CN116548388B - 标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法 - Google Patents
标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体公开了标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法。本发明基于荧光泛素化系统的细胞周期标记（fluorescent ubiquitination‑based cell cycle indicator,FUCCI）方法，利用造血干/祖细胞特异增强子驱动FUCCI表达，获得可稳定遗传的、特异标记造血干/祖细胞周期的斑马鱼模型。此外，本发明将FUCCI表达系统整合至Tg(cd41:eGFP)转基因品系，有利于更便捷地观察、分析造血干/祖细胞周期，可用于造血系统发育、血液系统疾病发病机制和药物筛选等方面的研究。
Description
技术领域
本发明属于生物技术领域，具体涉及利用Tol2转座技术，将造血干/祖细胞增强子驱动整合至斑马鱼基因组，活体标记斑马鱼造血干/祖细胞周期变化的方法。
背景技术
转基因载体的构建方法主要是通过分子克隆将组织特异性启动子、目的基因连入到真核表达载体中，为了便于观察目的基因的表达往往还会再将表达报告基因与目的基因融合。Tol2转座系统是目前斑马鱼常用的转基因构建系统，它发现于青鳉鱼的基因组中，是一种自主性转座子元件。Tol2转座子包括hobo、Ac和Tam3等成分，其mRNA能够编码六百多个氨基酸，转录出转座酶介导转座反应。将Tol2转座子质粒和转座酶mRNA混合物通过显微注射进入1细胞时期的斑马鱼胚胎中，转座酶能够将转座质粒中的目的片段引入到斑马鱼基因组中。通过Tol2转座子系统构建的斑马鱼转基因品系，外源基因整合效率高，且能稳定传代。
利用NEB公司的HiFi DNA Assembly技术构建表达载体，这种方法能够组装有15-80个碱基重叠的、不同大小的DNA片段。该反应的不同酶在同一缓冲液中，外切酶产生悬垂的3’单链，促进具有互补序列的片段退火，之后聚合酶填充在每个退火片段的缝隙，DNA连接酶将片段连接，最终形成完全密封的双链DNA分子。
FUCCI系统广泛应用于细胞周期的研究中。它是一种细胞周期依赖的泛素化调控系统，最初的FUCCI将mKO2和mAG荧光蛋白分别与人CDT1和GEMINI的泛素化功能域融合，这两种融合蛋白随转染细胞的细胞周期。此后，FUCCI系统应用至斑马鱼体内，mKO2和mAG荧光蛋白分别与斑马鱼Cdt1和Gemini相应的泛素化功能域融合，构建的Tg（EFIα:mKO2-zCdt1（1/190））和Tg(EF1α:mAG-zGem(1/100))转基因品系，实现了斑马鱼全身细胞的细胞周期标记。然而，尚未有斑马鱼活体成像模型，能在造血系统中特异标记细胞周期的变化。
本发明首次将小鼠340bpRunx1enhancer和129bpHbb2mini promoter片段融合，利用Tol2介导的转座系统，实现了在斑马鱼造血干/祖细胞中特异性地驱动基因表达。并且，本发明将在造血干/祖细胞中驱动FUCCI特异表达的质粒整合进斑马鱼基因组，获得标记斑马鱼造血干/祖细胞周期的转基因品系，借助斑马鱼胚胎透明、造血发育过程与小鼠、人等哺乳动物高度保守的特性，使活体、体内观察造血干/祖细胞周期变化成为可能，为造血干/祖细胞周期相关研究提供了有力的活体观察工具。
发明内容
本发明填补了活体观察造血系统细胞周期变化的动物模型缺口，提供了一种利用Tol2转座技术，制备标记造血干/祖细胞周期转基因斑马鱼的方法，为针对造血干/祖细胞周期相关研究提供了活体观察的实验模型。
为了实现上述目的，根据本发明的提供了一种标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法，包括以下步骤：
S1.将构建的斑马鱼造血干/祖细胞特异增强子驱动的质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)和Tol2 mRNA共同注射到1细胞期的Tg(cd41:eGFP)斑马鱼胚胎中；
S2.通过鉴定、筛选和培养获得稳定遗传的Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))转基因斑马鱼品系。
根据本发明的步骤S1中获取质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)方法，包括如下步骤：
