蘇國(guó)茂，林聰左，林貞武，黎幸有，梁日深，黃運(yùn)銀

( 1.仲愷農(nóng)業(yè)工程學(xué)院 動(dòng)物科技學(xué)院，廣東 廣州 510225；2.湖北生物科技職業(yè)學(xué)院，湖北 武漢 430070 )

蘇國(guó)茂1，林聰左1，林貞武1，黎幸有1，梁日深1，黃運(yùn)銀2

( 1.仲愷農(nóng)業(yè)工程學(xué)院 動(dòng)物科技學(xué)院，廣東 廣州 510225；2.湖北生物科技職業(yè)學(xué)院，湖北 武漢 430070 )

近年來，隨著分子生物技術(shù)的快速發(fā)展，應(yīng)用合適的線粒體DNA及核DNA分子標(biāo)記技術(shù)可在基因水平上有效解決魚類形態(tài)分類上懸而未決的難題[9-12]。線粒體DNA中細(xì)胞色素C氧化酶亞基Ⅰ(COⅠ)基因進(jìn)化適中，已被驗(yàn)證為魚類物種鑒定、分子系統(tǒng)發(fā)育研究中可靠的遺傳標(biāo)記。同時(shí)，COⅠ基因5′端一段長(zhǎng)度為648 bp的片段，能在基因水平上成功區(qū)分物種，已被廣泛應(yīng)用于物種的分子鑒定[13]。重組激活基因(RAGs)是脊椎動(dòng)物特異性免疫反應(yīng)的關(guān)鍵基因，它由RAG1和RAG2兩個(gè)基因組成，在引起V(D)J基因重排及淋巴系細(xì)胞發(fā)育過程中起到關(guān)鍵性作用[14-15]。其中，RAG2是可靠的核基因遺傳標(biāo)記，目前已經(jīng)廣泛應(yīng)用于硬骨魚類系統(tǒng)進(jìn)化分析的研究[16-18]。

1 材料與方法

1.1 材料來源

表1 試驗(yàn)材料的種類和采集地

1.2 總基因組DNA的提取

基因組DNA采用動(dòng)物組織DNA提取試劑盒(天根生化科技有限公司)進(jìn)行提取。提取的DNA溶解于100 μL滅菌水中，1%瓊脂糖凝膠電泳檢測(cè)提取質(zhì)量，-20 ℃保存?zhèn)溆谩?/p>

1.3 PCR擴(kuò)增及序列測(cè)定

[16, 20]，選擇用于擴(kuò)增COⅠ基因及RAG2基因片段序列的引物分別為:

COⅠ-F1: 5′-TCA ACY AAT CAY AAA GAT ATY GGC AC -3′；

COⅠ-R1: 5′-ACT TCY GGG TGR CCR AAR AAT CA -3′[20]；

RAG2-F1: 5′-GAG GGC CAT CTC CTT CTC CAA-3′；

RAG2-R3: 5′-GAT GGC CTT CCC TCT GTG GGT AC-3′[16]。

PCR反應(yīng)體系為50 μL，其中10×buffer 5 μL，dNTPs (各2.5 mmol/L)2 μL，上下游引物各1 μL，Ex Taq酶(1 U/μL) 2 μL。PCR反應(yīng)條件為：

COⅠ基因：94 ℃ 5 min，94 ℃ 30 s，60 ℃ 60 s，72 ℃ 90 s，35個(gè)循環(huán)，72 ℃ 10 min；

RAG2基因：94℃ 5 min，94 ℃ 30 s，55 ℃ 60 s，72℃ 90 s，35個(gè)循環(huán)，72 ℃ 10 min。

PCR產(chǎn)物經(jīng)1.5%瓊脂糖凝膠電泳分離，純化后送上海英駿生物技術(shù)有限公司進(jìn)行測(cè)序。

1.4 數(shù)據(jù)分析

測(cè)序所得DNA序列利用Clustal W[21]進(jìn)行比對(duì)，去除冗余序列。利用 Mega 5.0[22]計(jì)算序列的堿基組成及變異情況，基于Kimura′s 2-parameter模型計(jì)算各物種的遺傳距離。利用最大似然法分別基于COⅠ、RAG2及COⅠ+RAG2整合序列，以鱸形目裸頰鯛科的星斑裸頰鯛及金線魚科的深水金線魚作為外類群進(jìn)行分子系統(tǒng)進(jìn)化樹的構(gòu)建，進(jìn)化樹采用重復(fù)抽樣分析1000次的方法檢驗(yàn)各分支的置信度。

