彈涂魚多型卵黃蛋白原Vtg基因全長序列及其克隆方法與流程
本發明屬于分子生物學領域，具體涉及一種基于新型灘涂模式魚類-彈涂魚多型卵黃蛋白原(vitellogenin，vtg)基因全長序列及其克隆方法。
背景技術：
魚類的卵黃蛋白原(vitellogenin，Vtg)是在雌二醇(estradiol-17β，E2)作用下經肝臟合成后分泌到血液中的卵黃前驅蛋白。通常情況下，Vtg是雌魚在卵黃形成時期血液中出現的雌特異性血清蛋白，但雄魚和稚魚在外源性雌激素作用下同樣能誘導體內Vtg的生成。
以灘涂作為棲息地的魚類中，彈涂魚Periophthalmus modestus是典型的代表種。彈涂魚隸屬于鱸形目、蝦虎魚亞目、彈涂魚科、彈涂魚屬，棲息于河口咸淡水水域、近岸灘涂處或底質爛泥的低潮區，為沿岸暖水廣溫廣鹽性魚類(倪勇，2008)。彈涂魚喜穴居，攝食底棲硅藻、藍綠藻、橈足類及有機質的特點，這些特征決定了它對底質有很強的依存性。彈涂魚之用作海洋環境風險評估的指示生物，除了其體內多項生理生化指標對環境污染物敏感外(劉冰和周忠良，2007)，還具有其他一系列優勢：分布區域廣，易捕獲，易運輸，以底棲硅藻等為食，食物鏈較短，體內各項指標檢測能直觀地反映近岸環境的變化；較其他魚類而言，彈涂魚具有更強的定居性，能更加準確地反映棲息地的生態狀況；近年來彈涂魚作為養殖魚類發展也很快，取得的研究成果可以直接用于近岸養殖和水體質量評估。
隨著基因克隆和免疫生物化學分析在魚類Vtg研究上的廣泛應用，硬骨魚類Vtg被證實是由若干基因編碼的相關蛋白組成的一個家族，即“多型”蛋白，且不同類型Vtg對雌激素的感受性也存在差異(Matsubara等，2003；Ohkubo等，2004；Wang等，2005；Miracle等，2006)。針對不同類型Vtg建立特異的檢測系，是保證雌激素暴露檢測準確的第一步。然而傳統的針對單一型Vtg的檢測方法，未針對多型Vtg分離出相應蛋白和基因，因此往往特異性不佳，甚至存在多型Vtg混雜的情況，檢測結果的準確性亟待提高。
現在未見有關于彈涂魚多型vtg基因和其全長cDNA序列的報道。
技術實現要素：
本發明的目的在于克服上述現有技術存在的不足，提供一種彈涂魚多型卵黃蛋白原(vitellogenin，vtg)基因全長序列及其克隆方法。
本發明的目的是通過以下技術方案來實現的：
第一方面，本發明涉及一種彈涂魚卵黃蛋白原，其氨基酸序列如SEQ ID NO.2所示。
第二方面，本發明涉及一種編碼如前述的彈涂魚卵黃蛋白原的彈涂魚卵黃蛋白原基因，所述基因為編碼氨基酸序列如SEQ ID NO.2所示的彈涂魚卵黃蛋白原的彈涂魚VtgAa基因。
優選的，彈涂魚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，本發明涉及一種彈涂魚卵黃蛋白原，其氨基酸序列如SEQ ID NO.4所示。
第四方面，本發明涉及一種編碼如前述的彈涂魚卵黃蛋白原的彈涂魚卵黃蛋白原基因，所述基因為編碼氨基酸序列如SEQ ID NO.4所示的彈涂魚卵黃蛋白原的彈涂魚VtgAb基因。
優選的，彈涂魚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，本發明涉及一種彈涂魚卵黃蛋白原，其氨基酸序列如SEQ ID NO.6所示。
第六方面，本發明涉及一種編碼如前述的彈涂魚卵黃蛋白原的彈涂魚卵黃蛋白原基因，所述基因為編碼氨基酸序列如SEQ ID NO.6所示的彈涂魚卵黃蛋白原的彈涂魚VtgC基因。
優選的，彈涂魚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，本發明涉及一種前述的彈涂魚卵黃蛋白原在作為判斷性別和雌魚成熟度的指示蛋白中的用途。
第八方面，一種彈涂魚三型Vtg基因全長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，本發明還涉及一種克隆彈涂魚Vtg三型基因全長序列的方法，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、從性成熟期彈涂魚雌魚的肝臟組織中提取總RNA，進行反轉錄擴增；
S2、分別設計Vtg的簡并引物進行擴增反應，獲得VtgAa基因、VtgAb基因和VtgC基因片段；