S11.以小鼠基因组为模板，根据HiFi DNA重组要求设计合成引物HiFiRunx1enhancer-Hbb2mini promoter-F为SEQ ID NO.1:tagaaaagttgctCACTGCACCTGCTAGGTTC、Runx1enhancer-R为SEQ ID NO.2:CAAGGAGCGATGGAGGGATG，以及Hbb2minipromoter-F为SEQ ID NO.3:ccAATCTGCTCAGAGAGGAC、HiFiRunx1 enhancer-Hbb2minipromoter-R为SEQID NO.4: ccttagacaccatGGTAGCCTGCTTTTTTGTACAAACTTGGGATGTCTGTTTCTGAGGT所包含的核苷酸序列，进行PCR扩增，合成Runx1enhancer 以及Hbb2minipromoterDNA片段，进行DNA纯化。以纯化后的Runx1enhancer 和Hbb2mini promoter DNA片段混合物为模板，HiFiRunx1enhancer-Hbb2mini promoter-F和HiFiRunx1enhancer-Hbb2mini promoter-R为引物，进行PCR扩增，合成Runx1enhancer-Hbb2mini promoterDNA片段，纯化后得到纯化DNA片段；
S12.以pmTagBFP2-N质粒为模板，根据HiFi DNA重组要求设计合成上游引物HiFimTagBFP2-zGem(1/100)-F为SEQ ID NO.5：CCAAGTTTGTACAAAAAAGCAGGCTACCatggtgtctaagggcgaagagctga，mTagBFP2-R为SEQ ID NO.6：attaagcttgtgccccagtttgcta，进行PCR扩增，合成mTagBFP2 DNA片段，再进行DNA纯化。以斑马鱼受精后24小时cDNA为模板，根据HiFiDNA重组要求设计合成上游引物zGem (1/100)-F为SEQID NO.7: ATGAGTTCCATCAGAAGACCAA，下游引物HiFimTagBFP2-zGem(1/100)-R为SEQ ID NO.8:agaaagctgggtcCTCATAGGCCTCTTGTGTG，进行PCR扩增，合成zGem(1/100) DNA片段，进行DNA纯化。以纯化后的mTagBFP2 DNA片段和zGem(1/100) DNA片段混合物为模板，HiFi mTagBFP2-zGem(1/100)-F和HiFi mTagBFP2-zGem(1/100)-R为引物，进行PCR扩增，合成mTagBFP2-zGem(1/100) DNA片段，纯化后得到纯化DNA片段；
S13.以Tol2过表达质粒为模板，根据HiFi DNA重组要求设计合成上游引物vector-F为SEQ ID NO.9: gaggcctatgagGACCCAGCTTTCTTGTAC，下游引物vector-R为SEQID NO.10gcaggtgcagtgAGCAACTTTTCTATACAAAGTTG，进行PCR扩增，合成vector DNA片段，纯化后得到纯化DNA片段；
S14.将步骤S11、步骤S12、步骤13获得的纯化DNA片段，进行HiFi连接、转化、获得单克隆菌株，提取相应pRunx1：mTagBFP2-zGem(1/100)质粒；
S15.以pmKO2-N1质粒为模板，根据HiFi DNA重组要求设计合成上游引物HiFimKO2-zCdt1(1/190)-F为SEQ ID NO.11：acctaccaccATGGTGAGTGTGATTAAACCAG，mKO2-R为SEQ ID NO.12：GCTATGAGCTACTGCATCTTCTACC，进行PCR扩增，合成mKO2 DNA片段，再进行DNA纯化，以斑马鱼受精后24小时cDNA为模板，根据HiFi DNA重组要求设计合成上游引物zCdt1(1/190)-F为SEQ ID NO.13：ATGGCTCAAGCTCGTGTTAC，下游引物HiFi mKO2-zCdt1(1/190)-R为SEQ ID NO.14：atacgactcatcaCTCTTTGCGGGCAGTTTG，进行PCR扩增，合成zCdt1(1/190)DNA片段，进行DNA纯化。以纯化后的mKO2 DNA片段和zCdt1(1/190) DNA片段混合物为模板，HiFi mKO2-zCdt1(1/190)-F和HiFimKO2-zCdt1(1/190)-R为引物，进行PCR扩增，合成mKO2-zCdt1(1/190) DNA片段，纯化后得到纯化DNA片段；
S16.以pRunx1：mTagBFP2-zGem(1/100)质粒为模板，根据HiFi DNA重组要求设计合成上游引物Runx1-mTagBFP2-zGem(1/100) vector-F为SEQ ID NO.15：ccgcaaagagTGAGTCGTATTACGTAGAACCAG，下游引物Runx1-mTagBFP2-zGem(1/100) vector-R为SEQ IDNO.16：cactcaccatGGTGGTAGGTCCAGGGTTC，进行PCR扩增，合成Runx1-mTagBFP2-zGem(1/100)vector DNA片段，纯化后得到纯化DNA片段。
S17.将步骤S15、步骤S16获得的纯化DNA片段，进行HiFi连接、转化、获得单克隆菌株，提取相应pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)质粒。
