CN106906240A - 运用CRISPR‑Cas9系统敲除大麦VE合成通路中的关键基因HPT的方法 - Google Patents
运用CRISPR‑Cas9系统敲除大麦VE合成通路中的关键基因HPT的方法 Download PDFInfo
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Abstract
本发明涉及大麦转基因材料的构建，旨在提供一种运用CRISPR‑Cas9系统敲除大麦VE合成通路中的关键基因HPT的方法。该方法包括下述步骤：gRNA靶位点的选择、gRNA片段的克隆、GG(gRNA‑gRNA)片段的连接、连接产物的PCR扩增、纯化产物和目标载体的酶切、纯化产物和目标载体的连接反应、连接产物转化大肠杆菌感受态、农杆菌介导的大麦转基因、阳性转基因植株的筛选、突变体测序。本发明利用先进的基因编辑技术‑CRISPR‑Cas9系统对大麦VE合成通路中的关键基因(HPT)进行靶向基因编辑，获得有效的功能缺失突变体，为大麦中生物活性物质的研究创造条件。
Description
技术领域
本发明涉及大麦转基因材料的构建，特别涉及运用CRISPR-Cas9系统敲除HvHPT(MLOC_37476)基因的应用。
背景技术
维生素E(vitamin E，VE)是由光合生物合成的生育酚类化合物的总称。根据侧链是否饱和，VE可以分为生育酚(tocophero1)和生育三烯酚(tocotrieno1)两大类。根据芳香环上甲基位置和数目的不同，每类又可分为α，β，γ，δ四种形式，其中，α-生育酚活性最高。近些年来，由于生育三烯酚在某些方面更优越的生物学特性，备受人们关注。不仅表现在抗氧化活性，在降胆固醇、预防糖尿病、促进骨吸收、抗癌、神经保护等方面也有一定的作用。
大麦是世界上四大粮食作物之一，主要用于食品生产、动物饲养、啤酒制造等领域。另外，由于大麦含有丰富的生物活性物质如β-葡聚糖、酚类物质、维生素E等，也常被用作功能食品开发的原料。大麦谷粒，含有丰富的生育三烯酚，大概占VE总含量的70％，是研究生育三烯酚的好材料。因此利用基因工程手段调控大麦谷粒中的VE合成通路，可以提高大麦生育三烯酚的含量，从而起到增加大麦谷粒营养成分的作用。目前由于大麦突变体库的缺乏，限制了大麦VE合成通路中相关基因的功能研究。
近年来发展起来的以CRISPR-Cas9为代表的新一代基因组编辑技术，为植物基因工程带来了新的革命，已成为基因功能研究和作物品质改良的重要手段之一。运用基于CRISPR-Cas9的基因编辑技术改造相关基因，并通过自交、分子鉴定和后代筛选，获得“非转基因”大麦新材料，可为今后将成果应用于生产实践提供依据和技术支持。但是因为大麦基因转化效率低，稳定转基因材料的获得周期长，目前运用CRISPR-Cas9系统研究大麦基因的报导很少见。因此，应用CRISPR-Cas9技术敲除大麦VE合成通路中的关键基因(HvHPT)而获得的大麦突变体，可为HvHPT基因的功能研究提供可靠材料。
发明内容
本发明要解决的问题是，克服现有技术中的不足，提供一种运用CRISPR-Cas9系统敲除大麦HPT基因的编辑方法，以获得HPT基因突变的理想突变体。
为解决技术问题，本发明的解决方案是：
提供一种运用CRISPR-Cas9系统敲除大麦HPT基因的编辑方法，包括以下步骤：
(1)gRNA靶位点的选择
由于HPT基因位于大麦基因组的七号染色体上，根据CRISPR-Cas9技术的靶位点设计原则，应尽量将gRNA靶位点设计在外显子区并且要设计在基因的5’端(因该基因编码的是蛋白质，5’端编码的正好是蛋白质的功能区域)。
(2)gRNA片段的克隆
以质粒pGTR为模板，用PCR方法克隆四个片段L1、L2、L3、L4部分重叠的片段，引物序列如下，其中F和R分别代表正、反向引物：
L1-F：CGGGTCTCAGGCAGGATG GGCAGTCTGGGCAACAAAGCACCAGTGG
L1-R：CGGGTCTCACCCCTACCCTATTGCACCAGCCGGG
L2-F：TAGGTCTCCGGGGGTAGGGGTGTTTTAGAGCTAGAA
