser Ala Pro Tyr His Pro Arg Gly Ser Leu Lys Tyr Gln Phe

275 280 285

Ser Thr Glu Leu Leu Gln Ser Pro Leu Arg Ile Leu Lys Met Ser Asp

290 295 300

Val Lys Glu Gln Val Ala Asp Val Leu Asn Asn Leu Val Val Asn Asn

305 310 315 320

Arg Asp Lys Val His Glu Asp Ala Pro Leu Lys Phe Phe Glu Leu Ile

325 330 335

Leu Leu Leu Arg Ala Ser Asp Leu Thr Glu Leu Arg Asn Leu Leu Thr

340 345 350

Thr Tyr Lys Ser Arg Pro Leu Glu Arg Arg Trp Leu Met Asp Ala Ile

355 360 365

Ser Asn Thr Gly Thr Lys Ala Ala Leu Glu Ile Val Met Ala Glu Ile

370 375 380

Gln Lys Arg Glu Leu Ser Val Pro Glu Ala Ala Gln Val Leu Ile Gly

385 390 395 400

Thr Leu His Met Leu Lys Pro Thr Asp Glu Ile Ile Gln Lys Val Trp

405 410 415

Ser Tyr Ile Glu Gln Leu Ser Gln Glu Gln Gly Arg Tyr Glu Gln Val

420 425 430

Val Arg Lys Ala Leu Phe Leu Gly Tyr Gly Ser Ile Ile His Arg Gln

435 440 445

Ser Val Gln Arg Ala Glu Trp Asn Asp Arg Asp Ile Gln Arg Ile Gln

450 455 460

Ser Asp Phe Glu Arg Ala Phe Ala Glu Lys Asn Thr Gln Glu Leu Val

465 470 475 480

Leu Leu Ala Lys Val Met Ala Asn Ala Ala Gln Pro Trp Gly Tyr Lys

485 490 495

Pro Ile Thr Lys Leu Leu Pro Ile His Gly Thr Ala Gly Glu Gln Leu

500 505 510

Ser Gln Ser Val His Ile Glu Ala Ile Leu Ala Leu Arg Gly Ile Ala

515 520 525

Lys Gln Lys Pro Lys Glu Val Gln Asn Leu Ala Leu Gln Leu Phe Met

530 535 540

Asp Thr Thr Leu Gln Pro Glu Gln Arg Met Leu Ala Val Met Thr Leu

545 550 555 560

Phe Glu Thr Asn Pro Ser Met Ala Val Met Thr Asn Val Ile Asn Val

565 570 575

Val Lys Ser Asp Asn Ser Gln Pro Val Ile Ser Phe Thr Tyr Ser Leu

580 585 590

Ile Lys Ser Gln Ser Arg Ser Thr Ala Asn Pro Ser Val Ala Pro Glu

595 600 605

Ala Asn Ile Ala Leu Arg Leu Leu Gly Gln Arg Arg Gln Ser Met Lys

610 615 620

Leu Ser Lys Ala Phe Lys Ala Asp Phe Tyr Ser His Pro Leu Met Leu

625 630 635 640

Gly Ala Ala Ala Ser Ile Tyr Tyr Ile Asn Glu Ala Ala Thr Ile Leu

645 650 655

Pro Lys Ala Val Ile Ala Lys Thr Ser Ala Tyr Val Ala Gly Ala Ala

660 665 670

Ala Asp Val Phe Glu Ile Gly Val Arg Ser Glu Gly Phe Gln Glu Tyr

675 680 685

Phe Leu Lys Lys Glu Ser Ser Asp Val Ser Asp Arg Thr Thr Lys Met

690 695 700

Gln Arg Ile Ile Lys Ala Phe Thr Asn Trp Lys Ser Leu Pro Ile Ser

705 710 715 720

Lys Leu Leu Gly Ser Val Asn Val Lys Val Leu Gly Gln Glu Ile Ala

725 730 735

Phe Val Asp Ile Asp Glu Gln Leu Ile Glu Glu Ala Met Arg Ile Arg

740 745 750

Ser Glu Ile Asp Ile Lys Glu Tyr Gly Leu Asn Leu Leu Arg His Leu

755 760 765

Phe Gln Asn Gly Val Ser Ala His Leu Val Lys Ala Val Met Pro Ala

770 775 780

Glu Ile Arg Arg Val Met Pro Thr Ala Ala Gly Leu Pro Met Glu Leu

785 790 795 800

Ala Phe Tyr Thr Val Ala Val Thr Ala Ala Asn Val Gln Ala Arg Phe

805 810 815

Gln Ala Asn Leu Pro Gln Asn Phe His Val Ser Asp Leu Leu Lys Gln

820 825 830

Lys Thr Asn Ile Glu Ala Asp Ile Lys Pro Ser Met Ala Leu Asn Thr

835 840 845

Phe Ala Val Met Gly Met Asn Thr Asp Ile Val Gln Ala Ala Ile Val

850 855 860

Ser Arg Ala Lys Val Gln Val Asn Val Pro Ala Lys Ile Ala Ala Ser

865 870 875 880

Leu Asp Leu Ala Glu Asn Asn Phe Lys Ile Ser Ala Leu Pro Val Arg

885 890 895

Leu Ser Glu Asn Val Ala Val Ala Val Asp Val Asp Thr Leu Ala Ile

900 905 910

Ala Arg Gln Ala Lys Arg Val Thr Pro Leu Ile Pro Glu Asp Ala Ser

915 920 925

Pro Gln Ala Ser Ser Glu Thr Ser Ser Ser Ala Ser Ala Ser Asn Ser

930 935 940

Arg Glu Ile Leu Gly Asn Met Gln Gln Val Gln Asp Arg Pro Leu Pro

945 950 955 960

Thr Thr Arg Val Pro Arg Ser Asp Lys Lys Phe Cys Thr Val Thr Ala

965 970 975

Gly Val Lys Ile Cys Ile Asn Ile Ser Ser Ser Asn Ala Lys Phe Ile

980 985 990

Thr Asp Ser Ala Leu Ser Arg Leu Ala Gly Lys His Ala Val Leu Met

995 1000 1005

Ser Val Glu Gln Ser Glu Ser Asp Asn Val Glu Lys Trp Glu Met

1010 1015 1020

Glu Leu Gln Leu Gly Ala Lys Ala Ala Asn Lys Leu Ile Lys Asn

1025 1030 1035

Ile Asn Leu Asp Met Asp Glu Val Leu Glu Gly Thr Pro Ile Leu

1040 1045 1050

Ser Lys Leu Lys Arg Ile Leu Asn Pro Ser Met Lys Asn Asn Thr

1055 1060 1065

Ser Ser Ser Ser Ser Ser Ser Ser Arg Ser Arg Val Arg Ser Ser

1070 1075 1080

His Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser

1085 1090 1095

His Met Ala Gly Lys Val Ile Ser Thr Met Gly Lys Ile Ile Gly

1100 1105 1110

Val Asn Gln Lys Arg Ser Ser Ser Ser Ser Ser Ser Ser Arg Ser

1115 1120 1125

Gln Gln Asn Arg Lys Arg Gln Arg Ser Thr Val Ser Ser Leu Ser

1130 1135 1140

Ser Leu Phe Ser Ala Ser Ser Ser Ser Ser Gln Phe Phe Pro Lys

1145 1150 1155

Ser Gln Arg Gln Ser Ser Arg Ser Lys Phe Gln Pro Asn His Gln

1160 1165 1170

Lys Thr Thr Ser Lys Arg His Ser Gly Ser Ala Ser Ser Ala Arg