根据本发明的步骤S1中pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)质粒终浓度为40ng/μL，Tol2 mRNA终浓度为35ng/μL，总体积V=1nL。
根据本发明的步骤S2中稳定遗传的标识造血干/祖细胞周期的转基因斑马鱼品系获得步骤如下：
S22.将质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)与Tol2 mRNA共同注射到1细胞期的Tg(cd41:eGFP)斑马鱼胚胎中，在受精后33小时-4天，于共聚焦显微镜下挑选并拍摄在cd41:eGFP细胞中有mTagBFP2、mKO2特异荧光表达的胚胎，作为Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)嵌合表达的F0代；
S23.将F0代成鱼与野生型斑马鱼外交，进行可遗传性检测，获得在造血干/祖细胞中稳定表达可指示细胞周期变化荧光的F1代斑马鱼。
根据本发明的质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)通过FUCCI系统的在斑马鱼体内进行泛素化功能域融合。
本发明的有益效果是：
（1）本发明首次将小鼠340bpRunx1enhancer和129bpHbb2mini promoter片段融合，利用Tol2介导的转座系统，实现了在斑马鱼造血干/祖细胞中特异性地驱动基因表达。
（2）本发明实现了活体标记斑马鱼造血干/祖细胞周期，获得可稳定遗传的转基因品系。
本发明所提供的特异标识造血干/祖细胞周期的转基因斑马鱼模型的制备方法，得到的转基因品系可用造血发育、血液系统疾病中造血干/祖细胞周期相关分子机制的研究。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1：pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)质粒图谱。
图2：Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)；cd41:eGFP)F1胚胎荧光表达图。
图3：Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)；cd41:eGFP)F1胚胎EDU染色荧光表达图。
具体实施方式
下面结合附图和实施例对本发明作进一步的详细说明。可以理解的是，此处所描述的具体实施例仅仅用于解释本发明，而非对本发明的限定。另外还需要说明的是，为了便于描述，附图中仅示出了与本发明相关的部分而非全部试验结果。
实施例1获取质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)的方法，如图1所示，包括如下步骤：
S11.以小鼠基因组为模板，根据HiFi DNA重组要求设计合成引物HiFiRunx1enhancer-Hbb2mini promoter-F为SEQ ID NO.1:tagaaaagttgctCACTGCACCTGCTAGGTTC、Runx1enhancer-R为SEQ ID NO.2:CAAGGAGCGATGGAGGGATG，以及Hbb2minipromoter-F为SEQ ID NO.3:ccAATCTGCTCAGAGAGGAC、HiFiRunx1 enhancer-Hbb2minipromoter-R为SEQID NO.4: ccttagacaccatGGTAGCCTGCTTTTTTGTACAAACTTGGGATGTCTGTTTCTGAGGT所包含的核苷酸序列，进行PCR扩增，合成Runx1enhancer 以及Hbb2minipromoterDNA片段，进行DNA纯化。以纯化后的Runx1enhancer 和Hbb2mini promoter DNA片段混合物为模板，HiFiRunx1enhancer-Hbb2mini promoter-F和HiFiRunx1enhancer-Hbb2mini promoter-R为引物，进行PCR扩增，合成Runx1enhancer-Hbb2mini promoterDNA片段，纯化后得到纯化DNA片段；
S12.以pmTagBFP2-N质粒为模板，根据HiFi DNA重组要求设计合成上游引物HiFimTagBFP2-zGem(1/100)-F为SEQ ID NO.5：CCAAGTTTGTACAAAAAAGCAGGCTACCatggtgtctaagggcgaagagctga，mTagBFP2-R为SEQ ID NO.6：attaagcttgtgccccagtttgcta，进行PCR扩增，合成mTagBFP2 DNA片段，再进行DNA纯化。以斑马鱼受精后24小时cDNA为模板，根据HiFiDNA重组要求设计合成上游引物zGem (1/100)-F为SEQID NO.7: ATGAGTTCCATCAGAAGACCAA，下游引物HiFi mTagBFP2-zGem(1/100)-R为SEQ ID NO.8: agaaagctgggtcCTCATAGGCCTCTTGTGTG，进行PCR扩增，合成zGem(1/100) DNA片段，进行DNA纯化。以纯化后的mTagBFP2 DNA片段和zGem(1/100) DNA片段混合物为模板，HiFi mTagBFP2-zGem(1/100)-F和HiFimTagBFP2-zGem(1/100)-R为引物，进行PCR扩增，合成mTagBFP2-zGem(1/100) DNA片段，纯化后得到纯化DNA片段；