S3、以彈涂魚總RNA為模板根據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、分別設計特異性全長引物，擴增獲得Vtg三型基因全長序列。
優選的，步驟S2中，VtgAa基因對應的簡并引物為如SEQ ID NO.7所示的vtgAa-degenerate-F1和如SEQ ID NO.8所示的vtgAa-degenerate-R1；VtgAb基因對應的簡并引物為如SEQ ID NO.9所示的vtgAb-degenerate-F1和如SEQ ID NO.10所示的vtgAb-degenerate-R1；VtgC基因對應的簡并引物為如SEQ ID NO.11所示的vtgC-degenerate-F1和如SEQ ID NO.12所示的vtgC-degenerate-R1。
優選的，步驟S3中，VtgAa基因對應的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基因對應的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基因對應的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。
優選的，步驟S4中，VtgAa基因對應的特異性全長引物為如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基因對應的特異性全長引物為如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基因對應的特異性全長引物為如SEQ ID NO.23所示的vtgC-full primer-F1和如SEQ ID NO.24所示的vtgC-full primer-R1。
與現有技術相比，本發明具有如下有益效果：
1、本發明獲得了彈涂魚Vtg家族中三型Vtg基因全長CDS序列，填補了彈涂魚Vtg基因家族的空白。
2、彈涂魚是我國東南沿海廣泛分布的常見河口性魚類，Vtg是了解河口咸淡水水域、近岸灘涂處或底質爛泥的低潮區環境雌激素污染的重要生物標志物，獲得多型Vtg基因全長，對于更準確指示相應水域污染狀況，以及更深入探究水生生物在污染水域的蛋白表達等問題具有重要意義。
3、本發明通過設計特異性引物，建立了彈涂魚三型Vtg和ef1-α(內參)基因的實時熒光定量PCR方法。運用熒光定量PCR法檢測了Vtg三型基因在性成熟不同時期的表達變化，結果表明：未成熟的幼魚體內沒有該基因的表達，只有在雌魚中才有表達，且隨著卵巢的成熟，表達量呈現上升趨勢。
附圖說明
圖1為彈涂魚三型卵黃蛋白原VtgAa、VtgAb、VtgC基因的cDNA全長電泳圖；
其中，M：Marker，1：VtgAa，2：VtgAb，3：VtgC；
圖2為彈涂魚三型卵黃蛋白原VtgAa、VtgAb、VtgC基因在不同發育階段表達水平的差異。
具體實施方式
下面結合實施例對本發明進行詳細說明。以下實施例將有助于本領域的技術人員進一步理解本發明，但不以任何形式限制本發明。應當指出的是，對本領域的普通技術人員來說，在不脫離本發明構思的前提下，還可以做出若干調整和改進。這些都屬于本發明的保護范圍。
實施例
本實施例涉及一種彈涂魚多型卵黃蛋白原(vitellogenin，vtg)基因、編碼蛋白及其應用、克隆cDNA全長序列的方法。
VtgAa基因：本發明的彈涂魚多型VtgAa基因，其特征在于VtgAa序列全長5223bp，包含起始密碼子ATG、終止密碼子TAG及polyA。該基因的開放閱讀框(ORF)位于3～5000bp，長4998bp，共編碼1665個氨基酸，其中信號肽為15個氨基酸。根據推算，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構：1個vitellogenin結構域(位于編碼氨基酸肽鏈的24～594號氨基酸)、1個VWFD結構域(位于編碼氨基酸肽鏈的1423～1555號氨基酸)，1個多聚絲氨酸區域(位于編碼氨基酸肽鏈的1064～1195號氨基酸)，另外還含有1個蛋白水解酶切位點(RSRR)。將該推導氨基酸序列與其他魚類Vtg氨基酸序列比對后發現，該氨基酸序列包含LvH、LvL、Pv和β組分4個部分。
本發明所述彈涂魚VtgAa基因的核苷酸序列如SEQ ID NO.1所示。
