CN111876414A - 一种改良酵母上游激活元件及其在鱼类中的应用 - Google Patents
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Abstract
本发明公开了一种改良酵母上游激活元件及其在鱼类中的应用，是由UAS‑1、间隔序列、UAS‑2、间隔序列、UAS‑3、间隔序列、UAS‑4、间隔序列、UAS‑5、间隔序列、启动子依次组成的非重复序列，其中UAS‑1、UAS‑2、UAS‑3、UAS‑4、UAS‑5的碱基序列均不相同。本发明不仅能够被GAL4蛋白特异识别，而且其CpG位点不易发生甲基化，增强了鱼类中目的基因诱导表达率，从而提高目的蛋白的表达产量，很好的促进GAL4/UAS系统在鱼类中的应用。
Description
技术领域
本发明涉及一种改良酵母上游激活元件及其在鱼类中的应用，属于分子生物学领域。
背景技术
GAL蛋白家族在酵母的半乳糖代谢中起着重要作用。GAL4作为GAL蛋白家族的一员，其作用类似于原核生物乳糖操纵子的一个转录激活子。酵母中原始的GAL4基因编码含有881个氨基酸的蛋白，该蛋白主要包括N端的DNA结合域和C端的转录激活域（Traven etal., 2006）。上游激活序列UAS（upstream active sequence）是酵母中类似真核生物增强子的一段序列，位于靶基因启动子的上游，其序列特征为：5'-CGGRNNRCYNYNYNCNCCG-3'（R代表嘌呤，Y代表嘧啶，N代表任意脱氧核苷酸），GAL4因子以同源二聚体的形式特异识别并结合UAS，并促使UAS下游基因的转录表达（Giniger et al., 1985）。
转基因技术是研究基因功能的有力工具，但是以往的利用特异性启动子或热休克蛋白启动子驱动外源基因表达的方法具有一定的局限性，比如研究的基因如果是致死基因，那么就无法建立转基因家系。利用GAL4/UAS系统，可以分别建立含有GAL4的家系和UAS驱动靶基因表达的家系，只有这两种家系杂交才能调控靶基因的表达，因此GAL4/UAS系统在转基因技术中的应用，可以弥补上述不足。在GAL4/UAS系统的推广应用中，GAL4因子被逐步的改造和优化，使之具有更好的诱导精度和和诱导效率。GAL4蛋白之所以能够特异结合并诱导UAS下游基因的转录表达，是因为GAL4具有能够特异结合UAS的DNA结合域及转录激活域。因此对GAL4蛋白的优化改造，主要是针对GAL4因子含有的这两个功能域。Distel 等在斑马鱼中将GAL4蛋白进行了优化，首先将GAL4的DNA结合域与四环素转录激活子（tTA）的转录激活域融合，其次在5’端融合Kozak序列以增强转录和翻译的效率，在3’端融合兔子的β球蛋白的UTR区以增强mRNA的稳定性，该优化后的GAL4蛋白（KalTA4）在斑马鱼中具有很好的诱导活性和诱导精度，且能够建立含有KalTA4的稳定遗传的斑马鱼家系（Distel etal., 2009），UAS对GAL4/UAS系统诱导效率影响研究，主要集中在UAS拷贝数多少对其诱导效率的研究上。UAS发生甲基化对GAL4/UAS系统诱导效率影响在不同物种中被发现，已经成为共识（Halpern et al., 2008）。UAS序列之所以会发生甲基化，是因为UAS序列是一种重复序列，且中间含有容易发生甲基化的CpG位点，重复序列的CpG位点常常会发生甲基化修饰，而UAS序列甲基化后将严重影响GAL4/UAS系统的诱导表达效率。本发明针对UAS容易发生甲基化影响UAS下游基因的表达的现状，设计一种全新的非重复UAS序列，很大程度生减轻了甲基化症状。
发明内容
针对上述现有技术存在的不足，本发明的目的是提供一种不易被甲基化的改良型UAS序列，以保证GAL4/UAS系统的诱导表达效率，从而促进GAL4/UAS系统在鱼类中的应用。
为实现上述目的，本发明采用的技术方案之一是：一种改良酵母上游激活元件，其特征在于，该上游激活元件为非重复序列。
优选地，上述改良酵母上游激活元件是由UAS-1、间隔序列、UAS-2、间隔序列、UAS-3、间隔序列、UAS-4、间隔序列、UAS-5、间隔序列、启动子依次组成的非重复序列，其中UAS-1、UAS-2、UAS-3、UAS-4、UAS-5的碱基序列均不相同。
优选地，上述改良酵母上游激活元件的多个间隔序列的长度不一致。
优选地，上述改良酵母上游激活元件，其碱基序列如SEQ ID NO.1所示。
本发明的技术方案之二是：上述改良酵母上游激活元件在提高鱼类中目的基因诱导表达率的应用，其改良酵母上游激活元件的碱基序列如SEQ ID NO.1所示。
一种提高鱼类中目的基因诱导表达率的方法，其特征在于，构建重组表达载体CK5nrG，将本发明的改良酵母上游激活元件克隆到鱼类目的基因的上游；然后显微注射鱼类1个细胞时期的受精卵，获得P0代转基因个体，再通过测交筛选获得杂合子F1代；在杂合子F1中KalTA4诱导改良酵母上游激活元件下游基因的高效表达；其中重组表达载体CK5nrG的碱基序列如SEQ ID NO.5所示。
与现有技术相比，本发明的有益效果：设计非重复序列的改良酵母上游激活元件，不仅能够被GAL4蛋白特异识别，而且其CpG位点不易发生甲基化，增强了鱼类中目的基因诱导表达率，从而提高目的蛋白的表达产量，很好的促进GAL4/UAS系统在鱼类中的应用。
附图说明
图1为现有转基因载体CK5G（两侧含转座子Tol2元件，CMV启动子驱动KalTA4（改良型GAL4）表达框，优化之前5xUAS驱动EGFP表达框）；
图2为本发明转基因载体CK5nrG（两侧含有转座子Tol2元件，CMV启动子驱动KalTA4（改良型GAL4）表达框，优化之后5xnrUAS驱动EGFP表达框）；
图3为KalTA4诱导5xUAS驱动EGFP表达；