S13.以Tol2过表达质粒为模板，根据HiFi DNA重组要求设计合成上游引物vector-F为SEQ ID NO.9: gaggcctatgagGACCCAGCTTTCTTGTAC，下游引物vector-R为SEQID NO.10gcaggtgcagtgAGCAACTTTTCTATACAAAGTTG，进行PCR扩增，合成vector DNA片段，纯化后得到纯化DNA片段；
S14.将步骤S11、步骤S12、步骤13获得的纯化DNA片段，进行HiFi连接、转化、获得单克隆菌株，提取相应pRunx1：mTagBFP2-zGem(1/100)质粒；
S15.以pmKO2-N1质粒为模板，根据HiFi DNA重组要求设计合成上游引物HiFimKO2-zCdt1(1/190)-F为SEQ ID NO.11：acctaccaccATGGTGAGTGTGATTAAACCAG，mKO2-R为SEQ ID NO.12：GCTATGAGCTACTGCATCTTCTACC，进行PCR扩增，合成mKO2 DNA片段，再进行DNA纯化，以斑马鱼受精后24小时cDNA为模板，根据HiFi DNA重组要求设计合成上游引物zCdt1(1/190)-F为SEQ ID NO.13：ATGGCTCAAGCTCGTGTTAC，下游引物HiFi mKO2-zCdt1(1/190)-R为SEQ ID NO.14：atacgactcatcaCTCTTTGCGGGCAGTTTG，进行PCR扩增，合成zCdt1(1/190)DNA片段，进行DNA纯化。以纯化后的mKO2 DNA片段和zCdt1(1/190) DNA片段混合物为模板，HiFi mKO2-zCdt1(1/190)-F和HiFimKO2-zCdt1(1/190)-R为引物，进行PCR扩增，合成mKO2-zCdt1(1/190) DNA片段，纯化后得到纯化DNA片段；
S16.以pRunx1：mTagBFP2-zGem(1/100)质粒为模板，根据HiFi DNA重组要求设计合成上游引物Runx1-mTagBFP2-zGem(1/100) vector-F为SEQ ID NO.15：ccgcaaagagTGAGTCGTATTACGTAGAACCAG，下游引物Runx1-mTagBFP2-zGem(1/100) vector-R为SEQ IDNO.16：cactcaccatGGTGGTAGGTCCAGGGTTC，进行PCR扩增，合成Runx1-mTagBFP2-zGem(1/100)vector DNA片段，纯化后得到纯化DNA片段。
S17.将步骤S15、步骤S16获得的纯化DNA片段，进行HiFi连接、转化、获得单克隆菌株，提取相应pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)质粒。
实施例2.获取稳定遗传的标识造血干/祖细胞周期的转基因斑马鱼品系的步骤如下：
S22.将质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)与Tol2 mRNA共同注射到1细胞期的Tg(cd41:eGFP)斑马鱼胚胎中，在受精后33小时-4天，于共聚焦显微镜下挑选并拍摄在cd41:eGFP细胞中有mTagBFP2、mKO2特异荧光表达的胚胎，作为Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)嵌合表达的F0代；
S23.将F0代成鱼与野生型斑马鱼外交，进行可遗传性检测，获得在造血干/祖细胞中稳定表达可指示细胞周期变化荧光的F1代斑马鱼。
其中质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)质粒终浓度为40ng/μL，Tol2 mRNA终浓度为35ng/μL，总体积V=1nL。
实施例3：验证Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))标记的处于细胞周期的细胞与cd41:eGFP造血干/祖细胞荧光融合。
在共聚焦显微镜下拍摄F1代Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))受精后33小时、2天、3天和4天胚胎的荧光表达情况，如图2所示，其中Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))标记的mTagBFP2-zGem (1/100)荧光在受精后33小时便与cd41:eGFP造血干/祖细胞荧光融合，在2天、3天和4天Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))标记的mTagBFP2-zGem (1/100) 荧光和mKO2-zCdt1(1/190) 荧光与cd41:eGFP造血干/祖细胞荧光逐渐融合。说明Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))可标记造血干/祖细胞。
