CN102477429A - 一种拟穴青蟹etfb基因及其克隆、检测方法和应用 - Google Patents
一种拟穴青蟹etfb基因及其克隆、检测方法和应用 Download PDFInfo
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- CN102477429A CN102477429A CN201110382739XA CN201110382739A CN102477429A CN 102477429 A CN102477429 A CN 102477429A CN 201110382739X A CN201110382739X A CN 201110382739XA CN 201110382739 A CN201110382739 A CN 201110382739A CN 102477429 A CN102477429 A CN 102477429A
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Abstract
本发明涉及蟹类主要代谢器官中表达的基因序列，具体是一种拟穴青蟹ETFB基因及其克隆、检测方法和应用。本发明通过5’-RACE和3’-RACE技术克隆得到拟穴青蟹ETFB基因的cDNA全长序列，本发明基因的获得可以为后续通过RNAi，基因表达实验研究基因功能以及通过真核表达纯化获得具有生物活性的蛋白质，从结构生物学角度研究所述基因转录表达的蛋白产物提供前提条件，并最终为阐明蟹类ETFB基因及其表达的蛋白产物在线粒体电子传递链和能量代谢的功能及其发生机制奠定基础。
Description
技术领域
本发明涉及蟹类主要代谢器官中表达的基因序列，更具体涉及一种拟穴青蟹电子转移黄素蛋白β亚基基因ETFB的核苷酸序列、氨基酸序列及其克隆方法、检测方法和应用。
背景技术
黄素蛋白是一类以黄素核苷酸作为辅酶或辅基、参与线粒体呼吸链的组成，通过辅酶或辅基可接受和提供两个质子和两个电子，起到氧化还原酶或电子传递体的作用。电子转移黄素蛋白（Electron Transfer Flavoprotein，ETF）是一类以黄素腺嘌呤二核苷酸（flavin adenine dinucleotide，FAD）为辅酶的蛋白质。目前ETF已经成功地从哺乳动物如人、鼠和猪中，以及一些细菌中如脱氮副球菌、埃氏巨球形菌和食甲基嗜甲基菌中鉴定出来。在这些生物中，ETF是由30KDa的α亚基和28KDa的β亚基构成异源二聚体，每个亚基包含一个黄素腺嘌呤二核苷酸（flavin adenine dinucleotide，FAD）和一个腺苷一磷酸（Adenosine Monophosphate，AMP）。ETF是线粒体和细菌异化途径及呼吸链中至少11种脱氢酶的重要电子传递体，典型途径为通过ETF-泛醌氧化还原酶将电子从最初的脱氢酶转移至泛醌，以保证电子传递链持续运转产生质子势能。
蟹类生长所需的能量代谢受多种内外因素的调节，归根结底，它是受到相关基因的控制调节。在有关哺乳类研究中表明：ETF为核基因组编码，在细胞质内合成后再转移至线粒体加工处理为成熟肽，通过ETF-泛醌氧化还原酶将电子转移至泛醌，使得多种一级脱氢酶和泛醌-细胞色素C发生联系，从而保证电子传递链的运转生成生命活动的所需能量。在细菌中的研究也发现类似的机制。在蟹类中，目前还没有鉴定出电子传递黄素蛋白基因，如能找到呼吸链中联系电子转移和脱氢酶中的基因，则可以通过多种研究手段，如RNA干扰（RNAi），基因过表达，启动子分析，真核表达与具生物活性蛋白质分离纯化，体外生化功能分析，从而为阐明蟹类ETFB基因在电子传递机制和能量代谢的功能及其发生机制奠定理论基础。
发明内容
本发明旨在提供一种通过5’-RACE和3’-RACE技术克隆得到拟穴青蟹ETFB基因，还提供了所述ETFB基因的克隆方法、检测方法和应用。
为实现本发明的发明目的，发明人提供如下技术方案：
一种拟穴青蟹ETFB基因，具有如SEQ ID No. 1所示的核苷酸序列。
一种拟穴青蟹ETFB基因，它编码的蛋白质具有如SEQ ID No.2所示的氨基酸序列。