L2-R：CGGGTCTCACATACTGTTCCTTGCACCAGCCGGG
L3-F：TAGGTCTCCTATGCCGAAACGGTTTTAGAGCTAGAA
L3-R：CGGGTCTCACAGTATCGTGTGTGCACCAGCCGGG
L4-F：TAGGTCTCCACTGCAAGCTTCGTTTTAGAGCTAGAA
L4-R：
TAGGTCTCCAAACGGATGAGCGACAGCAAACAAAAAAAAAAGCACCGACTCG
PCR体系为：Phusion酶0.5μL；5×Phusion HF Buffer 10μL；上下游引物各2.5μL；dNTPs 4μL；pGTR plasmid 0.5μL；三蒸水30μL，共50μL体系。
PCR反应条件为：预变性95℃，5min；变性95℃，30s；退火60℃，30s；延伸72℃，30s；共33个循环；最后72℃延伸10min；
PCR反应后，取5-10μL产物，用2％的琼脂糖凝胶电泳检测后，纯化回收目的片段，测定四个PCR产物L1、L2、L3、L4的浓度；
(3)GG(gRNA-gRNA)片段的连接
根据测定的PCR产物浓度，将四个片段等量混合，T7酶连接反应与BsaI酶切反应同时进行；取L1、L2、L3、L4各2μL，与10μL T7ligase buffer、1μLBsaI-HF、0.5μL T7ligase、0.5μL水混合；在PCR仪中进行如下反应：37℃，5min；20℃，10min；30-50个循环；
(4)连接产物的PCR扩增；
连接反应结束后，取连接产物1μL，加19μL水稀释，将稀释后的产物作为模板，进行PCR扩增；PCR结束后，取5μL产物进行电泳检测，并将产物纯化；产物大小为500bp；
引物序列如下，其中F和R分别代表正、反向引物：
S1-F：CGGGTCTCAGGCAGGATGGGCAGTCTGGGCA
S1-R：TAGGTCTCCAAACGGATGAGCGACAGCAAAC
(5)纯化产物和目标载体的酶切
FokI酶切纯化产物暴露黏性末端，同时BsaI酶切空载体pRGEB32；
酶切体系为50μL，包括底物5μL、FokI或BsaI酶5μL、Buffer(cutsmart)10μL；底物包括GG纯化产物和空载体pRGEB32；
酶切时间3-4h，酶切温度37℃；用2％琼脂糖凝胶检测酶切产物，并回收目标产物，测定浓度；
(6)纯化产物和目标载体的连接反应
取酶切回收的GG纯化产物与pRGEB32载体等量混合(50ng)，T4DNA ligase 1μL，10×T4DNA ligase Buffer 1μL，加三蒸水至20μL，4℃连接过夜；
(7)连接产物转化大肠杆菌感受态
将连接后的载体转化大肠杆菌感受态细胞，涂板，37℃过夜；挑取单菌落，摇菌6-8h，提取质粒，PCR鉴定目标片段是否连入载体；鉴定正确的质粒送去测序，将测序结果正确的质粒，电转化农杆菌AGL1；
(8)农杆菌介导的大麦转基因
以Golden Promise野生型大麦的幼胚为外植体材料，以AGL1农杆菌进行感染转化，经潮霉素抗性筛选，抗性愈伤组织分化再生获得转基因植株；
(9)阳性转基因植株的筛选
提取转基因植株的基因组DNA，在三个gRNA序列的两侧设计引物，对目的片段进行PCR扩增，利用琼脂糖凝胶电泳和垂直聚丙烯凝胶电泳检测突变体；
(10)突变体测序
将以上突变株系PCR产物进行纯化回收，连接T载体测序，确认获得敲除了大麦HPT基因的突变材料。
发明原理描述：
VE的合成通路比较复杂，生育酚和生育三烯酚有共同的合成前体尿黑酸(homogentisate，HGA)。其中生育三烯酚合成的限速步骤是由尿黑酸牻牛儿基转移酶(homogentisate geranylgeranyl transferase，HGGT)催化HGA和牻牛儿焦磷酸(geranylgeranyl diphosphate，GGDP)的合成反应；而生育酚合成的限速步骤是由尿黑酸植基转移酶(homogentisate phytyltransferase，HPT)催化HGA和植基二磷酸(phytyldiphosphate，PDP)的合成反应。HGGT、HPT分别是生育三烯酚、生育酚合成过程中的关键基因，因此可以通过敲除生育酚合成通路中的关键限速酶基因HPT来调节代谢流，从而起到提高或者分离生育三烯酚组分的作用。