实施例4：验证 Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))标记的处于细胞周期的mTagBFP2-zGem(1/100)造血干/祖细胞与EDU染色荧光融合。
对受精后33小时F1代Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))胚胎进行EDU染色（可标记处于S期的细胞），结果显示，mTagBFP2-zGem(1/100)荧光，cd41:eGFP荧光与EDU染色荧光融合，如图3所示，说明Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))可标记造血干/祖细胞的细胞周期。
说明质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)通过FUCCI系统的在斑马鱼体内进行泛素化功能域融合。
本发明首次将小鼠340bpRunx1enhancer和129bpHbb2mini promoter片段融合，利用Tol2介导的转座系统，实现了在斑马鱼造血干/祖细胞中特异性地驱动基因表达。
本发明实现了活体标记斑马鱼造血干/祖细胞周期，获得可稳定遗传的转基因品系。
本发明所提供的特异标识造血干/祖细胞周期的转基因斑马鱼模型的制备方法，得到的转基因品系可用造血发育、血液系统疾病中造血干/祖细胞周期相关分子机制的研究。
本领域技术人员会理解，本发明不限于这里的实施例，对本领域技术人员来说能够进行各种明显的变化、重新调整和替代而不会脱离本发明的保护范围。因此，虽然通过以上实施例对本发明进行了较为详细的说明，但是本发明不仅仅限于以上实施例，在不脱离本发明构思的情况下，还可以包括更多其他等效实施例，而本发明的范围由所附的权利要求范围决定。
Claims (4)
1.一种标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法，其特征在于，包括如下步骤：
S1.将构建的斑马鱼造血干/祖细胞特异增强子驱动的质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)和Tol2 mRNA共同注射到1细胞期的Tg(cd41:eGFP)斑马鱼胚胎中；
S2.通过鉴定、筛选和培养获得稳定遗传的Tg(Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190))转基因斑马鱼品系,其中转基因斑马鱼品系获得步骤如下：
S22.将质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)与Tol2 mRNA共同注射到1细胞期的Tg(cd41:eGFP)斑马鱼胚胎中，在受精后33小时-4天，于共聚焦显微镜下挑选并拍摄在cd41:eGFP细胞中有mTagBFP2、mKO2特异荧光表达的胚胎，作为Runx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)嵌合表达的F0代；
S23.将F0代成鱼与野生型斑马鱼外交，进行可遗传性检测，获得在造血干/祖细胞中稳定表达可指示细胞周期变化荧光的F1代斑马鱼。
2.根据权利要求1所述的标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法，其特征在于，步骤S1中获取质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)方法，包括如下步骤：
S11.以小鼠基因组为模板，根据HiFi DNA重组要求设计合成引物HiFi Runx1enhancer-Hbb2mini promoter-F为SEQ ID NO.1:tagaaaagttgctCACTGCACCTGCTAGGTTC、Runx1 enhancer-R为SEQ ID NO.2:CAAGGAGCGATGGAGGGATG，以及Hbb2 mini promoter-F为SEQ ID NO.3:ccAATCTGCTCAGAGAGGAC、HiFi Runx1 enhancer-Hbb2 mini promoter-R为SEQID NO.4: ccttagacaccatGGTAGCCTGCTTTTTTGTACAAACTTGGGATGTCTGTTTCTGAGGT所包含的核苷酸序列，进行PCR扩增，合成Runx1enhancer 以及Hbb2 mini promoterDNA片段，进行DNA纯化，以纯化后的Runx1 enhancer 和Hbb2 mini promoter DNA片段混合物为模板，HiFi Runx1 enhancer-Hbb2mini promoter-F和HiFi Runx1 enhancer-Hbb2minipromoter-R为引物，进行PCR扩增，合成Runx1 enhancer-Hbb2 mini promoterDNA片段，纯化后得到纯化DNA片段；