1175 1180 1185

Thr Phe Glu Asp Ile Arg Lys Gln Asn Lys Phe Leu Gly Asn Thr

1190 1195 1200

Val Glu Pro Val Phe Ala Leu Ile Leu Arg Ala Val Arg Ala Asn

1205 1210 1215

Asn Asn Asn Pro Leu Gly Tyr Gln Ile Ala Ala Tyr Lys Asp Gly

1220 1225 1230

Asp Arg Val Gln Met Ile Met Ala Ala Leu Ala Ser His Asp Asn

1235 1240 1245

Trp Arg Leu Cys Ala Asp Ala Ile Lys Leu Ser Lys Asn Lys Ala

1250 1255 1260

Ala Ala Lys Ile Ala Trp Gly Glu Lys Cys Gln Lys Tyr Glu Thr

1265 1270 1275

Met Ile Thr Ala Glu Ser Gly Arg Val Gln Asn Lys Gly Gln Ser

1280 1285 1290

Gln Asp Ala Ala Arg Val Arg Val Ala Trp Lys Arg Leu Pro Thr

1295 1300 1305

Ala Val Val Lys Asp Val Lys Met Ile Tyr Asn Ser Ile Ile Ala

1310 1315 1320

Pro Tyr Leu Ser Ser Gly Tyr Leu Gln Lys Arg Ser Asp Ala Thr

1325 1330 1335

Arg Gln Ile Ser Phe Thr Val Val Val Glu Pro Lys Lys Gln Leu

1340 1345 1350

Gly Phe Ile Trp Lys Ser Pro Ala Phe Val Tyr Arg Ser Asn Val

1355 1360 1365

Pro Leu Pro Ile Thr Leu Pro Ile Asp Glu Leu Lys Glu Val Leu

1370 1375 1380

Pro Phe Asp Glu Met Leu Asp Asn Ala His Tyr Leu Leu Ala Lys

1385 1390 1395

Thr Thr Gly Ile Gln Cys Arg Phe Asn Glu Gly Gln Leu Thr Thr

1400 1405 1410

Ser Ser Lys Arg Gln His Lys Asn Tyr Met Pro Asn Ser Cys Tyr

1415 1420 1425

Gln Leu Leu Ala Gln Asp Cys Thr Asp Glu Leu Lys Phe Ile Val

1430 1435 1440

Leu Leu Lys Lys Asp Ser Ala Gly Arg Tyr Met Val Asn Val Lys

1445 1450 1455

Ile Gly Thr Arg Asp Ile Asp Met Phe Phe Asn Gly Glu Arg Pro

1460 1465 1470

Ala Val Met Ile Asn Gly Lys Glu Ile Ala Arg Glu Arg Leu Pro

1475 1480 1485

Tyr Asp Arg Asp Ser Val Thr Ile Leu Leu Met Asn Gly Lys Leu

1490 1495 1500

Phe Leu Arg Ala Leu Asp Phe Gly Ile Ala Glu Leu Gln Phe Ser

1505 1510 1515

Ala Ser Glu Val Thr Ile Asn Val Pro Glu Tyr Leu Arg Asn Lys

1520 1525 1530

Val Cys Gly Leu Cys Gly Gln Gly Asn Gly Asp Arg Arg Asn Asp

1535 1540 1545

Tyr Arg Met Pro Asn Gly Arg Ile Thr Asp Asn Pro Ile Ser Phe

1550 1555 1560

Ala His Ser Trp Thr Leu Pro Ser Gln Ser Cys Ser Asp Glu Thr

1565 1570 1575

Glu Cys Arg Leu Thr His Glu Ser Ile Glu Leu Glu Arg Glu Ile

1580 1585 1590

Asn Asp His Gly Val Pro Ser Lys Cys Phe Ser Val Asp Ser Val

1595 1600 1605

Leu Arg Cys Arg Pro Gly Cys Thr Pro Thr Lys Thr Thr Met Ser

1610 1615 1620

Ser Val Ser Phe His Cys Arg Pro Leu Asn Asp Asn Ser Gln Val

1625 1630 1635

Ser Asp Ile Arg Asn Arg Ser Ile Asp Met Thr Glu Ser Val Glu

1640 1645 1650

Ala His Leu Asp Cys Ser Cys Thr Ser Gln Cys Ala

1655 1660 1665

<210> 3

<211> 5198

<212> DNA

<213> Periophthalmus modestus（VtgAb）

<400> 3

acatggggac attcaccagc catgagggtg tttgtgctcg cgctcactgt ggcccttgca 60

gtggctaacc aagtcaattt ggttcctgag tttgatgtag gaaagacctc tgtgtacaat 120

tatgaagcag agattctggg aggtcttcca gaggaaggtc ttgcaagagc aggtctgaaa 180

atccaaagca aagttttgat ccatgcacaa gctcaggata acttcttctt gaagctcaca 240

gactctaaga tctttgagta caatggtgtc tggccaaatg atgcatacac tgagtctgct 300

aaattaaacg aagccctggc tgctcagctc cagacccctg tcaggtttga gtatgctaac 360

ggcatcgtgg gaaaagtgta tgcaccaaat ggcatgtcca ccaccatcct gaacatctgt 420

aaagccatca tcaacatcct ccagctgaac atcaagaaaa ctcaggacta ctacgacctg 480

caggaggctg ggccacatgg tgtatgtctt acccactatg tcatcactga ggacaagagt 540

gctggtcgct acttcatcac aaaagaaaag gatctgacca aatgccagga caaaattgta 600

aaagacattg gtttggctta catgaagaaa taccatgaat gtcgtgctag atcaaagacc 660

ttgaatggtg taacagtgac aaactacatc ctgaagccca cagctgctgg tagtactcag 720

atcgaggaag tgaaaactaa agagatcgtt gagtactctc catttgatgt tctccaaggc 780

gctgcccaca tagaagctaa acaaaagctg agactgatgg atatcacagc tgtaccagtt 840

gaacccatca ctgctcagta tctccagcgt gggtccctgc agtatgagtt tggtactgaa 900

ctcctccaga atccagtgca gttgctgaga atcgaaaatg tggatactca gattgttgag 960

ttactggagc acctgaagag ctatgatgtt gaaaaggttc atgaagatgc cccactaaag 1020

tttattgaac tcatccaact catgcgcatg cttgactaca gtgcaattga cactttgtgg 1080

acaaacaata aagaaaaacc tattcacaga ctctggatcc tgagtgctat ccctgttctg 1140

ggaagtcatg tttctctgaa gttcatcagg aatcacttac catggactgc acaggaatca 1200

tttacagaag caacgaatgc tttagtgtcc accttccaca tggtaacagc tgaccaagct 1260

gccataaatg aagctaagga gtttctgaat cccatttttg agaaggagaa gagtatggga 1320

gtgaaagatt tcaaattctt catttaccgg gacatagtta tgcttggcta tggtaccctg 1380

gttgctaaac actgcgtgga agaaagggat tgcactgttg atctggtcaa gccactccat 1440

aactatgcag ttgaaaatgc agctaaccca gagaggctta tcattgctct taaagctatt 1500

ggtaacgctg gacatgttgg aagtttgaag actattcaga agttcatccc tgggattggt 1560

atcgctgctg aacatctgcc tctgcatttg aagattcagg ctgtcgctgc actgaagaac 1620

atggcaaaga aagaacctag gatggtccaa ccagttgtcc tgagtctttt cagaaacacc 1680