CRISPR-Cas系统可定点修饰(删除、添加、激活、抑制)靶细胞中特定的基因序列，为靶向性编辑基因组序列提供行之有效的技术手段。但是，目前尚无运用该技术敲除大麦VE合成通路中的关键基因HPT的报道。
与现有技术相比，本发明的有益效果在于：
本发明利用先进的基因编辑技术-CRISPR-Cas9系统对大麦VE合成通路中的关键基因(HPT)进行靶向基因编辑，获得有效的功能缺失突变体，为大麦中生物活性物质的研究创造条件。
附图说明
图1为靶向大麦HPT基因的3个gRNA位点示意图；
图2为琼脂糖凝胶电泳检测大片段缺失突变体；
图3为PAGE凝胶电泳检测突变体；
图4为突变株系目的片段测序结果的比较(点状虚线表示缺失碱基，单下划线表示插入碱基)。
具体实施方式
实施例1大麦VE合成通路中的关键限速酶基因敲除株系HPT的获得与鉴定。
本发明所用大麦品种为Hordeum vulgare L.,cv.Golden promise，申请人承诺：从本专利申请之日起20年内向公众发放该大麦品种，以用于实现、利用本发明所述技术方案。
1.gRNA靶位点的选择
由于HPT基因位于大麦基因组的七号染色体上，根据CRISPR-Cas9技术的靶位点设计原则，本发明将gRNA靶位点设计在外显子区并且要设计在HPT基因的5’端(因该基因编码的是蛋白质，5’端编码的正好是蛋白质的功能区域)。如图1所示。
2.gRNA片段的克隆及载体构建
2.1以质粒pGTR为模板，用PCR方法克隆四个片段L1、L2、L3、L4部分重叠的片段，引物序列如下：
L1-F：CGGGTCTCAGGCAGGATG GGCAGTCTGGGCAACAAAGCACCAGTGG(如SEQ ID NO：1所示)
L1-R：CGGGTCTCACCCCTACCCTATTGCACCAGCCGGG(如SEQ ID NO：2所示)
L2-F：TAGGTCTCCGGGGGTAGGGGTGTTTTAGAGCTAGAA(如SEQ ID NO：3所示)
L2-R：CGGGTCTCACATACTGTTCCTTGCACCAGCCGGG(如SEQ ID NO：4所示)
L3-F：TAGGTCTCCTATGCCGAAACGGTTTTAGAGCTAGAA(如SEQ ID NO：5所示)
L3-R：CGGGTCTCACAGTATCGTGTGTGCACCAGCCGGG(如SEQ ID NO：6所示)
L4-F：TAGGTCTCCACTGCAAGCTTCGTTTTAGAGCTAGAA(如SEQ ID NO：7所示)
L4-R：
TAGGTCTCCAAACGGATGAGCGACAGCAAACAAAAAAAAAAGCACCGACTCG(如SEQ ID NO：8所示)
PCR扩增L1片段体系如下：
| Phusion酶 | 0.5μL |
| 5×Buffer | 10μL |
| L1-F | 2.5μL |
| L1-R | 2.5μL |
| 三蒸水 | 30μL |
| dNTPs | 4μL |
| 模板(pGTR plasmid) | 0.5μL |
| 总计 | 50μL |
PCR扩增L2片段体系如下：
| Phusion酶 | 0.5μL |
| 5×Buffer | 10μL |
| L2-F | 2.5μL |
| L2-R | 2.5μL |
| 三蒸水 | 30μL |
| dNTPs | 4μL |
| 模板(pGTR plasmid) | 0.5μL |
| 总计 | 50μL |
PCR扩增L3片段体系如下：
PCR扩增L4片段体系如下：
| Phusion酶 | 0.5μL |
| 5×Buffer | 10μL |
| L4-F | 2.5μL |
| L4-R | 2.5μL |
| 三蒸水 | 30μL |
| dNTPs | 4μL |
| 模板(pGTR plasmid) | 0.5μL |
| 总计 | 50μL |
PCR反应程序为：