图4为KalTA4诱导5xnrUAS驱动EGFP表达；
图5为KalTA4诱导5xUAS及5xnrUAS驱动EGFP表达强度（无显著性差异P<0.05）；
图6为P0代CK5G转基因斑马鱼中5xUAS甲基化率（48.33%）；
图7为P0代CK5nrG转基因斑马鱼中5xnrUAS甲基化率（40.00%）；
图8为F1代CK5G转基因斑马鱼中KalTA4诱导5xUAS驱动EGFP表达；
图9为F1代CK5nrG转基因斑马鱼中KalTA4诱导5xnrUAS驱动EGFP表达；
图10为F1代CK5G转基因斑马鱼中5xUAS甲基化率（64.17%）；
图11为F1代CK5nrG转基因斑马鱼中5xnrUAS甲基化率（46.67%）。
具体实施方式
本发明实施实例中的方法未经特别说明，均为本领域技术人员熟知的常规方法。本发明中未经特别说明的生物试剂，均来自TAKARA公司。现结合具体实施例来说明本发明的技术方案和技术效果，但并不是限制本发明保护范围的依据。
实施例一
1、基因合成改良酵母上游激活元件
改良酵母上游激活元件5xnrUAS DNA序列由上海英骏生物技术有限公司直接合成，且在5xnrUAS序列上游和下游分别添加XmaI和HindIII限制性内切酶酶切位点，以方便CK5nrG转基因载体的构建。且5xnrUAS DNA序列如SEQ ID NO.1：
TcccccgggctgcagcggagtactgtcctccgAGCGGATTAGAAGCCACCGGATCCGGGTGACAGCCCTCCGTCTTCACGGGATACTCTACACCGTAGGGTTCCGGAGTACTGTCCTCCGagtctagagggtatataatggatcccat cgcgtctcagcctcactttaagcttgaga；
序列SEQ ID NO.1注释：1-15 XmaI酶切位点；16-32：UAS-1；33-34：间隔序列；35-51：UAS-2; 52-55：间隔序列；56-72：UAS-3；73-78：间隔序列；79-95：UAS-4；96-103：间隔序列；104-120：UAS-5；121-145：间隔序列；146-167：E1b启动子；168-177：HindIII酶切位点。
2、构建表达载体CK5nrG
（1）CK5G转基因表达载体的构建：以转基因表达载体TG6（-Tol2-CMV-RFP-pA-5xUAS-EGFP-pA-Tol2-）为初始载体进行构建（Zhang, Y., Chen, J., Cui, X., Luo, D., Xia,H., & Dai, J., et al. (2015). A controllable on-off strategy for thereproductive containment of fish. Scientific Reports, 5(5), 73-82.）；然后用NEB公司的限制性核酸内切酶BglII和XmaI对转基因载体TG6进行双酶切，条件为37℃、水浴4小时；然后酶切产物进行琼脂糖凝胶电泳纯化，具体操作为：用电泳仪（北京六一仪器 DYY-4C型）进行1%的琼脂糖凝胶电泳，电压约110伏，电流约100毫安，电泳时间30分钟后利用琼脂糖凝胶DNA回收试剂盒（TAKARA）回收凝胶中较大的一条DNA片段（-Tol2-骨架序列-Tol2-pA-EGFP-5xUAS-），放入-20℃待用；
（2）CMV启动子DNA的获得：以TG6转基因载体为模板，引物如下：
SEQ ID NO.2， CMV-F：5’- GGAAGATCTAATCTCGACGCGCGTAATAC-3’（ 下划线标出部分GGAAGATCTAAT为BglII限制性DNA内切酶酶切位点及其保护碱基）
SEQ ID NO.3，CMV-R：5’-CAGGCTTGCTCGATGGATGAGAGCAGTTTCATGGTGGCGGCGAATTGAGGTCCAGGGTTCTC-3’
然后利用KOD PLUS高保真酶(TAKARA)在PCR仪（Veriti）上进行PCR扩增，条件为：95℃、3 min一个循环；95℃、30s，55℃、 30s，68℃、 2min共 30个循环；68℃、 5min一个循环，PCR产物利用琼脂糖电泳纯化（方法同上）后放入-20℃保存备用；
（3）KalTA4-pA序列由上海英骏生物技术有限公司直接合成。KalTA4-pA序列来自转基因载体TK5xC（Distel, M., Wullimann, M. F., Köster, R. W., & Dawid, I. B.(2009). Optimized gal4 genetics for permanent gene expression mapping inzebrafish. Proceedings of the National Academy of Sciences of the United States of America, 106(32), 13365-70.），另外在pA序列下游增加XmaI酶切位点序列。利用连接PCR（Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., & Pease, L.R. (1989). Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension. Gene, 77(1), 61-68.），将上述CMV启动子和KalTA4-pA合成序列连成一条DNA序列，即CMV-KalTA4-pA。用NEB公司的限制性核酸内切酶BglII和XmaI对-CMV-KalTA4-pA-进行双酶切，条件及操作同步骤（1）；
