CN108060166B - 一种长牡蛎gata2/3启动子及其重组表达载体及应用 - Google Patents
一种长牡蛎gata2/3启动子及其重组表达载体及应用 Download PDFInfo
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Abstract
本发明涉及分子生物学技术，具体的说是一种长牡蛎gata2/3启动子及其重组表达载体及应用。启动子为SEQ ID No.1所示的碱基序列。所述启动子为与SEQ ID No.1所示的碱基序列具有80％或80％以上同源性的碱基序列，且具有启动子功能。所述启动子在制备重组表达载体中的应用。将重组表达载体导入贝类卵细胞后，启动目的基因的表达，实现在贝类幼虫中表达的效果。
Description
技术领域
本发明涉及分子生物学技术，具体的说是一种长牡蛎gata2/3启动子及其重组表达载体及应用。
背景技术
在贝类早期发育研究中，目前尚无可用的启动子。Gata2/3属于GATA 转录因子家族的一个亚家族，在贝类中是一种重要的贝壳发生相关基因。 gata2/3启动子可以在贝类幼虫中驱动报告基因的表达，为贝类的发育研究提供便利。
发明内容
本发明的目的在于提供一种长牡蛎gata2/3启动子及其重组表达载体及应用。
为实现上述目的，本发明采用技术方案为：
一种长牡蛎gata2/3启动子，启动子为SEQ ID No.1所示的碱基序列。
所述启动子为与SEQ ID No.1所示的碱基序列具有80％或80％以上同源性的碱基序列，且具有启动子功能。
一种长牡蛎gata2/3启动子的应用，所述启动子在制备重组表达载体中的应用。
一种长牡蛎gata2/3启动子的应用，所述启动子在贝类幼虫中启动目的基因表达。
所述目的基因是可在细胞中转录或翻译的功能基因或RNA序列，如编码蛋白的各种功能基因(如EGFP)、反义RNA或其它具有调节功能的长 RNA序列(如LnRNA)，在本例应用中是EGFP。
含长牡蛎gata2/3启动子的重组表达载体。
一种含长牡蛎gata2/3启动子的重组表达载体的应用，所述重组表达载体在贝类早期胚胎中启动目的基因中的应用。
本发明所具有的优点：
将重组表达载体导入贝类卵细胞后，启动目的基因的表达，实现在贝类幼虫中表达的效果。
附图说明
图1为本发明实施例提供的pEGFP-1质粒示意图。
图2为本发明实施例提供的pEGFP-1质粒中的多克隆位点图。
图3为本发明实施例提供的注射重组载体的笠贝早期幼虫中gata2/3 启动子驱动GFP表达的结果图，其中，绿色荧光表明该启动子以及重组载体在笠贝早期胚胎中是有效表达的。
具体实施方式
下面结合附图和实施例对本发明做进一步解释说明。
实施例1
启动子扩增：
使用海洋动物组织基因组DNA提取试剂盒(TIANGEN，目录号：DP324)提取长牡蛎(Crassostrea gigas)基因组DNA，根据长牡蛎基因组数据库 (http://www.oysterdb.com)设计长牡蛎基因启动子特异性引物。 (上游引物cg-gata2/3PmF，加16碱基的载体pEGFP-1同源末端1，下游引物cg-gata2/3PmR，加16碱基的载体pEGFP-1同源末端2) 以上述提取的长牡蛎的gDNA为模板，使用Premix TaqTM(LA TaqTM Version 2.0)(Takara，目录号：RR900Q)聚合酶进行PCR扩增。如表 1所示。
表1基因启动子扩增的PCR体系
| 组成 | 体积 |
| Premix Taq(LA TaqVersion 2.0) | 25μl |
| DNA模板 | 2μl |
| 上游引物Cggata2/3PmF(10μM) | 2μl |
| 下游引物Cggata2/3PmR(10μM) | 2μl |
| 超纯水 | 19μl |
PCR扩增程序为：98℃预变性2min；98℃变性20s，58℃退火20s，72 ℃延伸2min30s，进行35个反应循环；72℃延伸7min。
上游引物cg-gata2/3PmF：CGCGGGCCCGGGATCCATTTACAATGTGCGTACTCTGTG，其中下划线代表16碱基的载体pEGFP-1同源末端1；下游引物 cg-gata2/3PmR：GGCGACCGGTGGATCCTTTATTTTAGGTCCAGAAGAAAGAAATC，其中下划线代表16碱基的载体pEGFP-1同源末端2。
PCR扩增产物经1.0％琼脂糖凝胶电泳分离，得到大小为2000bp左右的条带，使用Thermo Scientific的GeneJET Gel Extraction Kit(目录号：K0692)进行纯化回收，即得cggata2/3启动子碱基序列如序列表 1所示。
实施例2
cggata2/3::GFP重组载体构建：
将pEGFP-1质粒(中国科学院海洋研究所张晓军提供，该质粒还可以从优宝生物(http://www.youbio.cn/)订购，货号：VT1106，其碱基序列如序列2中所示)用BamHI酶切线性化，将如上述得到的PCR回收产物 cggata2/3启动与线性化的pEGFP-1进行In-Fusion连接(In-Fusion HD Cloning Kit，TaKaRa，目录号：638909)，转化大肠杆菌，挑取阳性转化子测序(如cggata2/3启动子序列所示)，证明准确，具体为：
In-Fusion连接体系：
| 组成 | 体积 |
| PCR回收产物(cggata2/3启动子) | 1.5μl |
| 超纯水 | 3.5μl |
| 线性化pEGFP-1 | 3μl |
| Infusion Mix | 2μl |
| 总体积 | 10μl |
将上述记载的两片段按照上述表中记载的连接体系混合，50℃水浴中连接15min，经过连接后用感受态细胞DH5α进行转化。取出-80℃保存感大肠杆菌受态，立即静置于冰中；将适量体积的待转化DNA(每100μL感受态加入50ng DNA，体积不要超过感受态细胞体积的5％)与感受态轻轻混匀，冰上静置20min；42℃热激90s，然后冰上放置2-3min。加入900 μL的LB液体培养基(不含抗生素)，37℃150rpm振荡培养1-1.5h； 5000rpm离心3min，弃上清，用100μL LB液体培养基重悬浮菌体。将重悬液涂于卡那霉素抗性LB平板上，37℃倒置培养12-20h。选取单菌落，进行菌落PCR鉴定。空载质粒的产物大小约为100bp，重组载体产物长度约为2200bp。鉴定用引物：pEGFP-1-F,CGAGCTCAAGCTTCGAATTCTG； pEGFP-1-R,CGCCGTCCAGCTCGACCAG。获得含有pEGFP-1+cggata2/3表达载体的重组大肠杆菌，命名为cggata2/3::GFP(参见图1)。对 cggata2/3::GFP表达载体中的cggata2/3启动子进行测序，测序结果如 cggata2/3启动子所示。测序结果表明，获得的cggata2/3::GFP重组表达载体中cggata2/3启动子序列正确。
用无内毒素质粒小提中量试剂盒(目录号：DP118-02)从大肠杆菌 cggata2/3::GFP中提取重组载体cggata2/3::GFP。
实施例3
贝类早期幼虫中GFP的表达
将提取的重组载体cggata2/3::GFP配置成注射液：重组载体50ng/uL, 酚红0.05％。
将性成熟的笠贝放入70ml新鲜海水的培养杯中(每杯一只)，待其产卵排精后，分别收集精子和卵细胞。