aagattgact ctgagctgcg tatgatttct gctgctatgc tgtttgagac aaatccctcc 1740

atggctctgg tgaatgtgat cgcggcctct gtgctgaatg agcccaatat gcaggtcgcc 1800

agctttgtgt acacctacat gaaggccctg gccaaaagca cagcccctga cctgctttct 1860

gttgctactg cctgtaatgt ggctgtgagg attataactc cgaaactggg ctcattgaat 1920

atgcgctaca gcagagccct gtatgtgaat gcctaccaaa acaagtggat gatgggagtg 1980

gctggaagtg cgttcattat caacaacgca gccagcatct ttcccagagc catggtggcc 2040

aaagcgcgtg cctatgctgc tggggcttat actgatgttt tagagtttgg ggtacgggct 2100

gaaggactcc aggaggtgct tctgaagttt aatgagtatc cagatactaa agacagaatt 2160

gctaagatga agaacattct caagacgctc aatgactgga gggctgaagc aacccccaag 2220

cctctggcct cagcttacat caaattcttt gaccaggaag ttgccttcac caacattgac 2280

aaggacctta tggattacat tgctgatgtc ttcagaagct cagaaattct caacacagga 2340

aaggccttct tgcgtcaact gctgtctgga gttaatatgc gtcatatgca ccccatgctt 2400

ctgtctgagg ccagacgcgt cctgcccact actattggtc tgcccatgga gctgagtttt 2460

tacactgcag ctgtggctgg ggcctctgtc aacattcaag ctactgtaac accccctcta 2520

cctgaggact tccgtccaag gcagcttctt gagtctgaca tcaaccttag ggcttcgatc 2580

attccaagtg ttgccatgca cacctatgct gtgatgggag tcaatacaca ccttgtccag 2640

gctgctctga tgtccagggc aaaaatccat gcagttgttc ctgctaaatt tgaggcaaga 2700

cttgacatca acaaaggcaa cttcaagctg gatctgctgc cagctgagag cattgacaaa 2760

gtcggctctg tacgtgttga tactgtggct gttgtcagaa acgtggaaga tctcagagat 2820

gccaaaacca ttcctgtgat cccagctgac caagcaacca agggctcctc aaaaagctcc 2880

tcaaaaggct cctcaaaaag ctcctccaat agctcctcaa aaggctttgt ctccgggggc 2940

ttgtcatcag agctgctctc ctctgtcgtg tcacacaaaa tgaccaacaa aattaggatt 3000

tcaaaagcct atgagaagaa gatatgtggc ttatttgaga cccttggatt caaagcgtgc 3060

accgtgattc agtcttataa tgctcagtcc atcaatgact ctcctttgta cgccacgatt 3120

ggaaaacacc aaatttttgt tgaagttgcc cgagttggtg aaagtccagt ggagaaaatt 3180

gaggttgaga ttcaatttgg agacaaagct gcagacatca tccaaaaggt caccatcaca 3240

caggcagagg agatcctgga ggacaagagc gtcctgatga agctgagaaa gatcctggct 3300

cctgtaccaa aaaacaccac taactcctcc agctcctcca gtcgctcaca ctcaggtcta 3360

agctccagtg ccagctcctc ctcaaagtcc agcagcagta gcagcagcag cagcagcagt 3420

caggccagcc gcaagtctag actccgggtg acgctcgacc agaagagcag cagcagcagc 3480

tccagcagta gatcttcatc cagcttaagc tcctccagac attctcctgt ggatcaagag 3540

atgtacgagg tcaagtttaa cagggatcac aaacaccagc attctcgcgc cagtactcat 3600

cagtcccgcc agagcaagag tagcgcacag agttttgagg atatctacaa caaggccaga 3660

ttcctgtcaa atacagtggc acctagtctg gctattgtca tccgtgctaa gagagctgac 3720

cacaagactc agggatacca ggttgcagct tacgtcgaca aagacaacag gagaattcag 3780

atcatctttg ccaaattggc tgatgccgag cactctgtaa tttgtgctga tggtgtcgct 3840

ctgagcaacc acaaactgat ggccagagtt gcttggggtc aggactgtaa gcagtactcc 3900

accgacgtcg ttgctgagac tggccgagtg aatgaaaaca tggctcttcg tctgaaactg 3960

agttgggaaa aatttccaag agcatttaaa cactacatct ccaagatgtc tcgtcttgca 4020

tactcccttc agcaatatgg ggccagcatt gcaaagacca gagaggtagt taagcagatg 4080

aaactcacag tggtgcttgc ttctgagaca aaacttgata ttctgctgag gaccccaaag 4140

aaactcatct acagcctggg tatacgtctt cccttttgcc tgcctttgaa agaaacagct 4200

tctcaactgc agtcctacaa caactgggct tcaaagattg tctacctgat ttccagtggc 4260

aacaacatga cctgtactat gaagcaaaat gtcctgacca gtttcaacaa ggcaaggtgt 4320

gaaagctcca tgcctaaatc ctgctaccag gttctggccc aggactgcac tgagaaactc 4380

cgtttcattg ttctagtgag gagagaaggc ccacaggcac agcgcgaact ccgtgtgctc 4440

atctcacact tgactctaga cattgcacag aggaacggcc atgttgtggt aacagtgaat 4500

ggaaaagagc aacaagtcac agaggagact cagttggccc ctgacgtgaa aattaaacgc 4560

gaagccaatg gtgtggttct ctatgcccat gagcttggtc ttaaggaact ctactttgat 4620

agtcaaatcc aaaaggttgt agtttctgac ctcatgaagg gaaagacctg tggcttgtgc 4680

ggccgcgctg atggggagta tagacaggag tatcgcacac ctagtaaccg cgttgtctct 4740

gatgctgtta gccatgccca cagttggact ctgtctggta atacctgccg caatgaggaa 4800

ggctgcaagc ttcagcatgt atccgtgaag ctggagggga agttcagtca ctatggcgta 4860

ccttcaaagt gctacacagt tgagccagtt ctgcgttgta ttcctggctg taaccctctg 4920

aagaccaaaa ccatcagagt tggctaccac tgcatacctt atgattcaaa cctgtcctct 4980

gcagacaata tcttcaacaa gagtaaggat ctggagctgg acacagtcgc ccacgaggac 5040

tgccagtgca cgcctcagtg tgcctgatct tactatcttg tctgttaagt aatgtttgat 5100

caatcacttt gttgtaaatt taaataataa actcctgaag caagtcaaaa aaaaaaaaaa 5160

aaaaaaaaaa aaagtactct gcgttgatac cactgctt 5198

<210> 4

<211> 1681

<212> PRT

<213> Periophthalmus modestus（VtgAb）

<400> 4

Met Arg Val Phe Val Leu Ala Leu Thr Val Ala Leu Ala Val Ala Asn

1 5 10 15

Gln Val Asn Leu Val Pro Glu Phe Asp Val Gly Lys Thr Ser Val Tyr

20 25 30