（4）将上述步骤（3）酶切后的片段（-Tol2-骨架序列-Tol2-pA-EGFP-5xUAS-）和CMV-KalTA4-pA用T4 DNA连接酶（NEB）进行4℃过夜连接，得到CK5G(-Tol2 -CMV-KalTA4-pA-5xUAS-EGFP-pA-Tol2-)转基因载体，其中CMV为巨细胞病毒基因启动子（589bp，Clontech公司产品）；eGFP为绿色荧光基因（720bp，Clontech公司产品）；pA为猴空泡病毒40的poly A终止序列（51bp，Clontech公司产品，且其CK5G具体碱基序列如SEQ ID NO.4：
Cagaggtgtaaaaagtactcaaaaattttactcaagtgaaagtacaagtacttagggaaaattttactcaattaaaagtaaaagtatctggctagaatcttacttgagtaaaagtaaaaaagtactccattaaaattgtacttgagtattagatctaatctcgacgcgcgtaatacgactcactatagggcgaattgggtactagggataacagggtaatgggccccccctcgagatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcgggatccccgggtaccgagctcgaattcagggaagaggaagtctgctgacctgcggagacgtggaggagaaccctggacctcaattcgccgccaccatgaaactgctctcatccatcgagcaagcctgcgacatttgtcggcttaagaagctgaaatgctccaaggaaaagccgaaatgtgccaaatgcctgaagaacaattgggaatgtcgttactctcccaaaaccaagcgaagtccactcacaagggctcatctgaccgaagtggagagcaggctagagagactggaacaactctttttgctcatcttccctagagaggaccttgacatgatcctcaagatggattctctccaggatattaaagcccttttgactggcttattcgtccaggacaatgtgaacaaagacgctgtgacagaccgattggcaagtgtcgagaccgatatgcctctgacactgagacagcacagaatcagcgctacttcctcaagcgaagagtcttctaacaagggacagagacagctgactgtttcgagcaggtcgaccccgtccccggccgacgccctggacgacggcgacctggacatgctgcctgctgatgctctcgatgatttcgatctggatatgctcccggccgacgccctggacgactacgacctggacatcctcccgggtaactaagtaaggatctcgaccaattcctcgacggatcgtagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaattctagttgtgggtcgacgccgctctagaactagtggatcccccgggctgcagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagcggagtactgtcctccgagtctagagggtatataatggatcccatcgcgtctcagcctcactttaagcttgatatcgaattcgccgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaactcgagcctctagaactatagtgagtcgtattactcgaccaattcctcgacggatcgtagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaattcgcggccgccaccgcggattaccctgttatccctaagctccagcttttgttccctttagtgagggttaattgcgcgtcgagccgggcccaagtgatctccaaaaaataagtactttttgactgtaaataaaattgtaaggagtaaaaagtacttttttttctaaaaaaatgtaattaagtaaaagtaaaagtattgatttttaattgtactcaagtaaagtaaaaatccccaaaaataatacttaagtacagtaatcaagtaaaattactcaagtactttacacctctg
对上述CK5G碱基序列的注释：1-150: Tol2-Left；151-235：间隔序列；236-810：CMV启动子；811-896：间隔序列；897-1493：KalTA4; 1494-1527：间隔序列；1528-1723：Poly A；1724-1778：间隔序列；1779-1918：5xUAS-E1b；1919-1945：间隔序列；1946-2662：EGFP；2663-2724：间隔序列；2725-2920：Poly A; 2921-3013：间隔序列；3014-3213：Tol2-Right。