PCR反应后，取5-10μL产物，用2％的琼脂糖凝胶电泳检测后纯化回收目的片段，测定产物浓度。L1，L2，L3，L4产物大小约为130bp，200bp，200bp，150bp。
2.2 GG(gRNA-gRNA)片段的连接。
按照上步测定的产物浓度，将4个片段等量混合，T7酶连接。反应体系如下：
| 试剂 | 体积(μL) |
| L1 | 2 |
| L2 | 2 |
| L3 | 2 |
| L4 | 2 |
| 2×T7ligase buffer | 10 |
| BsaI-HF | 1 |
| T7ligase | 0.5 |
| 三蒸水 | 0.5 |
| 总体积 | 20 |
以上连接反应在PCR仪中进行：37℃，5min；20℃，10min；30-50个循环。
2.3连接产物的PCR扩增
连接反应结束后，取连接产物1μL，加19μL水稀释，将稀释后的产物作为模板，
以S1-F、S1-R为引物进行PCR扩增。
S1-F：CGGGTCTCAGGCAGGATGGGCAGTCTGGGCA(如SEQ ID NO：9所示)
S1-R：TAGGTCTCCAAACGGATGAGCGACAGCAAAC(如SEQ ID NO：10所示)
PCR体系如下：
| 试剂 | 体积(μL) |
| 稀释后的GG产物 | 2.5 |
| S1-F | 2.5 |
| S1-R | 2.5 |
| 2×TaqMix | 25 |
| 三蒸水 | 17.5 |
| 总体积 | 50 |
PCR反应程序为：
取5-10μL PCR产物电泳检测，产物大小约为500bp，并将产物纯化。
2.4将上一步纯化的产物，FokI酶切暴露黏性末端，同时BsaI酶切空载体pRGEB32。
酶切体系为50μL，包括底物5μL、FokI或BsaI酶5μL、Buffer(cutsmart)10μL；底物包括GG纯化产物和空载体pRGEB32；
酶切时间3-4h，作用温度37℃；用2％琼脂糖凝胶检测酶切产物，并回收目标产物，测定浓度；
2.5酶切后的GG连接产物与目标载体pRGEB32的连接。
| 试剂 | 体积(μL) |
| 酶切后的GG连接产物 | 50ng |
| 酶切后的载体pRGEB32 | 50ng |
| T4DNA ligase | 1 |
| 10×T4ligase buffer | 1 |
| 三蒸水 | 补至10 |
| 总体积 | 10 |
3.将连接后的载体转化大肠杆菌感受态细胞，涂板，37℃过夜；挑取单菌落，摇菌6-8h，提取质粒，PCR鉴定目标片段是否连入载体；鉴定正确的质粒送去测序。
PCR鉴定及测序引物为U3-F，UGW-gRNA-R。序列如下：
U3-F：AGTACCACCTCGGCTATCCACA(如SEQ ID NO：11所示)
UGW-gRNA-R：CGCGCTAAAAACGGACTAGC(如SEQ ID NO：12所示)
4.将测序正确的质粒，电转化农杆菌AGL1。
5.农杆菌介导的大麦的遗传转化
以野生型大麦(Hordeum vulgare L.,cv.Golden Promise)的幼胚为材料诱导愈伤组织，以AGL1农杆菌进行感染转化，经潮霉素抗性筛选，抗性愈伤组织分化再生获得转基因植株；
6.阳性转基因植株的筛选
6.1琼脂糖凝胶电泳检测大片段缺失突变体
以叶片为材料，提取转基因植株的基因组DNA，在3个gRNA位点的两侧设计引物进行PCR扩增，1％琼脂糖凝胶电泳检测。(结果见图2)引物序列如下所示：
H1-F：ACCTTTCAGTCAGTGGCTTTGAACT(如SEQ ID NO：13所示)
H2-R：ACCTCCAGCAATCCAGTAAG(如SEQ ID NO：14所示)
6.2 PAGE胶检测小片段变化的突变体
以叶片为材料，提取转基因植株的基因组DNA，在每个gRNA位点两侧设计引物进行PCR。在常规PCR反应结束后，再对PCR产物进行高温变性、复性反应，PCR程序和变性复性步骤如下表：
引物如下所示：
H1-F：ACCTTTCAGTCAGTGGCTTTGAACT(如SEQ ID NO：13所示)
H1-R：TTACAAGAGGCGTTGCTGGTTCATT(如SEQ ID NO：15所示)