本发明所述的拟穴青蟹电子转移黄素蛋白β亚基基因（即拟穴青蟹ETFB基因）是从拟穴青蟹的肝胰脏中鉴定出的一种电子转移黄素蛋白β亚基基因。所述的电子转移黄素蛋白β亚基基因是通过5’-RACE和3’-RACE技术克隆得到的一个基因，其cDNA核苷酸序列和编码的蛋白质序列如下。该基因cDNA全长1030bp，自18bp到782bp区段为其开放阅读框，编码254个氨基酸，5’端非编码区长17bp，3’端非编码区长248bp，包含1个mRNA半衰调节基序（ATTTA），多聚腺苷酸加尾信号AATAAA和多聚腺苷酸尾巴。在GenBank中进行Blast同源性测定，确定该cDNA编码电子转移黄素蛋白β亚基基因。
拟穴青蟹电子转移黄素蛋白β亚基基因ETFB核苷酸序列如下（SEQ ID NO.1）：
ggccattcaccgatacaATGTCTCTCCGGGTACTTGTTGGCGTCAAGAGAGTGATTGATTATGCCGTGAAGATTCGGGTCCGGCCAGACAAGCTTGGGGTGGTGACAGATGGCGTGAAACACTCCATGAACCCTTTCGATGAGATAGCCATTGAGGAGGCCGTGCGGCTCAAGGAGAAGAAGATTGCAAAGGAGGTGGTGGCTGTGTCATGTGGCCCAACCCAGGCACAGGAGACCATCCGCACTGCCCTGGCCATGGGGGCTGACAGAGGTATCCACGTAGAGATTCCAGCCGAGGAGGTTGAGAAACTAGAACCATTGCATGTAGCCAAGATTATGGCCAAGCTGGTGGAGCAGGAGAAGGCTGACTTAGTTGTGCTTGGCAAACAGGCTATTGATGATGACTCCAATGCCACTGCCCAGATGACAGCTTCCATCCTTGACTGGCCTCAGGCAACCTTTGCTTCCAAGATTGAGAAGACTGATGGTGAGCTGCAGGTGACAAGAGAGGTGGATGGAGGTCTGGAGACAATCAAAGTGAAGTTGCCAGCTGTGGTCTCAGCTGATCTCCGCCTCAATGAGCCCCGATATGCCACCCTGCCCAACATCATGAAAGCTAAGAAGAAGAAAATTGCTAAGATGAAGGCTGCTGACCTTGGAGTAGATACAACTTCTCACTTTGAAGTTTTGGAGGTGGCTGATCCACCTGTGAGGGAGGCTGGCATCAAAGTGGAGGATGTGGACACTCTTATTACAAAGTTGAAGGAGACTGGGCGCATTTAGcacagttaatcggggacgtacagtgtgactgtggttggaggaattgcaccatacctgtacacctgccaccaatcaatgcaatatacctattttctttgtgggcaagctatccttaatgaatgtgctacttttcagagatttctttaatacacaatctgtatttatattttcttgtacaaaacattctatagaaacaggtttaataaaatatataaaacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa。
拟穴青蟹电子转移黄素蛋白β亚基基因ETFB编码的氨基酸序列如下（SEQ ID NO.2）：
MSLRVLVGVKRVIDYAVKIRVRPDKLGVVTDGVKHSMNPFDEIAIEEAVRLKEKKIAKEVVAVSCGPTQAQETIRTALAMGADRGIHVEIPAEEVEKLEPLHVAKIMAKLVEQEKADLVVLGKQAIDDDSNATAQMTASILDWPQATFASKIEKTDGELQVTREVDGGLETIKVKLPAVVSADLRLNEPRYATLPNIMKAKKKKIAKMKAADLGVDTTSHFEVLEVADPPVREAGIKVEDVDTLITKLKETGRI。
本发明还提供了一种拟穴青蟹ETFB基因的克隆方法，所述的克隆方法为利用5’-RACE和3’-RACE技术克隆拟穴青蟹电子转移黄素蛋白β亚基基因cDNA全长，包括以拟穴青蟹肝胰脏反转录产物为模板，利用3-GP1和3-GP2引物进行3’-RACE反应，扩增得到拟穴青蟹ETFB的3’端序列；以5-GP1和5-GP2引物进行5’-RACE反应，扩增得到ETFB的5’序列，其中：所述的3-GP1引物具有如SEQ ID No.3所示的核苷酸序列，3-GP2引物具有如SEQ ID No.4所示的核苷酸序列；5-GP1引物具有如SEQ ID No.5所示的核苷酸序列，5-GP2引物具有如SEQ ID No.6所示的核苷酸序列。