（5）CK5nrG转基因表达载体的构建：CK5G转基因表达载体为初始载体，用NEB公司的限制性核酸内切酶XmaI和HindIII对转基因载体CK5G进行双酶切，条件为37℃、水浴4小时。然后酶切产物进行琼脂糖凝胶电泳纯化，具体操作为：用电泳仪（北京六一仪器 DYY-4C型）进行1%的琼脂糖凝胶电泳，电压约110伏，电流约100毫安，电泳时间30分钟后利用琼脂糖凝胶DNA回收试剂盒（TAKARA）回收凝胶中的载体骨架DNA片段，放入-20℃待用。5xnrUASDNA序列由上海英骏生物技术有限公司直接合成，且在5xnrUAS序列上游和下游分别添加XmaI和HindIII限制性内切酶酶切位点。用NEB公司的限制性核酸内切酶XmaI和HindIII对5xnrUAS序列进行双酶切，条件及割胶回收同步骤（1）。将上述酶切后的CK5G骨架片段和5xnrUAS序列用T4 DNA连接酶（NEB）进行4℃过夜连接，得到CK5nrG(-Tol2 -CMV-KalTA4-pA-5xnrUAS-EGFP-pA-Tol2-)转基因载体。其中CK5nrG的碱基序列如SEQ ID NO.5：
CagaggtgtaaaaagtactcaaaaattttactcaagtgaaagtacaagtacttagggaaaattttactcaattaaaagtaaaagtatctggctagaatcttacttgagtaaaagtaaaaaagtactccattaaaattgtacttgagtattagatctaatctcgacgcgcgtaatacgactcactatagggcgaattgggtactagggataacagggtaatgggccccccctcgagatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatcgggatccccgggtaccgagctcgaattcagggaagaggaagtctgctgacctgcggagacgtggaggagaaccctggacctcaattcgccgccaccatgaaactgctctcatccatcgagcaagcctgcgacatttgtcggcttaagaagctgaaatgctccaaggaaaagccgaaatgtgccaaatgcctgaagaacaattgggaatgtcgttactctcccaaaaccaagcgaagtccactcacaagggctcatctgaccgaagtggagagcaggctagagagactggaacaactctttttgctcatcttccctagagaggaccttgacatgatcctcaagatggattctctccaggatattaaagcccttttgactggcttattcgtccaggacaatgtgaacaaagacgctgtgacagaccgattggcaagtgtcgagaccgatatgcctctgacactgagacagcacagaatcagcgctacttcctcaagcgaagagtcttctaacaagggacagagacagctgactgtttcgagcaggtcgaccccgtccccggccgacgccctggacgacggcgacctggacatgctgcctgctgatgctctcgatgatttcgatctggatatgctcccggccgacgccctggacgactacgacctggacatcctcccgggtaactaagtaaggatctcgaccaattcctcgacggatcgtagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaattctagttgtgggtcgacgccgctctagaactagtggatcccccgggctgcagcggagtactgtcctccgAGCGGATTAGAAGCCACCGGATCCG GGTGACAGCCCTCCGTCTTCACGGGATACTCTACACCGTAGGGTTCCGGAGTACTGTCCTCCGagtctagagggtatataatggatcccatcgcgtctcagcctcactttaagcttgatatcgaattcgccgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaactcgagcctctagaactatagtgagtcgtattactcgaccaattcctcgacggatcgtagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaattcgcggccgccaccgcggattaccctgttatccctaagctccagcttttgttccctttagtgagggttaattgcgcgtcgagccgggcccaagtgatctccaaaaaataagtactttttgactgtaaataaaattgtaaggagtaaaaagtacttttttttctaaaaaaatgtaattaagtaaaagtaaaagtattgatttttaattgtactcaagtaaagtaaaaatccccaaaaataatacttaagtacagtaatcaagtaaaattactcaagtactttacacctctg
上述CK5nrG碱基序列的具体注释：1-150: Tol2-Left；151-235：间隔序列；236-810：CMV启动子；811-896：间隔序列；897-1493：KalTA4; 1494-1527：间隔序列；1528-1723：PolyA；1724-1778：间隔序列；1779-1930：5xUAS-E1b；1931-1957：间隔序列；1958-2674：EGFP；2675-2736：间隔序列；2737-2932：Poly A; 2933-3025：间隔序列；3026-3225：Tol2-Right；
3、表达载体显微注射斑马鱼胚胎及转基因鱼的筛选