所獲得的全長VtgAa cDNA序列的編碼區為從第3個核苷酸到5000個核苷酸。
本發明所述彈涂魚VtgAa基因的氨基酸序列如SEQ ID NO.2所示。
VtgAb基因：本發明的彈涂魚Vtg Ab基因，其特征在于序列全長5198bp，包含起始密碼子ATG、終止密碼子TGA及polyA。開放閱讀框(ORF)位于3～5048bp，長5046bp，共編碼1681個氨基酸，其中信號肽為15個氨基酸。根據推算，其編碼蛋白分子量為187kDa。將該推導氨基酸序列與其他魚類Vtg氨基酸序列比對后發現，該氨基酸序列同樣包含LvH、LvL、Pv和β組分4個部分。
本發明所述彈涂魚VtgAb基因的核苷酸序列如SEQ ID NO.3所示。
所獲得的全長VtgAb cDNA序列的編碼區從第3個核苷酸到5048個核苷酸。
本發明所述彈涂魚VtgAb基因的氨基酸序列如SEQ ID NO.4所示。
VtgC基因：本發明彈涂魚VtgC基因，其特征在于序列全長3770bp，包含起始密碼子ATG、終止密碼子TAA。開放閱讀框(ORF)位于7～3732bp，長3726bp，共編碼1241個氨基酸，其中信號肽為15個氨基酸。根據推算，其編碼蛋白分子量為141kDa。將該推導氨基酸序列與其他魚類Vtg氨基酸序列比對后發現，該氨基酸序列僅包含LvH和LvL兩個部分。
本發明所述彈涂魚VtgC基因的核苷酸序列如SEQ ID NO.5所示。
所獲得的全長VtgC cDNA序列的編碼區從第7個核苷酸到3732個核苷酸。
本發明所述彈涂魚VtgC基因的氨基酸序列如SEQ ID NO.6所示。
本發明通過設計特異性引物，引物為如SEQ ID NO.25～32所示，建立了彈涂魚三型Vtg和ef1-α(內參)基因的實時熒光定量PCR方法。運用熒光定量PCR法檢測了Vtg三型基因在性成熟不同時期的表達變化，結果表明：未成熟的幼魚體內沒有該基因的表達，只有在雌魚中才有表達，且隨著卵巢的成熟，表達量呈現上升趨勢。
克隆cDNA全長序列的方法：從性成熟期彈涂魚雌魚的肝臟組織中提取總RNA，進行反轉錄擴增，設計不同類型Vtg的簡并引物，獲得相應基因片段；在此基礎上，采用SMART RACE技術獲得5’和3’端未知序列；最后設計全長上下游引物，克隆獲得相應的全長cDNA序列。用Macvector軟件確認完整開放閱讀框(ORF)并分析其各功能域。通過完整氨基酸序列比對建立系統分析圖，確定其Vtg類型。
在實施過程中，克隆cDNA全長序列的技術方案可采用如下具體步驟：
1、動物組織器官樣品采集：
采集新鮮的懷卵彈涂魚雌魚的肝臟，剪碎后置于RNAlater中保存用于總RNA提取。
2、彈涂魚總RNA的提取及反轉錄擴增
按照ISOGEN II試劑盒操作說明提取總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凝膠電泳檢測總RNA完整性；用Thermo Fisher NanoDrop2000分光光度計測定總RNA濃度及純度。
(8)將上述總RNA按照VILOTM cDNA Synthesis Kit(Invitrogen)反轉錄成cDNA模板。
注：以上所述離心管，勻漿棒等均為RNase free處理。
2、簡并引物擴增各型Vtg基因片段
Vtg基因屬在序列上具有保守性，根據庸鰈、真鯛、鯔魚、食蚊魚、矛尾刺蝦虎魚三種vtg基因即VtgAa/VtgAb/VtgC基因CDS序列，用Blast進行序列比對，根據Vtg基因的保守序列分別設計三對簡并引物，序列如下：
PCR擴增反應體系為10μl體系：F和R端引物(10μM)各1μl，cDNA模板0.5μl，ddH2O2.5μl，Master Mix 5μl，反應條件為95℃預變性5min，35個循環(95℃，30s；54℃，30s；72℃，1min)，72℃延伸10min。
4、PCR產物的回收和純化
取5μl PCR反應物進行1.5％的瓊脂糖凝膠電泳，用于確認中間序列擴增產物。將目標條帶產物用GENECLEAN Turbo Kit(MP Biomedicals Europe，France)割膠純化回收。
5、PCR擴增目的片段的連接轉化
(1)連接：在0.2ml PCR管中加入PCR產物3μl，pGEM-T EasyVector(Promega，USA)1μl，T4ligase 1μl，Buffer 5μl，輕輕混勻后，4℃靜置過夜。
