史 寶 孫冉冉, 2 柳學(xué)周, 2 徐永江 姜 燕 王 濱 張正榮

史 寶1孫冉冉1, 2柳學(xué)周1, 2①徐永江1姜 燕1王 濱1張正榮1

(1. 青島海洋科學(xué)與技術(shù)試點國家實驗室海洋漁業(yè)科學(xué)與食物產(chǎn)出過程功能實驗室 農(nóng)業(yè)農(nóng)村部海洋漁業(yè)可持續(xù)發(fā)展重點實驗室 中國水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所 青島 266071; 2. 大連海洋大學(xué)水產(chǎn)與生命學(xué)院 大連 116023)

肌纖維體積決定了肌肉的生長發(fā)育, 骨骼肌纖維粗肌絲的基本組成單位是肌球蛋白。肌球蛋白是動物體內(nèi)一種重要的功能性馬達(dá)蛋白, 在肌肉運動、細(xì)胞器運動以及肌肉收縮和信號傳導(dǎo)等方面發(fā)揮著重要調(diào)控作用(Heissler, 2016)。肌球蛋白是由2條肌球蛋白重鏈(MyHC)和4條肌球蛋白輕鏈(MyLC)組成。MyHC具有ATP酶活性, 可將其水解為ADP釋放能量, 并且含有肌動蛋白結(jié)合位點, 為肌肉收縮提供動力從而直接影響魚類的生長(Knight, 2000; Campion, 2012)。與哺乳動物肌肉生長的模式不同, 魚類肌纖維生長模式始終存在增生和增粗, 魚類這種生長現(xiàn)象被稱為非限制性生長。研究表明表達(dá)量高低影響肌纖維的增生, 與魚類的非限制性生長有關(guān)(Biga, 2009; 陳之航等, 2017)。在翹嘴鱖(),在快長組與慢長組實驗魚中的差異表達(dá)提示其在翹嘴鱖胚胎及早期生長發(fā)育過程中發(fā)揮重要作用(陳之航等, 2017)。對大西洋鮭()的研究表明魚體特定生長率與表達(dá)量的高低有關(guān)(Hevr?y, 2006)。對脊尾白蝦()的研究發(fā)現(xiàn)在仔蝦第一天的表達(dá)量顯著高于胚胎發(fā)育的其他時期(王佳佳等, 2019)。

1 材料與方法

1.1 材料及取樣

1.2 總RNA的提取與cDNA第一鏈的合成

1.3 MyHC基因cDNA的RACE克隆

根據(jù)克隆得到的核心片段序列及RACE試劑盒要求設(shè)計RACE特異引物(表1)。5′RACE反應(yīng)的操作參照SMARTerTMRACE cDNA Amplification Kit試劑盒方法進(jìn)行。5′RACE的首次PCR反應(yīng)體系為25μL: 10×Advantage 2 PCR Buffer 2.5μL, 50×dNTP Mix 2μL, 50×Advantage 2 Polymerase Mix 0.5μL, 5′RACE模板cDNA 1μL, 正向引物UPM 1μL、反向引物MyHC-5′-R1 0.5μL, PCR-Grade Water 17.5μL。PCR反應(yīng)條件為94°C 30s, 65°C 30s, 72°C 3min, 共25個循環(huán)。取首次PCR產(chǎn)物, 加入Tricine-EDTA buffer進(jìn)行10倍稀釋后作為巢式PCR的模板, NUP為正向引物、MyHC-5′-R2為內(nèi)側(cè)反向引物, 進(jìn)行目標(biāo)基因的5′RACE第二次擴增, PCR反應(yīng)條件同上。3′RACE的操作參照SMARTerTMRACE cDNA Amplification Kit試劑盒方法進(jìn)行, 3′RACE的PCR反應(yīng)體系及反應(yīng)條件與5′RACE相似。RACE產(chǎn)物純化、克隆和測序方法同上。

1.4 反轉(zhuǎn)錄實時熒光定量PCR(qRT-PCR)

Tab.1 Sequence of specific primers used for cloning and quantitative real-time PCR analyses in S. aureovittata

1.5 序列結(jié)構(gòu)及系統(tǒng)進(jìn)化分析

1.6 數(shù)據(jù)處理

實驗數(shù)據(jù)用平均值±標(biāo)準(zhǔn)差表示, 并用 Origin 8.0作圖。統(tǒng)計分析采用單因素方差分析(One-way ANOVA), 使用SPSS 19.0軟件中Duncan's Multiple Range Test 比較基因相對表達(dá)水平的差異, 以<0.05作為顯著性差異。