将重组转基因载体CK5G和CK5nrG分别溶于ST 溶液 (88 mmol/l NaCl, 10 mmol/lTris-HCl, pH 7.5) 中，调节其终浓度为100ng/μl，采用显微注射的方法（Zhu Z, Li G,He L, et al. Novel gene transfer into the fertilized eggs of goldfish(Carassius auratus L. 1758). Z angew Ichthyol, 1985, 1:31-34），在第一次卵裂前将DNA溶液导入斑马鱼受精卵动物极，DNA注射剂量0.5-1 nl/卵，完成显微操作后的受精卵置于28.5℃水温孵化和养殖。养殖两天后在荧光显微镜下（Olympus SZX12型）筛选绿色荧光的胚胎，即为P0代CK5G和CK5nrG转基因斑马鱼。按斑马鱼养殖手册(Westerfield M.(1993). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish(Brachydanio rerio). University of Oregon Press, Eugene, OR.)，在循环水养殖系统中将P0代CK5G和CK5nrG转基因斑马鱼分别养殖至性成熟后，分别与野生型斑马鱼杂交，在荧光显微镜（Olympus SZX12型）下筛选通体表达EGFP的胚胎，获得杂合子F1代。
4、检测目的DNA甲基化概率
用亚硫酸氢盐测序法进行DNA甲基化效率的测定，具体操作为：利用动物组织提取试剂盒（Omega公司）提取P0代及F1代CK5G和CK5nrG转基因斑马鱼肌肉组织基因组DNA。利用DNA甲基化测定试剂盒（Invitrogen）处理0.5微克上述基因组DNA，条件为：首先98℃水浴10分钟，然后64℃水浴2.5小时，最后4℃冰箱冷藏18小时。利用PCR技术以处理后的基因组DNA为模板，分别扩增CK5G转基因斑马鱼基因组中的5xUAS-E1b序列和CK5nrG转基因斑马鱼基因组中的5xnrUAS-E1b序列，所用引物均为5’-TTTTTTTTATTGTATTTTAGTTGTGGT-3’和5’-TAAAATTTTCATCA-ATCAAATCC-3’为引物，然后利用pfu高保真酶(TAKARA)在PCR仪（Veriti）上进行PCR扩增，条件为：95℃ 3min一个循环；95℃ 30s、53℃ 30s、68℃ 30s 共30个循环；68℃ 3min一个循环，PCR产物利用琼脂糖电泳纯化（方法同上）后连入pMD-18T载体(TAKARA)，条件为：4℃过夜。连接产物转化大肠杆菌DH5α后涂在含有氨苄青霉素的LB固体培养基上，37℃培养16小时后随机挑取10个菌落进行扩大培养。然后用质粒提取试剂盒（Invitrogen）分别提取质粒后送上海英骏生物技术有限公司进行测序。然后通过与原始序列比对分析确定所测序列的甲基化效率。
通过实验比较原始UAS序列和本发明的改良酵母上游激活元件的表达量差异，具体如图3、4、5、8、9所示。
通过实验比较原始UAS序列和本发明的改良酵母上游激活元件的甲基化程度，具体如图6、7、10、11所示。结果说明，在转基因P0代中，5xnrUAS甲基化率显著低于5xUAS，改良后的5xnrUAS甲基化降低程度明显；在转基因F1代中，5xnrUAS甲基化率仍然显著低于5xUAS，改良后的5xnrUAS随着传代的进行其甲基化程度降低更加明显; UAS甲基化会导致UAS下游基因的镶嵌型表达，本发明的 UAS下游基因镶嵌型表达明显改善。本申请的技术受到湖南省自然科学青年基金2018JJ3371和湖南文理学院校级项目16BSQD47的资助。
序列表
<110> 湖南文理学院
<120> 一种改良酵母上游激活元件及其在鱼类中应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 177
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcccccgggc tgcagcggag tactgtcctc cgagcggatt agaagccacc ggatccgggt 60
gacagccctc cgtcttcacg ggatactcta caccgtaggg ttccggagta ctgtcctccg 120
agtctagagg gtatataatg gatcccatcg cgtctcagcc tcactttaag cttgaga 177
<210> 2
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggaagatcta atctcgacgc gcgtaatac 29
<210> 3
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caggcttgct cgatggatga gagcagtttc atggtggcgg cgaattgagg tccagggttc 60
tc 62
<210> 4
<211> 3213
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cagaggtgta aaaagtactc aaaaatttta ctcaagtgaa agtacaagta cttagggaaa 60
attttactca attaaaagta aaagtatctg gctagaatct tacttgagta aaagtaaaaa 120
agtactccat taaaattgta cttgagtatt agatctaatc tcgacgcgcg taatacgact 180
cactataggg cgaattgggt actagggata acagggtaat gggccccccc tcgagatcaa 240
ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 300
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 360