2 結(jié)果與分析

2.1 序列分析

RAG2基因部分序列比對(duì)分析得到一致序列為729 bp，編碼243個(gè)氨基酸。堿基A、T、G、C平均含量分別為23.9%、28.2%、21.6%和26.3%，4種堿基含量相差不大，分布平均。其中A+T的含量(52.1%)高于G+C含量(47.9%)。密碼子第1、2位，4種堿基含量均相差不大，而密碼子第3位，C-3含量最高，為36.1%，A-3含量最低，為16.8%，表現(xiàn)出反A偏倚。序列保守位點(diǎn)613個(gè)，約占84.4%；變異位點(diǎn)114個(gè)，約占15.7%；簡(jiǎn)約性信息位點(diǎn)85個(gè)，約11.7%。由此可知，與CO Ⅰ基因相比，RAG2基因相對(duì)保守，物種間序列差異較小。

COⅠ基因與RAG2基因序列中轉(zhuǎn)換與顛換之比值分別為2.61與3.12 (Kimura 2-parameter模型)，兩個(gè)基因序列轉(zhuǎn)換明顯大于顛換，顯示其位點(diǎn)沒有突變飽和，可將兩基因序列所有轉(zhuǎn)換與顛換信息應(yīng)用于系統(tǒng)發(fā)育樹的構(gòu)建。同時(shí)，COⅠ及RAG2兩基因同質(zhì)性檢驗(yàn)分析得出P=1.0，說明兩基因之間不存在異質(zhì)性，可將兩序列合并進(jìn)行系統(tǒng)進(jìn)化分析。

2.2 種間及種內(nèi)的遺傳距離

2.3 科魚類分類及分子系統(tǒng)樹

表2 基于 Kimura-2-Parameter模型計(jì)算7種科魚類COⅠ基因種間遺傳距離

表3 基于 Kimura-2-Parameter模型計(jì)算7種科魚類RAG2基因種間遺傳距離

圖2 7種科魚類基于COⅠ序列利用最大似然法構(gòu)建的分子系統(tǒng)進(jìn)化樹

圖3 7種科魚類基于RAG2序列利用最大似然法構(gòu)建的分子系統(tǒng)進(jìn)化樹

圖4 7種科魚類基于COⅠ與RAG2合并序列利用最大似然法構(gòu)建的分子系統(tǒng)進(jìn)化樹

3 討 論

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MolecularPhylogeneticAnalysisofFishesinTeraponidaefromChinaSeaBasedonMitochondrialCOⅠandNuclearRAG2GeneSequences

SU Guomao1, LIN Congzuo1, LIN Zhenwu1, LI Xingyou1, LIANG Rishen1, HUANG Yunyin2

( 1.College of Animal Technology,Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; 2.Hubei Vocational College of Bio-Technology, Wuhan 430070, China )

The 651 bp of mitochondrial DNA COⅠ sequences and 726 bp of nuclear DNA RAG2 sequences from 25 individuals of 5 Teraponidae fish collected from China Sea were determined in this study. No insertion and deletion existed in the two gene fragments. Base composition and genetic distances among and within species were calculated using Mega 5.0 and the molecular phylogenetic trees were constructed using maximum likelihood methods. The results showed that in the molecular phylogenetic tree,Teraponputawas first separated and rooted at the base of the tree, the next was speciesT.jarbua, which also formed an independent branch and located outside the other Teraponidae clusters. ThreeTeraponspecies did not formed an monophyletic group, the lastTeraponspeciesT.therapswas clustered together withRhynchopelatesoxyrhynchusandMesopristescancellatus;HelotessexlineatusandPelatesquadrilineatuswere clustered tightly as sister species, indicating that there were close phylogenetic relationships between the two species. These phylogenetic results might in accordance with the view of Lee and Tsai′s report thatT.therapsshould be removed from genusTeraponandHeloteswas combined withPelates.

Phylogeny; COⅠ gene; RAG2 gene; Teraponidae

2016-12-20；

2017-02-27.

廣東省自然科學(xué)基金資助項(xiàng)目(2016A030310236)；廣東省教育廳青年創(chuàng)新人才類項(xiàng)目(2014KQNCX16)；廣東省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目(201611347067).

蘇國(guó)茂(1992-)，男，碩士研究生；研究方向：水產(chǎn)動(dòng)物種質(zhì)資源.E-mail：1091109070@qq.com.通訊作者：黃運(yùn)銀(1972-)，男，工程師，碩士；研究方向：水產(chǎn)養(yǎng)殖.E-mail：379830153@qq.com.

S917

A

1003-1111(2017)05-0652-06