本发明还提供了一种拟穴青蟹ETFB基因的检测方法，所述的检测方法至少包括使用一对半定量PCR引物，所述的一对半定量PCR引物由上游引物和下游引物组成，其中，所述的半定量PCR上游引物（ETFB-RT-F）具有如SEQ ID NO.7所示的核苷酸序列；半定量PCR下游引物（ETFB-RT-R）具有如SEQ ID NO.8所示的核苷酸序列。
本发明还提供了所述的一种拟穴青蟹ETFB基因在拟穴青蟹线粒体电子传递链运转中的应用。
本发明通过基于5’-RACE和3’-RACE技术的同源克隆策略，PCR扩增测序以及Blast比对，克隆到拟穴青蟹电子转移黄素蛋白β亚基基因ETFB的全长序列；通过对拟穴青蟹电子转移黄素蛋白β亚基基因ETFB设计有效的表达引物，从而利用半定量PCR技术分析所述基因在正常组织中的表达图谱。发明人运用半定量RT-PCR技术检测了ETFB基因在拟穴青蟹中四个主要器官（肝胰脏、卵巢、心脏和肌肉）中的表达谱，结果显示在所检测的四个器官中均具有较高水平的ETFB基因表达。
与现有技术相比，本发明具有如下优点：
本发明基因的获得可以为后续通过RNAi，基因表达实验研究基因功能以及通过真核表达纯化获得具有生物活性的蛋白质，从结构生物学角度研究所述基因转录表达的蛋白产物提供前提条件，并最终为阐明蟹类ETFB基因及其表达的蛋白产物在线粒体电子传递链和能量代谢的功能及其发生机制奠定基础。
附图说明
图1为半定量RT-PCR检测拟穴青蟹电子转移黄素蛋白β亚基基因ETFB在四个组织中的分布表达。
具体实施方式
下面结合实施例和说明书附图，更具体地说明本发明的内容。应当理解，本发明的实施并不局限于下面的实施例，对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。
在本发明中，若非特指，所有的份、百分比均为重量单位，所有的设备和原料等均可从市场购得或是本行业常用的。若无特别指明，实施例采用的方法为本领域通用技术。
主要材料、试剂和仪器设备：手术刀，眼科剪，镊子，解剖盘，充氧泵，水箱，1.5ml离心管，微量移液器、微量移液器枪头，一次性培养皿，陶瓷研钵。脱氧核糖核苷酸dNTP，热聚合Taq酶，Trnzol-A+总RNA提取试剂盒，5’-Full RACE Kit（TaKaRa），3’-Full RACE Core Set Ver.2.0 (TaKaRa)， Quant cDNA第一链合成试剂盒以及DH5α感受态细胞，小型离心机（QspinTM，BAYGENE），涡旋振荡器（QL-866型，QILINEBEIER），核酸蛋白仪（SmartSpaceTM Plus，Bio-Rad），超净工作台（SW-CJ-1G型，名牌之星），恒温培养箱（PH070A型，上海一恒科学仪器有限公司），-80℃超低温冰箱（Thermo scientific），荧光定量PCR系统（ABI 7300型），pH计(Mettler Toledo 320PH Meter)、高压蒸气灭菌锅（SANYO）、高速冷冻离心机(Centrifuge 5417R，eppendorf)、电泳仪（DYY-6C型，北京六一）、水浴锅（上海精宏）、凝胶成像系统(Bio-Rad GD2000)、PCR仪(ABI Veriti 96well Thermal Cycler)、自动测序仪（ABI 3730型）。
本发明实施例所用的常规药品氯仿（分析纯），异丙醇（分析纯），氯霉素，氯化钠（分析纯）购自国药集团，异丙基硫代-β-D-半乳糖苷（IPTG），5-溴-4-氯-3-吲哚-β-D-半乳糖苷（X-gal），胰蛋白胨，酵母提取物，琼脂糖，溴化乙锭，焦碳酸二乙酯（DEPC）等购自Takara公司，液氮为浙江省舟山亿洋气体有限公司产品，脱氧核糖核苷酸dNTP，热聚合Taq酶，Trnzol-A+总RNA提取试剂盒，RealMasterMix(SYBRGreen)试剂，Quant cDNA第一链合成试剂盒以及DH5α感受态细胞购自TIANGEN公司，SMARTTM cDNA Library Construction Kit购自Clontech公司，质粒文库测序由华大基因公司完成，引物由南京金斯瑞生物科技有限公司合成。
实验样本拟穴青蟹购自浙江省舟山市定海区南珍菜场。