Asn Tyr Glu Ala Glu Ile Leu Gly Gly Leu Pro Glu Glu Gly Leu Ala

35 40 45

Arg Ala Gly Leu Lys Ile Gln Ser Lys Val Leu Ile His Ala Gln Ala

50 55 60

Gln Asp Asn Phe Phe Leu Lys Leu Thr Asp Ser Lys Ile Phe Glu Tyr

65 70 75 80

Asn Gly Val Trp Pro Asn Asp Ala Tyr Thr Glu Ser Ala Lys Leu Asn

85 90 95

Glu Ala Leu Ala Ala Gln Leu Gln Thr Pro Val Arg Phe Glu Tyr Ala

100 105 110

Asn Gly Ile Val Gly Lys Val Tyr Ala Pro Asn Gly Met Ser Thr Thr

115 120 125

Ile Leu Asn Ile Cys Lys Ala Ile Ile Asn Ile Leu Gln Leu Asn Ile

130 135 140

Lys Lys Thr Gln Asp Tyr Tyr Asp Leu Gln Glu Ala Gly Pro His Gly

145 150 155 160

Val Cys Leu Thr His Tyr Val Ile Thr Glu Asp Lys Ser Ala Gly Arg

165 170 175

Tyr Phe Ile Thr Lys Glu Lys Asp Leu Thr Lys Cys Gln Asp Lys Ile

180 185 190

Val Lys Asp Ile Gly Leu Ala Tyr Met Lys Lys Tyr His Glu Cys Arg

195 200 205

Ala Arg Ser Lys Thr Leu Asn Gly Val Thr Val Thr Asn Tyr Ile Leu

210 215 220

Lys Pro Thr Ala Ala Gly Ser Thr Gln Ile Glu Glu Val Lys Thr Lys

225 230 235 240

Glu Ile Val Glu Tyr Ser Pro Phe Asp Val Leu Gln Gly Ala Ala His

245 250 255

Ile Glu Ala Lys Gln Lys Leu Arg Leu Met Asp Ile Thr Ala Val Pro

260 265 270

Val Glu Pro Ile Thr Ala Gln Tyr Leu Gln Arg Gly Ser Leu Gln Tyr

275 280 285

Glu Phe Gly Thr Glu Leu Leu Gln Asn Pro Val Gln Leu Leu Arg Ile

290 295 300

Glu Asn Val Asp Thr Gln Ile Val Glu Leu Leu Glu His Leu Lys Ser

305 310 315 320

Tyr Asp Val Glu Lys Val His Glu Asp Ala Pro Leu Lys Phe Ile Glu

325 330 335

Leu Ile Gln Leu Met Arg Met Leu Asp Tyr Ser Ala Ile Asp Thr Leu

340 345 350

Trp Thr Asn Asn Lys Glu Lys Pro Ile His Arg Leu Trp Ile Leu Ser

355 360 365

Ala Ile Pro Val Leu Gly Ser His Val Ser Leu Lys Phe Ile Arg Asn

370 375 380

His Leu Pro Trp Thr Ala Gln Glu Ser Phe Thr Glu Ala Thr Asn Ala

385 390 395 400

Leu Val Ser Thr Phe His Met Val Thr Ala Asp Gln Ala Ala Ile Asn

405 410 415

Glu Ala Lys Glu Phe Leu Asn Pro Ile Phe Glu Lys Glu Lys Ser Met

420 425 430

Gly Val Lys Asp Phe Lys Phe Phe Ile Tyr Arg Asp Ile Val Met Leu

435 440 445

Gly Tyr Gly Thr Leu Val Ala Lys His Cys Val Glu Glu Arg Asp Cys

450 455 460

Thr Val Asp Leu Val Lys Pro Leu His Asn Tyr Ala Val Glu Asn Ala

465 470 475 480

Ala Asn Pro Glu Arg Leu Ile Ile Ala Leu Lys Ala Ile Gly Asn Ala

485 490 495

Gly His Val Gly Ser Leu Lys Thr Ile Gln Lys Phe Ile Pro Gly Ile

500 505 510

Gly Ile Ala Ala Glu His Leu Pro Leu His Leu Lys Ile Gln Ala Val

515 520 525

Ala Ala Leu Lys Asn Met Ala Lys Lys Glu Pro Arg Met Val Gln Pro

530 535 540

Val Val Leu Ser Leu Phe Arg Asn Thr Lys Ile Asp Ser Glu Leu Arg

545 550 555 560

Met Ile Ser Ala Ala Met Leu Phe Glu Thr Asn Pro Ser Met Ala Leu

565 570 575

Val Asn Val Ile Ala Ala Ser Val Leu Asn Glu Pro Asn Met Gln Val

580 585 590

Ala Ser Phe Val Tyr Thr Tyr Met Lys Ala Leu Ala Lys Ser Thr Ala

595 600 605

Pro Asp Leu Leu Ser Val Ala Thr Ala Cys Asn Val Ala Val Arg Ile

610 615 620

Ile Thr Pro Lys Leu Gly Ser Leu Asn Met Arg Tyr Ser Arg Ala Leu

625 630 635 640

Tyr Val Asn Ala Tyr Gln Asn Lys Trp Met Met Gly Val Ala Gly Ser

645 650 655

Ala Phe Ile Ile Asn Asn Ala Ala Ser Ile Phe Pro Arg Ala Met Val

660 665 670

Ala Lys Ala Arg Ala Tyr Ala Ala Gly Ala Tyr Thr Asp Val Leu Glu

675 680 685

Phe Gly Val Arg Ala Glu Gly Leu Gln Glu Val Leu Leu Lys Phe Asn

690 695 700

Glu Tyr Pro Asp Thr Lys Asp Arg Ile Ala Lys Met Lys Asn Ile Leu

705 710 715 720

Lys Thr Leu Asn Asp Trp Arg Ala Glu Ala Thr Pro Lys Pro Leu Ala

725 730 735

Ser Ala Tyr Ile Lys Phe Phe Asp Gln Glu Val Ala Phe Thr Asn Ile

740 745 750

Asp Lys Asp Leu Met Asp Tyr Ile Ala Asp Val Phe Arg Ser Ser Glu

755 760 765

Ile Leu Asn Thr Gly Lys Ala Phe Leu Arg Gln Leu Leu Ser Gly Val

770 775 780

Asn Met Arg His Met His Pro Met Leu Leu Ser Glu Ala Arg Arg Val

785 790 795 800

Leu Pro Thr Thr Ile Gly Leu Pro Met Glu Leu Ser Phe Tyr Thr Ala

805 810 815

Ala Val Ala Gly Ala Ser Val Asn Ile Gln Ala Thr Val Thr Pro Pro

820 825 830

Leu Pro Glu Asp Phe Arg Pro Arg Gln Leu Leu Glu Ser Asp Ile Asn

835 840 845

Leu Arg Ala Ser Ile Ile Pro Ser Val Ala Met His Thr Tyr Ala Val

850 855 860

Met Gly Val Asn Thr His Leu Val Gln Ala Ala Leu Met Ser Arg Ala

865 870 875 880

Lys Ile His Ala Val Val Pro Ala Lys Phe Glu Ala Arg Leu Asp Ile

885 890 895

Asn Lys Gly Asn Phe Lys Leu Asp Leu Leu Pro Ala Glu Ser Ile Asp

900 905 910

Lys Val Gly Ser Val Arg Val Asp Thr Val Ala Val Val Arg Asn Val

915 920 925

Glu Asp Leu Arg Asp Ala Lys Thr Ile Pro Val Ile Pro Ala Asp Gln

930 935 940

Ala Thr Lys Gly Ser Ser Lys Ser Ser Ser Lys Gly Ser Ser Lys Ser