2 結(jié)果

2.1 MyHC基因序列與結(jié)構(gòu)特征

2.2 MyHC的氨基酸序列比對及系統(tǒng)進(jìn)化樹分析

注：方框表示Src同源區(qū); 下劃線表示MYSc-class II; 雙下劃線表示Myosin tail1

圖2 黃條與其他動物MyHC tail1結(jié)構(gòu)域氨基酸序列的多重比較

2.3 MyHC組織表達(dá)及早期發(fā)育階段的表達(dá)分析

在胚胎發(fā)育的各個時期都能檢測到的表達(dá), 其中在16細(xì)胞期之前表達(dá)量較高, 隨后顯著下降(<0.05); 從原腸胚早期表達(dá)量開始再次升高, 胚體下包2/3期表達(dá)量顯著升高, 至孵化期表達(dá)量達(dá)到峰值(<0.05)(圖5)。在仔稚幼魚時期,在孵化后的20d之前表達(dá)水平較低, 20d后表達(dá)量顯著升高(<0.05), 在30d表達(dá)水平達(dá)到峰值, 隨后的35d到40d表達(dá)水平略有下降但仍保持較高表達(dá)趨勢至40d, 之后表達(dá)量顯著下降但仍維持在較高水平(<0.05) (圖6)。

圖3 基于MyHC氨基酸序列的系統(tǒng)進(jìn)化樹(NJ法, bootstraps=1000)

圖4 黃條MyHC在各組織的相對表達(dá)量

注：BR. 腦; P. 垂體; L. 肝臟; M. 肌肉; SP. 脾臟; K. 腎臟; GI. 鰓; H. 心臟; ST. 胃; I. 腸; HK. 頭腎;

柱上不同小寫字母表示顯著差異(<0.05)

3 討論

圖5 黃條MyHC在胚胎發(fā)育時期的表達(dá)

注：1. 受精卵; 2. 2細(xì)胞; 3. 4細(xì)胞; 4. 8細(xì)胞; 5. 16細(xì)胞; 6. 32細(xì)胞; 7. 多細(xì)胞; 8. 桑椹胚; 9. 高囊胚; 10. 低囊胚; 11. 原腸胚早期; 12. 原腸胚中期; 13. 原腸胚末期; 14. 神經(jīng)胚; 15. 胚體下包1/2; 16. 胚體下包2/3; 17. 胚體全包; 18. 孵化期; 柱上不同小寫字母表示顯著差異(<0.05); 以受精卵mRNA表達(dá)量為標(biāo)準(zhǔn)1

圖6 黃條MyHC在仔稚幼魚發(fā)育階段的表達(dá)

注：柱子上不同小寫字母表示顯著差異 (<0.05); 以孵化后1d表達(dá)量為標(biāo)準(zhǔn)1

4 結(jié)論

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MOLECULAR CLONING AND mRNA EXPRESSION OFGENE IN THE EARLY DEVELOPMENT OF YELLOWTAIL KINGFISH

SHI Bao1, SUN Ran-Ran1, 2, LIU Xue-Zhou1, 2, XU Yong-Jiang1, JIANG Yan1, WANG Bin1, ZHANG Zheng-Rong1

(1. Laboratory for Marine Fisheries Science and Food Production Processes Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; 2. College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China)

The myosin heavy chain (MyHC) is one of the major structural and contractile proteins of muscle. Study on the early growth and development and gene resources is relatively lacking in yellowtail kingfishTo understand the regulatory roles ofin the early growth and development of, the full-length cDNA sequences ofwas cloned by the RACE technology. The real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the expression patterns ofin different tissues, embryonic development stages, and larval and juvenile stages. The full length of the MyHC cDNA sequence was 6143bp, and the open reading frame was 5811bp, encoding 1936 amino acids. Additionally, common features were found in theof, including MYSc-class II, myosin tail l and SH3 domain. Molecular phylogenetic analysis confirmed thatwas closely related to greater amberjack.was expressed in various tissues, but it had the highest expression in muscle. Developmentally, there was a gradual increase inexpression before the 16-cell stage. And the expression ofincreased significantly in the embryo encircling 2/3 of yolk sac, and reached the peak in the hatching larva (<0.05). In the larval and juvenile stages,was significantly up-regulated on 20 days post hatching (dph), with peak expression occurring on 30 dph and decreased slightly between 35 and 45 dph (<0.05). Theexpression ofshowed a development-stage–specific characteristic. Therefore,plays a regulatory role in the embryo and early developmental stage of.

;; embryonic development; larval and juvenile development; gene cloning; expression analysis

* 青島海洋科學(xué)與技術(shù)國家實驗室海洋漁業(yè)科學(xué)與食物產(chǎn)出過程功能實驗室開放課題, 2017-3A01號; 國家重點研發(fā)計劃項目, 2018YFD0901204號, 2019YFD0900503號; 國家自然科學(xué)基金項目, 31772829號; 中國水產(chǎn)科學(xué)研究院院級基本科研業(yè)務(wù)費-農(nóng)業(yè)部海洋漁業(yè)可持續(xù)發(fā)展重點實驗室開放課題資助, 2019HY-XKQ01號; 國家海水魚產(chǎn)業(yè)技術(shù)體系項目, CARS-47號。史 寶, 博士, 副研究員, E-mail: shibao@ysfri.ac.cn

柳學(xué)周, 研究員, E-mail: liuxz@ysfri.ac.cn

2019-11-06,

2020-01-06

S917.4

10.11693/hyhz20191100205