实施例中未注明具体条件的实验方法，是按照常规条件，例如Sambrook等作者的分子克隆实验室手册（New York：Cold Spring Harbor Laboratory Press，1989）中所述的条件，或按照制造厂商说明书建议的条件。
实施例1
1.利用Trnzol-A+总RNA提取试剂盒（TIANGEN）提取拟穴青蟹肝胰脏总RNA。
取健康的拟穴青蟹（体重约为500g）在充氧水箱养殖7日后，解剖蟹体取出肝胰脏、卵巢、心脏和肌肉，液氮速冻放入-80℃冰箱备用。分别取备用的肝胰脏组织约100mg用液氮研磨后加入1ml的Trnzol-A+试剂匀浆（TIANGEN），按照该试剂盒操作手册所示实验方法提取总RNA。取2μl提取的总RNA，分别用分光光度计和电泳检测总RNA浓度和质量。
2.利用5’-RACE和3’-RACE技术克隆拟穴青蟹电子转移黄素蛋白β亚基基因cDNA全长序列。
通过比对其他物种电子转移黄素蛋白β亚基基因ETFB的核苷酸序列，找到保守区段，作为模板设计5-GP1/5-GP2引物和3-GP1/3-GP2引物。5’-RACE和3’-RACE实验使用5’-Full RACE Kit (TaKaRa)和3’-Full RACE Core Set Ver.2.0 (TaKaRa)试剂盒。具体操作方法见试剂盒说明书，简要如下:
以拟穴青蟹肝胰脏反转录产物为模板，利用3-GP1:ATGGGBGCWGACMGAGGYATCCACGT (SEQ ID No.3)和3-GP2:TGCCCAACATCATGAAAGCYAAGAAGA(SEQ ID No.4)进行3’-RACE反应，扩增得到ETFB的3’端序列；以5-GP1:CCTTTGATGCCAGCCTCCCTCACAGGT（SEQ ID No.5） 和5-GP2 :GGAGATCAGCTGAGACCACAGCTGGC（SEQ ID No.6）进行5’-RACE反应，扩增得到ETFB的5’序列。
3.对扩增得到RACE-PCR 产物克隆测序并拼接获得ETFB基因cDNA全长序列。
以1.%的琼脂糖凝胶电泳检测扩增产物并利用琼脂糖凝胶DNA回收试剂盒（TIANGEN）纯化回收PCR产物。将纯化回收的PCR产物同pGEM-T载体（Promega）在4℃条件下连接过夜，连接体系按说明书配制。将过夜连接产物转化入DH5α感受态细胞中。涂平板（内含氯霉素、异丙基硫代-β-D-半乳糖苷（IPTG）及5-溴-4-氯-3-吲哚-β-D-半乳糖苷（X-gal），37℃恒温培养箱中培养过夜。通过蓝白斑筛选含cDNA插入片段的阳性重组子，将挑取的每个单克隆子分别接种到1ml氯霉素抗性的LB培养液中，37℃摇菌2个小时，菌液PCR确认阳性克隆子，剔除假阳性克隆子。将经过菌液PCR鉴定的阳性克隆子寄送华大基因公司测序，测序在ABI 3730测序仪上进行。将所得的5’端序列和3’端序列在MEGA4.1软件（生物软件网，http://www.bio-soft.net/）中进行拼接得到ETFB基因cDNA全长序列。结果如SEQ ID NO.1和SEQ ID NO.2所示。
将测序所得序列在GenBank中进行Blast同源性测定，鉴定出拟穴青蟹电子转移黄素蛋白β亚基基因，该基因cDNA全长1030bp，自18bp到782bp区段为其开放阅读框，编码254个氨基酸，5’端非编码区长17bp，3’端非编码区长248bp，包含1个mRNA半衰调节基序（ATTTA），多聚腺苷酸加尾信号AATAAA和多聚腺苷酸尾巴。
4.正常组织中ETFB基因的半定量表达图谱。
为检测ETFB基因在正常拟穴青蟹主要代谢器官中的分布情况，分别取出保存的四个组织（肝胰脏、卵巢、心脏和肌肉）各约100mg，使用Trnzol-A+总RNA提取试剂盒（TIANGEN）抽提总RNA。取1μg总RNA用Quant cDNA第一链合成试剂盒合成cDNA第一链，作为半定量RT-PCR的模板。根据拟穴青蟹电子转移黄素蛋白β亚基基因ETFB设计一对特异引物，半定量PCR上游引物(ETFB-RT-F):TTCCAGCCGAGGAGGTTGAG(SEQ ID No.7)；半定量PCR下游引物(ETFB-RT-R): TGTTGGGCAGGGTGGCATAT(SEQ ID No.8)，对ETFB基因的cDNA进行扩增；根据已报道的拟穴青蟹β-actin基因作为内参基因（该序列收录于GenBank数据，登录号为HM217821），设计一对特异引物（上游引物(β-actin-RT-F)： TCACCAACTGGGACGACATG(SEQ ID No.9);下游引物(β-actin-RT-R)： ATAGCGTGAGGAAGGGCATA(SEQ ID No.10)。