945 950 955 960

Ser Ser Asn Ser Ser Ser Lys Gly Phe Val Ser Gly Gly Leu Ser Ser

965 970 975

Glu Leu Leu Ser Ser Val Val Ser His Lys Met Thr Asn Lys Ile Arg

980 985 990

Ile Ser Lys Ala Tyr Glu Lys Lys Ile Cys Gly Leu Phe Glu Thr Leu

995 1000 1005

Gly Phe Lys Ala Cys Thr Val Ile Gln Ser Tyr Asn Ala Gln Ser

1010 1015 1020

Ile Asn Asp Ser Pro Leu Tyr Ala Thr Ile Gly Lys His Gln Ile

1025 1030 1035

Phe Val Glu Val Ala Arg Val Gly Glu Ser Pro Val Glu Lys Ile

1040 1045 1050

Glu Val Glu Ile Gln Phe Gly Asp Lys Ala Ala Asp Ile Ile Gln

1055 1060 1065

Lys Val Thr Ile Thr Gln Ala Glu Glu Ile Leu Glu Asp Lys Ser

1070 1075 1080

Val Leu Met Lys Leu Arg Lys Ile Leu Ala Pro Val Pro Lys Asn

1085 1090 1095

Thr Thr Asn Ser Ser Ser Ser Ser Ser Arg Ser His Ser Gly Leu

1100 1105 1110

Ser Ser Ser Ala Ser Ser Ser Ser Lys Ser Ser Ser Ser Ser Ser

1115 1120 1125

Ser Ser Ser Ser Ser Gln Ala Ser Arg Lys Ser Arg Leu Arg Val

1130 1135 1140

Thr Leu Asp Gln Lys Ser Ser Ser Ser Ser Ser Ser Ser Arg Ser

1145 1150 1155

Ser Ser Ser Leu Ser Ser Ser Arg His Ser Pro Val Asp Gln Glu

1160 1165 1170

Met Tyr Glu Val Lys Phe Asn Arg Asp His Lys His Gln His Ser

1175 1180 1185

Arg Ala Ser Thr His Gln Ser Arg Gln Ser Lys Ser Ser Ala Gln

1190 1195 1200

Ser Phe Glu Asp Ile Tyr Asn Lys Ala Arg Phe Leu Ser Asn Thr

1205 1210 1215

Val Ala Pro Ser Leu Ala Ile Val Ile Arg Ala Lys Arg Ala Asp

1220 1225 1230

His Lys Thr Gln Gly Tyr Gln Val Ala Ala Tyr Val Asp Lys Asp

1235 1240 1245

Asn Arg Arg Ile Gln Ile Ile Phe Ala Lys Leu Ala Asp Ala Glu

1250 1255 1260

His Ser Val Ile Cys Ala Asp Gly Val Ala Leu Ser Asn His Lys

1265 1270 1275

Leu Met Ala Arg Val Ala Trp Gly Gln Asp Cys Lys Gln Tyr Ser

1280 1285 1290

Thr Asp Val Val Ala Glu Thr Gly Arg Val Asn Glu Asn Met Ala

1295 1300 1305

Leu Arg Leu Lys Leu Ser Trp Glu Lys Phe Pro Arg Ala Phe Lys

1310 1315 1320

His Tyr Ile Ser Lys Met Ser Arg Leu Ala Tyr Ser Leu Gln Gln

1325 1330 1335

Tyr Gly Ala Ser Ile Ala Lys Thr Arg Glu Val Val Lys Gln Met

1340 1345 1350

Lys Leu Thr Val Val Leu Ala Ser Glu Thr Lys Leu Asp Ile Leu

1355 1360 1365

Leu Arg Thr Pro Lys Lys Leu Ile Tyr Ser Leu Gly Ile Arg Leu

1370 1375 1380

Pro Phe Cys Leu Pro Leu Lys Glu Thr Ala Ser Gln Leu Gln Ser

1385 1390 1395

Tyr Asn Asn Trp Ala Ser Lys Ile Val Tyr Leu Ile Ser Ser Gly

1400 1405 1410

Asn Asn Met Thr Cys Thr Met Lys Gln Asn Val Leu Thr Ser Phe

1415 1420 1425

Asn Lys Ala Arg Cys Glu Ser Ser Met Pro Lys Ser Cys Tyr Gln

1430 1435 1440

Val Leu Ala Gln Asp Cys Thr Glu Lys Leu Arg Phe Ile Val Leu

1445 1450 1455

Val Arg Arg Glu Gly Pro Gln Ala Gln Arg Glu Leu Arg Val Leu

1460 1465 1470

Ile Ser His Leu Thr Leu Asp Ile Ala Gln Arg Asn Gly His Val

1475 1480 1485

Val Val Thr Val Asn Gly Lys Glu Gln Gln Val Thr Glu Glu Thr

1490 1495 1500

Gln Leu Ala Pro Asp Val Lys Ile Lys Arg Glu Ala Asn Gly Val

1505 1510 1515

Val Leu Tyr Ala His Glu Leu Gly Leu Lys Glu Leu Tyr Phe Asp

1520 1525 1530

Ser Gln Ile Gln Lys Val Val Val Ser Asp Leu Met Lys Gly Lys

1535 1540 1545

Thr Cys Gly Leu Cys Gly Arg Ala Asp Gly Glu Tyr Arg Gln Glu

1550 1555 1560

Tyr Arg Thr Pro Ser Asn Arg Val Val Ser Asp Ala Val Ser His

1565 1570 1575

Ala His Ser Trp Thr Leu Ser Gly Asn Thr Cys Arg Asn Glu Glu

1580 1585 1590

Gly Cys Lys Leu Gln His Val Ser Val Lys Leu Glu Gly Lys Phe

1595 1600 1605

Ser His Tyr Gly Val Pro Ser Lys Cys Tyr Thr Val Glu Pro Val

1610 1615 1620

Leu Arg Cys Ile Pro Gly Cys Asn Pro Leu Lys Thr Lys Thr Ile

1625 1630 1635

Arg Val Gly Tyr His Cys Ile Pro Tyr Asp Ser Asn Leu Ser Ser

1640 1645 1650

Ala Asp Asn Ile Phe Asn Lys Ser Lys Asp Leu Glu Leu Asp Thr

1655 1660 1665

Val Ala His Glu Asp Cys Gln Cys Thr Pro Gln Cys Ala

1670 1675 1680

<210> 5

<211> 3770

<212> DNA

<213> Periophthalmus modestus（VtgC）

<400> 5

ctcaccatgc aggccctact tctgtgctgc ctggtggctc tggccgtctg caacgagaat 60

gcacgctatg acctcaactt gaaccctaat aagatctatg agtaccagta tgaaggatgg 120

gtcaaatttg gcctgggcaa cccaaaccat gctgagtccg gtgcaagact gaagtctaaa 180

gtcacgattg agggtgcttc tgctgacact tttaatctga agttctccaa tgtagtcttt 240

gaggagttca atggtgtccc agaaagtaac tttgtggcag ctgaccagct cgcccaagag 300

attgctgatc agttggcaaa gcccattgag ttcacatact ctgcagggaa cgtgggtgag 360

atcaaagcct cctctgaggt ggccgatact gtgctcaaca ttatcagagc attcctgagc 420

tttttccaag tcacagtcaa aaccacacag gatatctacg agcttgagga ggttggcatc 480

catggaaagt gttttagcag ttatgcagtg aagataaatg accaggagaa agtcatggat 540

ctcactcaag tggtggatgt gaccaactgc agggaaaagg ctgcctttta ctttggaatg 600