S12.以pmTagBFP2-N质粒为模板，根据HiFi DNA重组要求设计合成上游引物HiFimTagBFP2-zGem(1/100)-F为SEQ ID NO.5：CCAAGTTTGTACAAAAAAGCAGGCTACCatggtgtctaagggcgaagagctga，mTagBFP2-R为SEQ ID NO.6：attaagcttgtgccccagtttgcta，进行PCR扩增，合成mTagBFP2 DNA片段，再进行DNA纯化，以斑马鱼受精后24小时cDNA为模板，根据HiFiDNA重组要求设计合成上游引物zGem (1/100)-F为SEQID NO.7: ATGAGTTCCATCAGAAGACCAA，下游引物HiFimTagBFP2-zGem(1/100)-R为SEQ ID NO.8:agaaagctgggtcCTCATAGGCCTCTTGTGTG，进行PCR扩增，合成zGem(1/100) DNA片段，进行DNA纯化，以纯化后的mTagBFP2 DNA片段和zGem(1/100) DNA片段混合物为模板，HiFi mTagBFP2-zGem(1/100)-F和HiFi mTagBFP2-zGem(1/100)-R为引物，进行PCR扩增，合成mTagBFP2-zGem(1/100) DNA片段，纯化后得到纯化DNA片段；
S13.以Tol2过表达质粒为模板，根据HiFi DNA重组要求设计合成上游引物vector-F为SEQ ID NO.9: gaggcctatgagGACCCAGCTTTCTTGTAC，下游引物vector-R为SEQ ID NO.10gcaggtgcagtgAGCAACTTTTCTATACAAAGTTG，进行PCR扩增，合成vector DNA片段，纯化后得到纯化DNA片段；
S14.将步骤S11、步骤S12、步骤13获得的纯化DNA片段，进行HiFi连接、转化、获得单克隆菌株，提取相应pRunx1：mTagBFP2-zGem(1/100)质粒；
S15.以pmKO2-N1质粒为模板，根据HiFi DNA重组要求设计合成上游引物HiFi mKO2-zCdt1(1/190)-F为SEQ ID NO.11：acctaccaccATGGTGAGTGTGATTAAACCAG，mKO2-R为SEQ IDNO.12：GCTATGAGCTACTGCATCTTCTACC，进行PCR扩增，合成mKO2 DNA片段，再进行DNA纯化，以斑马鱼受精后24小时cDNA为模板，根据HiFi DNA重组要求设计合成上游引物zCdt1 (1/190)-F为SEQ ID NO.13：ATGGCTCAAGCTCGTGTTAC，下游引物HiFi mKO2-zCdt1(1/190)-R为SEQ ID NO.14：atacgactcatcaCTCTTTGCGGGCAGTTTG，进行PCR扩增，合成zCdt1(1/190) DNA片段，进行DNA纯化，以纯化后的mKO2 DNA片段和zCdt1(1/190) DNA片段混合物为模板，HiFi mKO2-zCdt1(1/190)-F和HiFimKO2-zCdt1(1/190)-R为引物，进行PCR扩增，合成mKO2-zCdt1(1/190) DNA片段，纯化后得到纯化DNA片段；
S16.以pRunx1：mTagBFP2-zGem(1/100)质粒为模板，根据HiFi DNA重组要求设计合成上游引物Runx1-mTagBFP2-zGem(1/100) vector-F为SEQ ID NO.15：ccgcaaagagTGAGTCGTATTACGTAGAACCAG，下游引物Runx1-mTagBFP2-zGem(1/100) vector-R为SEQ ID NO.16：cactcaccatGGTGGTAGGTCCAGGGTTC，进行PCR扩增，合成Runx1-mTagBFP2-zGem(1/100)vector DNA片段，纯化后得到纯化DNA片段，
S17.将步骤S15、步骤S16获得的纯化DNA片段，进行HiFi连接、转化、获得单克隆菌株，提取相应pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)质粒。
3.根据权利要求1所述的标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法，其特征在于，步骤S1中pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)质粒终浓度为40ng/μL，Tol2 mRNA终浓度为35ng/μL，总体积V=1nL。
4.根据权利要求1所述的标记造血干/祖细胞周期的转基因斑马鱼模型的制备方法，其特征在于, 质粒pRunx1:mTagBFP2-zGem(1/100);mKO2-zCdt1(1/190)通过FUCCI系统的在斑马鱼体内进行泛素化功能域融合。
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