H2-F：CCACAACAAATCTACCGTCTC(如SEQ ID NO：16所示)
H2-R：ACCTCCAGCAATCCAGTAAG(如SEQ ID NO：14所示)
结果见图3。
7.突变株系的基因测序
将以上突变株系PCR产物进行纯化回收。大片段缺失的突变体可直接割胶回收测序，小片段变化的突变体则需连接T载体进行测序。测序公司为杭州擎科梓熙生物技术有限公司。测序结果见图4。
测序结果分析，发现了15#株系存在746bp的大片段缺失，获得了敲除大麦HPT基因的理想突变材料。
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Claims (1)
1.运用CRISPR-Cas9系统敲除大麦HPT基因的编辑方法，其特征在于，包括以下步骤：
(1)gRNA靶位点的选择
由于HPT基因位于大麦基因组的七号染色体上，根据CRISPR-Cas9技术的靶位点设计原则，选择gRNA靶位点在基因5’端的外显子区；
(2)gRNA片段的克隆
以质粒pGTR为模板，用PCR方法克隆四个部分重叠的片段L1、L2、L3、L4；其中，
PCR体系为：Phusion酶0.5μL；5×Phusion HF Buffer 10μL；上下游引物各2.5μL；dNTPs 4μL；pGTR plasmid 0.5μL；三蒸水30μL，共50μL体系；上下游引物的序列如SEQ IDNO：1～8所示：
PCR反应条件为：预变性95℃，5min；变性95℃，30s；退火60℃，30s；延伸72℃，30s；共33个循环；最后72℃延伸10min；
PCR反应后，取5～10μL产物，用2％的琼脂糖凝胶电泳检测后，纯化回收目的片段，测定四个PCR产物L1、L2、L3、L4的浓度；
(3)gRNA-gRNA片段的连接
根据测定的PCR产物浓度，将L1、L2、L3、L4四个片段等量混合，T7酶连接反应与BsaI酶切反应同时进行；取L1、L2、L3、L4各2μL，与10μL T7ligase buffer、1μLBsaI-HF、0.5μLT7ligase、0.5μL水混合；在PCR仪中进行如下反应：37℃，5min；20℃，10min；30-50个循环；
(4)连接产物的PCR扩增；
连接反应结束后，取连接产物1μL，加19μL水稀释，将稀释后的产物作为模板，进行PCR扩增；PCR结束后，取5μL产物进行电泳检测，并将产物纯化；产物大小为500bp；
PCR所用引物的序列如SEQ ID NO：9～10所示；
(5)纯化产物和目标载体的酶切
FokI酶切纯化产物暴露黏性末端，同时BsaI酶切空载体pRGEB32；
酶切体系为50μL，包括底物5μL、FokI或BsaI酶5μL、Buffer(cutsmart)10μL；底物包括GG纯化产物和空载体pRGEB32；
酶切时间3-4h，酶切温度37℃；用2％琼脂糖凝胶检测酶切产物，并回收目标产物，测定浓度；
(6)纯化产物和目标载体的连接反应
取酶切回收的GG纯化产物与pRGEB32载体等量混合(50ng)，T4DNA ligase 1μL，10×T4DNA ligase Buffer 1μL，加三蒸水至20μL，4℃连接过夜；
(7)连接产物转化大肠杆菌感受态
将连接后的载体转化大肠杆菌感受态细胞，涂板，37℃过夜；挑取单菌落，摇菌6-8h，提取质粒，PCR鉴定目标片段是否连入载体；鉴定正确的质粒送去测序，将测序结果正确的质粒，电转化农杆菌AGL1；
(8)农杆菌介导的大麦转基因
以Golden Promise野生型大麦的幼胚为外植体材料，以AGL1农杆菌进行感染转化，经潮霉素抗性筛选，抗性愈伤组织分化再生获得转基因植株；
(9)阳性转基因植株的筛选
提取转基因植株的基因组DNA，在三个gRNA序列的两侧设计引物，对目的片段进行PCR扩增，利用琼脂糖凝胶电泳和垂直聚丙烯凝胶电泳检测突变体；
(10)突变体测序
将以上突变株系PCR产物进行纯化回收，连接T载体测序，确认获得敲除了大麦HPT基因的突变材料。
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