gctacagccc ttgaggacaa ggactccaag cagaggggtg agtctacctt ctcaactgtt 660

aaatacacat acaacatcaa aggagcagag gatgcaggac tcatcactaa agcccaagcc 720

ctggagctgc agtacttcac tcccttcaat gtgaagagag gtgtcttcaa gatggaggcc 780

aggaaggagc tggtgctcat atctgagggg gccaaaactc agaacatccc caggattcag 840

gagagacagc tccagaacag aggaaacctc atatacaaag tggtgaagaa ctgggctaat 900

gtaccaatca caatgcagaa gatggacaag cctgtatcta aggctaaaga gctgatcaaa 960

cgtctggccc aagcaaacag acaccaaatt gacagtgcaa caactgaaga taccatcaaa 1020

ctgtaccagc tgctgcgagt cattcctttg gatcaactgg aaaagatgtg ggcagatgtg 1080

gaagggaact ctgatgagag gaactggttc ttacacactg ttgttgaggt taacgatgcc 1140

agaatcctga ctttcctgga acgtgttctt cgggaggaaa aattaaatca actggaagcc 1200

attactgtca ttgtgcgagc attcaaccac ctcgatgcaa ctccagagct gctcaagatg 1260

gcagagaaat tcctcaccat gacctacaac aatcctgtca taagaagaac tgtggtgctg 1320

tcctatggct ccctggtatt taaactttgt gcctacaaca acccctgccc agaggaggcc 1380

atcaggccac tcttgaacat ggcagaggag agtatcagaa ataacaatgt gatggagaag 1440

attctagtcc tgaaagcgct gggtaatgca ggtcatcctc gcagtctcaa aaccatcaac 1500

aagttcctgc ctgatgtgga ccctaacccg aaccgagtgg acagggaacc acgggtcgtg 1560

agcgcagctg tgcaggctat gagactgatc acagccaggg accctcatgg ggtgcagcag 1620

aggactctaa agctgttctt gaaccgtgag cttaaatctg agctcaggat gctggctctc 1680

atgatcatgt ttgacgctaa gccctctacg gccctagtct ccacggtgac cgctcacctg 1740

ctggaggaga gagacatgca ggtggtcaac ttcgcaaaca catactttag gaccctgtcc 1800

agagcaatga caccagacaa ccagtatcta tctactgctg ccagtgttgc tgtgaaaatc 1860

ttggccccga aatttggttg cctgagctac ctcggcagta gagcagtacg catggactgg 1920

ttccacgatg acttcctgat tggtaccgcc gctgaggcct tcatactgaa aagagcttca 1980

cagatcatcc cagctgaaat gatggtgaaa tggaactttt atttcattgg gaggatcctg 2040

cagctggtgg agttgggtat ccgcccagag ggactgagag atctgtttgg tgccagcatc 2100

gaaggattta gaggagatct gagcatgact gacttccagg ctatttttaa tgttctcaaa 2160

aagtgggaaa cactgccaaa tgacaagcct ttgttgagtg tttatacacg tgcatcagga 2220

caggagtttt acttcagtga tctaaacaag gactttgtgg agtggatgat gagggcactc 2280

agcccatctg caggaaaaga cagttttgtg tggagaatga ttgagcagct aatccaaggc 2340

ttctcatggc atggagtcct gccattcctg actgttgaag ctcgctatat ccaagccaca 2400

acccttggtc tcccactcga gatcagcaaa tactaccatg ttgtaaacag cctctcagtc 2460

aatgctcaag ctgcattgaa ccaacaagca gctcagaatg ttggagagtt gctttcttca 2520

gagactacag tgaagacaga tggcttcatt ggatacacca agaatttctg gcttttctac 2580

ggcatcaaca ctgacttatt ccagtccgct gttgagatta agaccataaa tcctgttgct 2640

atcccatgga attttgttgc aaagatcaat atccgtgagt ggaaagttga acttgagatc 2700

cctcaaagca acagagagtt tgaagtcttc tctttcagtt ccaatgcata tgccatctca 2760

aggaacatca ttaacccaga agcagctaaa aagatcccag tttttcccaa cgagcagaat 2820

gtgaaccaaa taaataaccc taagcctaat aagtggaatc cagttgtaaa catgtgcacc 2880

aagagcgccg cctatggagt atccctgtgc ttagagtacg agctgaagcg agagttctgc 2940

caggaggagt accccttgta ctacttcctc gggttcacac acgttgcctt caaagtagta 3000

ccagtccgac aaacttcagc tgatgagaag atccttgtgg agattaacgc agccccagac 3060

acagttgcaa tgaatgtacg ccagctcctt gccaatctga gaacaatgtc caaggaaggc 3120

cgtcaatcaa gcaccagcgc atctcaagag cagcaggaca gacaatcaag gggcatggag 3180

gctccacctg tgcctgtgct gaacttcaaa gtgctggcag tgagcagcag tcagaaggcc 3240

gtgggttacg aagcctcctt ctactacacg cctgcggagg agggccagag cactcagctc 3300

atcgtcagcc aggtgggaga ggccaccaac tggaagatgt gtgtggacac tgctgtggct 3360

gcagtggcta ggagccaatc cagagcaaag ctgtttaagg ctcacgctgc atggggagca 3420

gagtgtcagc cctactctgt gtctgtgagg gcctctggca gcatgaggga gctgcaggcc 3480

gtcttccgct gggaccagat gccagagacc atggcagcgc agggaagaag aattgccaga 3540

tacatcccag gtatggccat tcttacaggc tgcacccagg aggaggacag aaatgccacg 3600

caggaggttt ctgcgtcggt tattgctgct tcagcagaca gcatcaatgt gaagattaaa 3660

ctcccagagc ttacactgcg ctgtgaggct ctgccttttc caatggcagt ctccactaca 3720

acagaacaat aaacagcatt acacaggcag agacacactg tgccatggaa 3770

<210> 6

<211> 1241

<212> PRT

<213> Periophthalmus modestus（VtgC）

<400> 6

Met Gln Ala Leu Leu Leu Cys Cys Leu Val Ala Leu Ala Val Cys Asn

1 5 10 15

Glu Asn Ala Arg Tyr Asp Leu Asn Leu Asn Pro Asn Lys Ile Tyr Glu

20 25 30

Tyr Gln Tyr Glu Gly Trp Val Lys Phe Gly Leu Gly Asn Pro Asn His

35 40 45

Ala Glu Ser Gly Ala Arg Leu Lys Ser Lys Val Thr Ile Glu Gly Ala

50 55 60

Ser Ala Asp Thr Phe Asn Leu Lys Phe Ser Asn Val Val Phe Glu Glu

65 70 75 80

Phe Asn Gly Val Pro Glu Ser Asn Phe Val Ala Ala Asp Gln Leu Ala

85 90 95

Gln Glu Ile Ala Asp Gln Leu Ala Lys Pro Ile Glu Phe Thr Tyr Ser

100 105 110

Ala Gly Asn Val Gly Glu Ile Lys Ala Ser Ser Glu Val Ala Asp Thr

115 120 125

Val Leu Asn Ile Ile Arg Ala Phe Leu Ser Phe Phe Gln Val Thr Val