(2)轉化：加連接產物5μl于大腸桿菌DH5d感受態細胞中，輕彈混勻，冰浴30min；42℃熱激45s，立刻置于冰上2min；涂平板，將培養皿倒置放入37℃培養箱過夜。
6、陽性克隆的鑒定、測序及序列分析
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，無菌水4ul，用無菌牙簽取單一克隆后放入上述PCR管中，使之充分接觸后進行菌落PCR驗證。PCR反應條件如下：95℃預變性5min；35個循環(95℃變性30s，50℃退火30s，72℃延伸1min)；72℃延伸10min。確認包含目的片段的克隆，擴大培養，用T7和SP6引物測序。結果分別獲得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擴增彈涂魚Vtg5’端和3’端基因序列
利用Macvector設計RACE引物如下：
按照RACE 5’/3’Kit(Clontech)說明書進行，以彈涂魚總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反應條件如下：5個循環(94℃，30s；72℃，3min)，5個循環(94℃，30s；70℃，30s；72℃，3min)，25個循環(94℃，30s；68℃，30s；72℃，3min)。
5’RACE和3’RACE進行PCR反應后連接轉化步驟同5。
8、擴增Vtg三型基因全長序列
根據RACE法獲得的3’端和5’端cDNA序列，分別設計特異性全長引物如下：
配置如下PCR反應液：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.至總反應體積為50μl。PCR反應條件如下：95℃預變性1min；5個循環(95℃，30s；68℃，3min)，25個循環(94℃，30s；68℃，30s；72℃，3min)，68℃延伸3min。
PCR產物用1％瓊脂糖凝膠電泳檢測擴增效果如圖1所示，由圖1可知，以cDNA為模板，分別以SEQ ID19和SEQID20引物擴增獲得了預期的全長5000bp左右的VtgAa條帶；以SEQ ID21和SEQID22引物擴增獲得了預期的全長5000bp左右的VtgAb條帶；以SEQ ID23和SEQID24引物擴增獲得了預期的全長4000bp左右的VtgC條帶。其中，M：Marker，1：VtgAa，2：VtgAb，3：VtgC。
9、實時熒光定量PCR分析各型Vtg基因表達差異
分別從大腸桿菌中提取含有各型Vtg基因片段的重組質粒，在分光光度計上測量其DNA濃度和純度。以制備的質粒cDNA(目標基因和內參基因)為模板，稀釋成101至106拷貝數6個梯度，每個梯度2個重復，制作目的基因Vtg和ef1-α(內參)基因的標準曲線。根據標準曲線計算出目的基因的實際絕對定量。
實時熒光定量PCR反應采用Power SYBR Green PCR Master Mix(Applied Biosystems，USA)配置20μl反應體系：2μl cDNA，F and R primer(終濃度200nM)，SYBR Green Mix(10μl)，N.F.W。同時進行無反轉錄樣本對照和組間對照。PCR反應條件為：1個循環(50℃，2min；95℃，10min)，40個循環(95℃，15s；60℃，1min)。
根據已得到的3型卵黃蛋白原全長cDNA序列，利用Primer3Plus(http：//www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/)設計如下引物進行實時熒光定量PCR反應。
隨著卵巢的發育，Vtg濃度呈現出遞增的趨勢(圖2)，特別在卵黃形成中期至完全成熟期，表現出急速增長趨勢。從表達量來看，vtgAa＞vtgAb＞vtgC。
SEQUENCE LISTING
<110> 上海海洋大學
<120> 彈涂魚多型卵黃蛋白原Vtg基因全長序列及其克隆方法
<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
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