半定量RT-PCR反应体系为：ddH2O 17.3μl，10×PCR Buffer （TIAGEN）2.5μl，dNTP（2.5mM，TIANGEN）2μl，上下游引物各1μl，cDNA模板1μl，Taq酶（5U/μl，TIANGEN）0.2μl，反应总体积为25μl。反应条件设置为：95℃，3分钟；95℃，30秒，55℃，30秒，72℃，30秒，共30个循环；72℃，5分钟。反应结束后，取5μl PCR产物用1.5%的琼脂糖凝胶电泳检测并拍照保存。
发明人运用半定量RT-PCR技术检测了ETFB基因在拟穴青蟹中四个主要器官中的表达谱，结果如图1所示，在所检测的四个器官中具有较高水平的ETFB基因表达，这提示克隆到的ETFB基因确在拟穴青蟹四个主要器官中发挥持续作用，因而具备较高的转录活性和转录水平。
尽管发明人已经对本发明的技术方案做了较为详细的阐述和列举，应当理解，对于本领域一个熟练的技术人员来说，对上述实施例作出修改和/或变通或者采用等同的替代方案是显然的，都不能脱离本发明精神的实质，本发明中出现的术语用于对本发明技术方案的阐述和理解，并不能构成对本发明的限制。
SEQUENCE LISTING
<110> 浙江海洋学院
<120> 一种拟穴青蟹ETFB基因及其克隆、检测方法和应用
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ggccattcac cgatacaatg tctctccggg tacttgttgg cgtcaagaga gtgattgatt 60
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actccatgaa ccctttcgat gagatagcca ttgaggaggc cgtgcggctc aaggagaaga 180
agattgcaaa ggaggtggtg gctgtgtcat gtggcccaac ccaggcacag gagaccatcc 240
gcactgccct ggccatgggg gctgacagag gtatccacgt agagattcca gccgaggagg 300
ttgagaaact agaaccattg catgtagcca agattatggc caagctggtg gagcaggaga 360
aggctgactt agttgtgctt ggcaaacagg ctattgatga tgactccaat gccactgccc 420
agatgacagc ttccatcctt gactggcctc aggcaacctt tgcttccaag attgagaaga 480
ctgatggtga gctgcaggtg acaagagagg tggatggagg tctggagaca atcaaagtga 540
agttgccagc tgtggtctca gctgatctcc gcctcaatga gccccgatat gccaccctgc 600
ccaacatcat gaaagctaag aagaagaaaa ttgctaagat gaaggctgct gaccttggag 660
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agcacagtta atcggggacg tacagtgtga ctgtggttgg aggaattgca ccatacctgt 840
acacctgcca ccaatcaatg caatatacct attttctttg tgggcaagct atccttaatg 900
aatgtgctac ttttcagaga tttctttaat acacaatctg tatttatatt ttcttgtaca 960
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Val Lys Ile Arg Val Arg Pro Asp Lys Leu Gly Val Val Thr Asp Gly