130 135 140

Lys Thr Thr Gln Asp Ile Tyr Glu Leu Glu Glu Val Gly Ile His Gly

145 150 155 160

Lys Cys Phe Ser Ser Tyr Ala Val Lys Ile Asn Asp Gln Glu Lys Val

165 170 175

Met Asp Leu Thr Gln Val Val Asp Val Thr Asn Cys Arg Glu Lys Ala

180 185 190

Ala Phe Tyr Phe Gly Met Ala Thr Ala Leu Glu Asp Lys Asp Ser Lys

195 200 205

Gln Arg Gly Glu Ser Thr Phe Ser Thr Val Lys Tyr Thr Tyr Asn Ile

210 215 220

Lys Gly Ala Glu Asp Ala Gly Leu Ile Thr Lys Ala Gln Ala Leu Glu

225 230 235 240

Leu Gln Tyr Phe Thr Pro Phe Asn Val Lys Arg Gly Val Phe Lys Met

245 250 255

Glu Ala Arg Lys Glu Leu Val Leu Ile Ser Glu Gly Ala Lys Thr Gln

260 265 270

Asn Ile Pro Arg Ile Gln Glu Arg Gln Leu Gln Asn Arg Gly Asn Leu

275 280 285

Ile Tyr Lys Val Val Lys Asn Trp Ala Asn Val Pro Ile Thr Met Gln

290 295 300

Lys Met Asp Lys Pro Val Ser Lys Ala Lys Glu Leu Ile Lys Arg Leu

305 310 315 320

Ala Gln Ala Asn Arg His Gln Ile Asp Ser Ala Thr Thr Glu Asp Thr

325 330 335

Ile Lys Leu Tyr Gln Leu Leu Arg Val Ile Pro Leu Asp Gln Leu Glu

340 345 350

Lys Met Trp Ala Asp Val Glu Gly Asn Ser Asp Glu Arg Asn Trp Phe

355 360 365

Leu His Thr Val Val Glu Val Asn Asp Ala Arg Ile Leu Thr Phe Leu

370 375 380

Glu Arg Val Leu Arg Glu Glu Lys Leu Asn Gln Leu Glu Ala Ile Thr

385 390 395 400

Val Ile Val Arg Ala Phe Asn His Leu Asp Ala Thr Pro Glu Leu Leu

405 410 415

Lys Met Ala Glu Lys Phe Leu Thr Met Thr Tyr Asn Asn Pro Val Ile

420 425 430

Arg Arg Thr Val Val Leu Ser Tyr Gly Ser Leu Val Phe Lys Leu Cys

435 440 445

Ala Tyr Asn Asn Pro Cys Pro Glu Glu Ala Ile Arg Pro Leu Leu Asn

450 455 460

Met Ala Glu Glu Ser Ile Arg Asn Asn Asn Val Met Glu Lys Ile Leu

465 470 475 480

Val Leu Lys Ala Leu Gly Asn Ala Gly His Pro Arg Ser Leu Lys Thr

485 490 495

Ile Asn Lys Phe Leu Pro Asp Val Asp Pro Asn Pro Asn Arg Val Asp

500 505 510

Arg Glu Pro Arg Val Val Ser Ala Ala Val Gln Ala Met Arg Leu Ile

515 520 525

Thr Ala Arg Asp Pro His Gly Val Gln Gln Arg Thr Leu Lys Leu Phe

530 535 540

Leu Asn Arg Glu Leu Lys Ser Glu Leu Arg Met Leu Ala Leu Met Ile

545 550 555 560

Met Phe Asp Ala Lys Pro Ser Thr Ala Leu Val Ser Thr Val Thr Ala

565 570 575

His Leu Leu Glu Glu Arg Asp Met Gln Val Val Asn Phe Ala Asn Thr

580 585 590

Tyr Phe Arg Thr Leu Ser Arg Ala Met Thr Pro Asp Asn Gln Tyr Leu

595 600 605

Ser Thr Ala Ala Ser Val Ala Val Lys Ile Leu Ala Pro Lys Phe Gly

610 615 620

Cys Leu Ser Tyr Leu Gly Ser Arg Ala Val Arg Met Asp Trp Phe His

625 630 635 640

Asp Asp Phe Leu Ile Gly Thr Ala Ala Glu Ala Phe Ile Leu Lys Arg

645 650 655

Ala Ser Gln Ile Ile Pro Ala Glu Met Met Val Lys Trp Asn Phe Tyr

660 665 670

Phe Ile Gly Arg Ile Leu Gln Leu Val Glu Leu Gly Ile Arg Pro Glu

675 680 685

Gly Leu Arg Asp Leu Phe Gly Ala Ser Ile Glu Gly Phe Arg Gly Asp

690 695 700

Leu Ser Met Thr Asp Phe Gln Ala Ile Phe Asn Val Leu Lys Lys Trp

705 710 715 720

Glu Thr Leu Pro Asn Asp Lys Pro Leu Leu Ser Val Tyr Thr Arg Ala

725 730 735

Ser Gly Gln Glu Phe Tyr Phe Ser Asp Leu Asn Lys Asp Phe Val Glu

740 745 750

Trp Met Met Arg Ala Leu Ser Leu Ser Ala Gly Lys Asp Ser Phe Val

755 760 765

Trp Arg Met Ile Glu Gln Leu Ile Gln Gly Phe Ser Trp His Gly Val

770 775 780

Leu Pro Phe Leu Thr Val Glu Ala Arg Tyr Ile Gln Ala Thr Thr Leu

785 790 795 800

Gly Leu Pro Leu Glu Ile Ser Lys Tyr Tyr His Val Val Asn Ser Leu

805 810 815

Ser Val Asn Ala Gln Ala Ala Leu Asn Gln Gln Ala Ala Gln Asn Val

820 825 830

Gly Glu Leu Leu Ser Ser Glu Thr Thr Val Lys Thr Asp Gly Phe Ile

835 840 845

Gly Tyr Thr Lys Asn Phe Trp Leu Phe Tyr Gly Ile Asn Thr Asp Leu

850 855 860

Phe Gln Ser Ala Val Glu Ile Lys Thr Ile Asn Pro Val Ala Ile Pro

865 870 875 880

Trp Asn Phe Val Ala Lys Ile Asn Ile Arg Glu Trp Lys Val Glu Leu

885 890 895

Glu Ile Pro Gln Ser Asn Arg Glu Phe Glu Val Phe Ser Phe Ser Ser

900 905 910

Asn Ala Tyr Ala Ile Ser Arg Asn Ile Ile Asn Pro Glu Ala Ala Lys

915 920 925

Lys Ile Pro Val Phe Pro Asn Glu Gln Asn Val Asn Gln Ile Asn Asn

930 935 940

Pro Lys Pro Asn Lys Trp Asn Pro Val Val Asn Met Cys Ser Lys Ser

945 950 955 960

Ala Ala Tyr Gly Val Ser Leu Cys Leu Glu Tyr Glu Leu Lys Arg Glu

965 970 975

Phe Cys Gln Glu Glu Tyr Pro Leu Tyr Tyr Phe Leu Gly Phe Thr His

980 985 990

Val Ala Phe Lys Val Val Pro Val Arg Gln Thr Ser Ala Asp Glu Lys

995 1000 1005

Ile Leu Val Glu Ile Asn Ala Ala Pro Asp Thr Val Ala Met Asn

1010 1015 1020

Val Arg Gln Leu Leu Ala Asn Leu Arg Thr Met Ser Lys Glu Gly

1025 1030 1035

Arg Gln Ser Ser Thr Ser Ala Ser Gln Glu Gln Gln Asp Arg Gln

1040 1045 1050