将收集的卵细胞转入盛有5ml新鲜海水的培养皿中，放置于解剖镜下。将显微注射仪(WARNER，目录号：PLI-100A)输入气压维持在200pa。用 Microloader(eppendorf，目录号：5242956003)吸取0.5ul注射液注入注射针Femtotip II(eppendorf，目录号：5242957000)，将注射针连接于注射仪。设置注射参数：输入气压200pa，平衡气压3pa，注射压力 15pa，注射时间0.1s。注射体积依据酚红颜色指示，不超过卵体积的5％。将注射后的卵转移至12孔培养板，加入5ml新鲜海水，按体积5％的比例加入备好的精子，室温受精一小时后将精子洗脱，重新加入5ml新鲜海水。培养20hpf后检测重组载体的的表达情况，拍照记录(参见图3)。由图3可见注射重组载体的笠贝幼虫中GFP表达(绿色荧光)，证明该重组载体可以在贝类中启动目的基因表达。
gata2/3启动子序列：该启动子名称cgigata2/3,或简称cggata2/3启动子
ATTTACAATGTGCGTACTCTGTGAAGTAAGTGCATTTTCTAGATTTCCGTTTAATCCCTTTTTTCTCGATAAATG CATGATAATATCATTAAGAAAAATATCTTAGGATTTACAAGATGTTACACAGAACTGATGTATTTTCCTTTTTTA AGTATTCCCTTGGCCTTTTAGGTGCTTCCCAGAGAAATATCGAGAAAACAACACCCTCTAATACGCATGAAAC AATCCGCGGAGATTTTATTTATTTGCATTCGTAAATACCGGCTTTTCTCGCTAATAAGCGCTGGCACGAGTCGC CGCTAACAGGTCTTGATTATGAGTTGATATTTTCCAAATATCTCACAATATCTACTTGTAGTTAATGCCCTTCTA ATAGATATCGACAAAAAAAACCCCGATATCAGCCTTATTTCTCGTCCGATCGGTTCCACAGGTTAATCGGGAA GCATCAGCATTTTCCCCGGAATGAAGTTGGCCGATCATATCAAGGCCGGTAAATAAGAAAGCAGTTCTATCACAGATCTCGGTCTTTGGAATCGGTTGATAAAGGAATGGTGTGTACCTCTCGTGGTAATTGGTTGTATGCGAATG CTTTTATCTGATTCCCATTTTCACCTATTTACCATGTAAACCAAGATATAACACTTATCTCATGACGAGAAACTC CGGAGAAAACTAAAACAAAAATCTCTTTTTTTTCGTGAGTTCATAGAATTTAAAACAAGAGACTTTCATACTT TTATTGTTCTTTTAAATCTCTCTATTTTCAGATATGCATATATTTATTTTTAAAGTTTTGTCCTGGTATTGTTCGTA AATTTTTACATATGAATATAAACCAATATTTAATTCTTTAATAATATCTTGTTCATCTTGAGATAAATTTCATCTACAAATTGGTGTATTTTTGTATTTAAAAGATTAAAACGTGTGTTTCAAATTTACCAAAATTACGCTCAAAGTATTT GATTATCCTCAAATTATATCACACACGCTCAATTTTTCTCTGAGGCAAATATCCAATCATCTCATTGGAGAATTG TTTCTGGAGGCGGAATACTGAATATCAAGCCACGCCCCTCGCGGTTATTCACACCCTCATAGTGTTACGGATG GATAAACTGATTGATATAATTTGTAAATAGAATGGCTTCAGGGGGCGGGGTTTAATTACTCAACAAATCTGATT GGCTCTCTAGGCCTCGTCTCGCCAGCGTACAATGAATATGTAACCATAAGGCGCCGACAAAAAATAGAAAAA CTGAGTGAATTCGTATACATGAAACAAGCAAGCAAACCTATCAGCATATCGAAGTATTCTCTCTAACTGGTCG GATAAAAACATAAGATTAGAATACTTCCAACATTTTGTGTGTGTGGTAAACCTGTGCGATTCTGCAGCATGCA TTTGGGAAGCTTTTGGATAGTAAGTTTAGTTTTATGTGCTGGGTGCTTGAGCGCCGTGAATTTGGTCTACTCTC AATTGGAGGGTTTAGTGAAACAAGTTTGTAAAGCACGTGATCTCTAAGCGGATTGTTTGAGATAAGGAGACT TCATCTCTTGGACATTGAGTCTTATCTTCATCAGTTAAATATTTAATCAGATACTGTTAAAGCAAGGCAGTATTT AACTCCTTAACAGTAACTGTCAATTTCTATATCTGATTCTAAATTTCTTAATTTTGAAGATGCAAATTTAAATTA TAATTCTATATGATAATTCATAATACATTGTTCTTAATTTTTTGTGCATTTGAATTAATCTGTTCTTAGTCTGTTTA TTTATTTTCTTTATTTTATTTTTTCTTTCAGACAAAAGAACAAGTGTGAAAATTCAGAATTTTGTGAAATATTTT ACAGCCTTATTACAAACTGTAAAAACCATATAACTATAGTTTGGTACTCCTATATTGGATTTAACGAAGAAGGA ATTTGGACTTAGAATAAAAAAGAAAATAAAAAACTTGATTTCTTTCTTCTGGACCTAAAATAAA
pEGFP-1载体序列：
TAGTTATTACTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGG CCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC ACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCC GCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGAC GGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCA GAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCG GGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGACTCTAGATCATAATCAGCCATACCAC ATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCA ATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAA TAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTAAATTGTA AGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATC GGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGT CCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACG TGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGG AGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATG CGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTA AATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGA GTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAG CAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATC CCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGC CGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAA AGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGG CCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGT TCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGC AAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTC ACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCT CCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCAT TCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGAT GATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGA CGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCT GGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTG CTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGC GCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGC GACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTT TTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGG AGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGA ATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATA CCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGG GTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCTCAGGTTACTCATATATAC TTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCT TCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAG GTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCATGCAT。
序列表
<110> 中国科学院海洋研究所
<120> 一种长牡蛎gata2/3启动子及其重组表达载体及应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1993
<212> DNA
<213> cggata2/3启动子(cgigata2/3)
<400> 1
atttacaatg tgcgtactct gtgaagtaag tgcattttct agatttccgt ttaatccctt 60
ttttctcgat aaatgcatga taatatcatt aagaaaaata tcttaggatt tacaagatgt 120
tacacagaac tgatgtattt tcctttttta agtattccct tggcctttta ggtgcttccc 180
agagaaatat cgagaaaaca acaccctcta atacgcatga aacaatccgc ggagatttta 240
tttatttgca ttcgtaaata ccggcttttc tcgctaataa gcgctggcac gagtcgccgc 300
taacaggtct tgattatgag ttgatatttt ccaaatatct cacaatatct acttgtagtt 360
aatgcccttc taatagatat cgacaaaaaa aaccccgata tcagccttat ttctcgtccg 420
atcggttcca caggttaatc gggaagcatc agcattttcc ccggaatgaa gttggccgat 480
catatcaagg ccggtaaata agaaagcagt tctatcacag atctcggtct ttggaatcgg 540
ttgataaagg aatggtgtgt acctctcgtg gtaattggtt gtatgcgaat gcttttatct 600
gattcccatt ttcacctatt taccatgtaa accaagatat aacacttatc tcatgacgag 660
aaactccgga gaaaactaaa acaaaaatct cttttttttc gtgagttcat agaatttaaa 720
acaagagact ttcatacttt tattgttctt ttaaatctct ctattttcag atatgcatat 780
atttattttt aaagttttgt cctggtattg ttcgtaaatt tttacatatg aatataaacc 840
aatatttaat tctttaataa tatcttgttc atcttgagat aaatttcatc tacaaattgg 900
tgtatttttg tatttaaaag attaaaacgt gtgtttcaaa tttaccaaaa ttacgctcaa 960
agtatttgat tatcctcaaa ttatatcaca cacgctcaat ttttctctga ggcaaatatc 1020