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Val Lys His Ser Met Asn Pro Phe Asp Glu Ile Ala Ile Glu Glu Ala
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Val Arg Leu Lys Glu Lys Lys Ile Ala Lys Glu Val Val Ala Val Ser
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Cys Gly Pro Thr Gln Ala Gln Glu Thr Ile Arg Thr Ala Leu Ala Met
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Gln Ala Thr Phe Ala Ser Lys Ile Glu Lys Thr Asp Gly Glu Leu Gln
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Claims (5)
1.一种拟穴青蟹ETFB基因，具有如SEQ ID No. 1所示的核苷酸序列。
2.一种拟穴青蟹ETFB基因，它编码的蛋白质具有如SEQ ID No.2所示的氨基酸序列。
3.一种拟穴青蟹ETFB基因的克隆方法，其特征在于，所述的克隆方法为利用5’-RACE和3’-RACE技术克隆拟穴青蟹电子转移黄素蛋白β亚基基因cDNA全长，包括以拟穴青蟹肝胰脏反转录产物为模板，利用3-GP1和3-GP2引物进行3’-RACE反应，扩增得到拟穴青蟹ETFB的3’端序列；以5-GP1和5-GP2引物进行5’-RACE反应，扩增得到ETFB的5’序列，其中：所述的3-GP1引物具有如SEQ ID No.3所示的核苷酸序列，3-GP2引物具有如SEQ ID No.4所示的核苷酸序列；5-GP1引物具有如SEQ ID No.5所示的核苷酸序列，5-GP2引物具有如SEQ ID No.6所示的核苷酸序列。
4.一种拟穴青蟹ETFB基因的检测方法，其特征在于，所述的检测方法至少包括使用一对半定量PCR引物，所述的一对半定量PCR引物由上游引物和下游引物组成，其中，所述的半定量PCR上游引物具有如SEQ ID NO.7所示的核苷酸序列；半定量PCR下游引物具有如SEQ ID NO.8所示的核苷酸序列。
5.如权利要求1或2所述的一种拟穴青蟹ETFB基因在拟穴青蟹线粒体电子传递链运转中的应用。
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| CN103667320A (zh) * | 2013-11-04 | 2014-03-26 | 内蒙古科技大学 | 一种拟穴青蟹过氧素基因Sp-PX、其编码的氨基酸序列以及克隆方法 |
| CN115058430A (zh) * | 2022-06-12 | 2022-09-16 | 宁波大学 | 一种拟穴青蟹5-ht2受体基因及其应用 |
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| WO2002002580A2 (en) * | 2000-07-05 | 2002-01-10 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the etfb gene |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103667320A (zh) * | 2013-11-04 | 2014-03-26 | 内蒙古科技大学 | 一种拟穴青蟹过氧素基因Sp-PX、其编码的氨基酸序列以及克隆方法 |
| CN115058430A (zh) * | 2022-06-12 | 2022-09-16 | 宁波大学 | 一种拟穴青蟹5-ht2受体基因及其应用 |
| CN115058430B (zh) * | 2022-06-12 | 2023-06-09 | 宁波大学 | 一种拟穴青蟹5-ht2受体基因及其应用 |
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