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 420
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 480
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 540
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 600
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 660
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 720
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 780
gcagagctgg tttagtgaac cgtcagatcg ggatccccgg gtaccgagct cgaattcagg 840
gaagaggaag tctgctgacc tgcggagacg tggaggagaa ccctggacct caattcgccg 900
ccaccatgaa actgctctca tccatcgagc aagcctgcga catttgtcgg cttaagaagc 960
tgaaatgctc caaggaaaag ccgaaatgtg ccaaatgcct gaagaacaat tgggaatgtc 1020
gttactctcc caaaaccaag cgaagtccac tcacaagggc tcatctgacc gaagtggaga 1080
gcaggctaga gagactggaa caactctttt tgctcatctt ccctagagag gaccttgaca 1140
tgatcctcaa gatggattct ctccaggata ttaaagccct tttgactggc ttattcgtcc 1200
aggacaatgt gaacaaagac gctgtgacag accgattggc aagtgtcgag accgatatgc 1260
ctctgacact gagacagcac agaatcagcg ctacttcctc aagcgaagag tcttctaaca 1320
agggacagag acagctgact gtttcgagca ggtcgacccc gtccccggcc gacgccctgg 1380
acgacggcga cctggacatg ctgcctgctg atgctctcga tgatttcgat ctggatatgc 1440
tcccggccga cgccctggac gactacgacc tggacatcct cccgggtaac taagtaagga 1500
tctcgaccaa ttcctcgacg gatcgtagat ccagacatga taagatacat tgatgagttt 1560
ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 1620
attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt 1680
cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcta gttgtgggtc 1740
gacgccgctc tagaactagt ggatcccccg ggctgcagcg gagtactgtc ctccgagcgg 1800
agtactgtcc tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt 1860
actgtcctcc gagtctagag ggtatataat ggatcccatc gcgtctcagc ctcactttaa 1920
gcttgatatc gaattcgccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt 1980
ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg 2040
cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg 2100
caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt 2160
cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg 2220
ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga 2280
ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa 2340
ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta 2400
tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat 2460
cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg 2520
ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc 2580
caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct 2640
cggcatggac gagctgtaca agtaactcga gcctctagaa ctatagtgag tcgtattact 2700
cgaccaattc ctcgacggat cgtagatcca gacatgataa gatacattga tgagtttgga 2760
caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 2820
gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 2880
tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc cgccaccgcg 2940
gattaccctg ttatccctaa gctccagctt ttgttccctt tagtgagggt taattgcgcg 3000
tcgagccggg cccaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt 3060
aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg 3120
atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat 3180
caagtaaaat tactcaagta ctttacacct ctg 3213
<210> 5
<211> 3225
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cagaggtgta aaaagtactc aaaaatttta ctcaagtgaa agtacaagta cttagggaaa 60
attttactca attaaaagta aaagtatctg gctagaatct tacttgagta aaagtaaaaa 120
agtactccat taaaattgta cttgagtatt agatctaatc tcgacgcgcg taatacgact 180
cactataggg cgaattgggt actagggata acagggtaat gggccccccc tcgagatcaa 240
ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 300
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 360
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 420
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 480
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 540
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 600
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 660
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 720
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 780
gcagagctgg tttagtgaac cgtcagatcg ggatccccgg gtaccgagct cgaattcagg 840
gaagaggaag tctgctgacc tgcggagacg tggaggagaa ccctggacct caattcgccg 900
ccaccatgaa actgctctca tccatcgagc aagcctgcga catttgtcgg cttaagaagc 960
tgaaatgctc caaggaaaag ccgaaatgtg ccaaatgcct gaagaacaat tgggaatgtc 1020
gttactctcc caaaaccaag cgaagtccac tcacaagggc tcatctgacc gaagtggaga 1080
gcaggctaga gagactggaa caactctttt tgctcatctt ccctagagag gaccttgaca 1140
tgatcctcaa gatggattct ctccaggata ttaaagccct tttgactggc ttattcgtcc 1200
aggacaatgt gaacaaagac gctgtgacag accgattggc aagtgtcgag accgatatgc 1260
ctctgacact gagacagcac agaatcagcg ctacttcctc aagcgaagag tcttctaaca 1320
agggacagag acagctgact gtttcgagca ggtcgacccc gtccccggcc gacgccctgg 1380
acgacggcga cctggacatg ctgcctgctg atgctctcga tgatttcgat ctggatatgc 1440
tcccggccga cgccctggac gactacgacc tggacatcct cccgggtaac taagtaagga 1500