caatcatctc attggagaat tgtttctgga ggcggaatac tgaatatcaa gccacgcccc 1080
tcgcggttat tcacaccctc atagtgttac ggatggataa actgattgat ataatttgta 1140
aatagaatgg cttcaggggg cggggtttaa ttactcaaca aatctgattg gctctctagg 1200
cctcgtctcg ccagcgtaca atgaatatgt aaccataagg cgccgacaaa aaatagaaaa 1260
actgagtgaa ttcgtataca tgaaacaagc aagcaaacct atcagcatat cgaagtattc 1320
tctctaactg gtcggataaa aacataagat tagaatactt ccaacatttt gtgtgtgtgg 1380
taaacctgtg cgattctgca gcatgcattt gggaagcttt tggatagtaa gtttagtttt 1440
atgtgctggg tgcttgagcg ccgtgaattt ggtctactct caattggagg gtttagtgaa 1500
acaagtttgt aaagcacgtg atctctaagc ggattgtttg agataaggag acttcatctc 1560
ttggacattg agtcttatct tcatcagtta aatatttaat cagatactgt taaagcaagg 1620
cagtatttaa ctccttaaca gtaactgtca atttctatat ctgattctaa atttcttaat 1680
tttgaagatg caaatttaaa ttataattct atatgataat tcataataca ttgttcttaa 1740
ttttttgtgc atttgaatta atctgttctt agtctgttta tttattttct ttattttatt 1800
ttttctttca gacaaaagaa caagtgtgaa aattcagaat tttgtgaaat attttacagc 1860
cttattacaa actgtaaaaa ccatataact atagtttggt actcctatat tggatttaac 1920
gaagaaggaa tttggactta gaataaaaaa gaaaataaaa aacttgattt ctttcttctg 1980
gacctaaaat aaa 1993
<210> 2
<211> 4151
<212> DNA
<213> pEGFP-1载体序列(pEGFP-1)
<400> 2
tagttattac tagcgctacc ggactcagat ctcgagctca agcttcgaat tctgcagtcg 60
acggtaccgc gggcccggga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg 120
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 180
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 240
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 300
gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 360
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 420
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 480
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 540
cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 600
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 660
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 720
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 780
gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagatcataa 840
tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 900
tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 960
atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 1020
attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt 1080
aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1140
gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1200
gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1260
aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1320
gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1380
tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1440
gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1500
aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 1560
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 1620
taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga 1680
atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 1740
gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 1800
gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 1860
ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 1920
tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 1980
gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg 2040
tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 2100
ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 2160
ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 2220
gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 2280
gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 2340
gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 2400
gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 2460
catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 2520
gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg 2580
cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 2640
gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 2700
caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 2760
cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 2820
cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 2880
ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 2940
gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 3000
tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa 3060
cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc 3120
ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt 3180
ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag 3240
gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat 3300
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 3360
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 3420
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 3480
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 3540
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 3600
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 3660
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 3720
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 3780
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 3840
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 3900
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3960
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 4020
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 4080
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 4140
ccgccatgca t 4151
Claims (6)
1.一种长牡蛎gata2/3启动子，其特征在于：启动子为SEQ ID No.1所示的碱基序列。
2.一种按权利要求1所述的长牡蛎gata2/3启动子的应用，其特征在于：所述启动子在制备重组表达载体中的应用。
3.一种按权利要求1所述的长牡蛎gata2/3启动子的应用，其特征在于：所述启动子在贝类幼虫中启动目的基因中的应用。
4.按权利要求3所述的长牡蛎gata2/3启动子的应用，其特征在于：所述目的基因是在细胞中转录或翻译的功能基因或RNA序列。
5.含权利要求1所述的长牡蛎gata2/3启动子的重组表达载体。
6.一种权利要求5所述的含长牡蛎gata2/3启动子的重组表达载体的应用，其特征在于：所述重组表达载体在贝类幼虫中启动目的基因中的应用。
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Non-Patent Citations (4)
| Title |
|---|
| A GATA2/3 gene potentially involved in larval shell formation of the Pacific oyster Crassostrea gigas;Gang Liu等;《Dev Genes Evol》;20150711;第225卷(第4期);第253-257页 * |
| A SoxC gene related to larval shell development and co-expression analysis of different shell formation genes in early larvae of oyster;Gang Liu等;《Dev Genes Evol》;20170309;第227卷(第3期);第181-188页 * |
| Crassostrea gigas strain QD chromosome 4，CP048842.1;Qi,H.等;《NCBI Genbank》;20200227;第1-2页 * |
| 长牡蛎免疫系统的发生和发育机制的初步研究;宋小瑞;《中国博士学位论文全文数据库农业科技辑》;20160815;D052-11 * |
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