Ser Arg Gly Met Glu Ala Pro Pro Val Pro Val Leu Asn Phe Lys

1055 1060 1065

Val Leu Ala Val Ser Ser Ser Gln Lys Ala Val Gly Tyr Glu Ala

1070 1075 1080

Ser Phe Tyr Tyr Thr Pro Ala Glu Glu Gly Gln Ser Thr Gln Leu

1085 1090 1095

Ile Val Ser Gln Val Gly Glu Ala Thr Asn Trp Lys Met Cys Val

1100 1105 1110

Asp Thr Ala Val Ala Ala Val Ala Arg Ser Gln Ser Arg Ala Lys

1115 1120 1125

Leu Phe Lys Ala His Ala Ala Trp Gly Ala Glu Cys Gln Pro Tyr

1130 1135 1140

Ser Val Ser Val Lys Ala Ser Gly Ser Met Arg Glu Leu Gln Ala

1145 1150 1155

Val Phe Arg Trp Asp Gln Met Pro Glu Thr Met Ala Ala Gln Gly

1160 1165 1170

Arg Arg Ile Ala Arg Tyr Ile Pro Gly Met Ala Ile Leu Thr Gly

1175 1180 1185

Cys Thr Gln Glu Glu Asp Arg Asn Ala Thr Gln Glu Val Ser Ala

1190 1195 1200

Ser Val Ile Ala Ala Ser Ala Asp Ser Ile Asn Val Lys Ile Lys

1205 1210 1215

Leu Pro Glu Leu Thr Leu Arg Cys Glu Ala Leu Pro Phe Pro Met

1220 1225 1230

Ala Val Ser Thr Thr Thr Glu Gln

1235 1240

<210> 7

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> vtgAa-degenerate-F1

<400> 7

Ala Ala Arg Ala Thr Gly Ala Ala Gly Cys Gly Tyr Arg Thr Cys Met

1 5 10 15

Thr Asp Met Ala Gly Gly Cys Thr Cys Thr

20 25

<210> 8

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> vtgAa-degenerate-R1

<400> 8

Ala Ala Cys Trp Gly Gly Met Ala Gly Arg Arg Cys Thr Tyr Ser Asp

1 5 10 15

Ala Lys Cys Thr Thr Ala Ala Ala Ser Thr

20 25

<210> 9

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> vtgAb-degenerate-F1

<400> 9

Thr Thr Cys Thr Lys Gly Ala Gly Lys Thr Tyr Gly Gly Ala Gly Tyr

1 5 10 15

Ser Met Gly Ala Asp Cys Thr Gly

20

<210> 10

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> vtgAb-degenerate-R1

<400> 10

Gly Cys Ala Gly Cys Trp Gly Tyr Arg Ala Gly Arg Tyr Cys Tyr Thr

1 5 10 15

Cys Met Ala Cys Ala Thr Tyr Thr

20

<210> 11

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> vtgC-degenerate-F1

<400> 11

Thr Trp Met Arg Gly Cys Cys Val Tyr Gly Ser Thr Gly Arg Ala His

1 5 10 15

Ala Thr Gly Gly

20

<210> 12

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> vtgC-degenerate-R1

<400> 12

Ala Lys Tyr Cys Cys His Thr Cys His Gly Ser Arg Cys Gly Arg Ala

1 5 10 15

Tyr Ala Cys Cys

20

<210> 13

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAa-5'RACE-GSP1

<400> 13

cctggacctt ggctcttgag actatgg 27

<210> 14

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAa-3'RACE-GSP2

<400> 14

gtcacttgtt ccaaaatggt gtctccgc 28

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAb-5'RACE-GSP1

<400> 15

gccctggaca tcagagcagc ctg 23

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAb-3'RACE-GSP2

<400> 16

gctcaatgac tggagggctg aagc 24

<210> 17

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgC-5'RACE-GSP1

<400> 17

caatcaggaa gtcatcgtgg aaccagtc 28

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgC-3'RACE-GSP2

<400> 18

gcctgatgtg gaccctaacc cgaac 25

<210> 19

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAa-full primer-F1

<400> 19

cagccatgag agtcgttgta ctagcct 27

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAa-full primer-R1

<400> 20

caagcagtgg tatcagcgca gagta 25

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAb-full primer-F1

<400> 21

agccatgagg gtgtttgtgc tc 22

<210> 22

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAb-full primer-R1

<400> 22

aacagacaag atagtaagat caggcacact 30

<210> 23

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgC-full primer-F1

<400> 23

ctcaccatgc aggccctact tct 23

<210> 24

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgC-full primer-R1

<400> 24

ttccatggca cagtgtgtct ctg 23

<210> 25

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> ef1-α qPCR-F1

<400> 25

tggtgacagc aagaacaacc 20

<210> 26

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> ef1-α qPCR-R1

<400> 26

atgaacttgc aggcgatgtg 20

<210> 27

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAa-qPCR-F1

<400> 27

aaattgccag agagcgcttg 20

<210> 28

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAa-qPCR-R1

<400> 28

aacagcttgc cgttcatcag 20

<210> 29

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAb-qPCR-F1

<400> 29

aaactcacag tggtgcttgc 20

<210> 30

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgAb-qPCR-R1

<400> 30

ttcaaaggca ggcaaaaggg 20

<210> 31

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgC-qPCR-F1

<400> 31

acgagaatgc acgctatgac 20

<210> 32

<211>

<212> DNA

<213> Artificial Sequence

<220>

<223> vtgC-qPCR-R1

<400> 32

ttgcccaggc caaatttgac 20