tctcgaccaa ttcctcgacg gatcgtagat ccagacatga taagatacat tgatgagttt 1560
ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 1620
attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt 1680
cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcta gttgtgggtc 1740
gacgccgctc tagaactagt ggatcccccg ggctgcagcg gagtactgtc ctccgagcgg 1800
attagaagcc accggatccg ggtgacagcc ctccgtcttc acgggatact ctacaccgta 1860
gggttccgga gtactgtcct ccgagtctag agggtatata atggatccca tcgcgtctca 1920
gcctcacttt aagcttgata tcgaattcgc cgccaccatg gtgagcaagg gcgaggagct 1980
gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 2040
cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 2100
ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 2160
cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 2220
catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 2280
gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 2340
catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 2400
ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 2460
ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 2520
catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 2580
gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 2640
cgggatcact ctcggcatgg acgagctgta caagtaactc gagcctctag aactatagtg 2700
agtcgtatta ctcgaccaat tcctcgacgg atcgtagatc cagacatgat aagatacatt 2760
gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt 2820
tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac 2880
aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg 2940
gccgccaccg cggattaccc tgttatccct aagctccagc ttttgttccc tttagtgagg 3000
gttaattgcg cgtcgagccg ggcccaagtg atctccaaaa aataagtact ttttgactgt 3060
aaataaaatt gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa 3120
gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa aataatactt 3180
aagtacagta atcaagtaaa attactcaag tactttacac ctctg 3225
Claims (6)
1.一种改良酵母上游激活元件，其特征在于，该上游激活元件为非重复序列。
2.根据权利要求1所述的改良酵母上游激活元件，其特征在于，上述改良酵母上游激活元件是由UAS-1、间隔序列、UAS-2、间隔序列、UAS-3、间隔序列、UAS-4、间隔序列、UAS-5、间隔序列、基本启动子依次组成的非重复序列，其中UAS-1、UAS-2、UAS-3、UAS-4、UAS-5的碱基序列均不相同。
3.根据权利要求2所述的改良酵母上游激活元件，其特征在于，上述改良酵母上游激活元件的多个间隔序列的长度不一致。
4.根据权利要求3所述的改良酵母上游激活元件，其特征在于，其碱基序列如SEQ IDNO.1所示。
5.根据权利要求4所述的改良酵母上游激活元件在提高鱼类中目的基因诱导表达率的应用。
6.一种提高鱼类中目的基因诱导表达率的方法，其特征在于，构建重组表达载体CK5nrG，将改良酵母上游激活元件克隆到鱼类目的基因的上游，其中重组表达载体CK5nrG的碱基序列如SEQ ID NO.5所示；然后显微注射鱼类1个细胞时期的受精卵，获得P0代转基因个体，再通过测交筛选获得杂合子F1代；在P0代及杂合子F1中KalTA4诱导改良酵母上游激活元件下游基因的高效表达。
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