CN107266541B - 玉米转录因子ZmbHLH167及其应用 - Google Patents

玉米转录因子ZmbHLH167及其应用 Download PDF

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CN107266541B
CN107266541B CN201710467168.7A CN201710467168A CN107266541B CN 107266541 B CN107266541 B CN 107266541B CN 201710467168 A CN201710467168 A CN 201710467168A CN 107266541 B CN107266541 B CN 107266541B
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CN107266541A (zh
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宋任涛
冯帆
祁巍巍
朱晨光
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University of Shanghai for Science and Technology
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Abstract

本发明涉及玉米转录因子ZmbHLH167及其应用。该基因为SEQ ID NO：1所示的碱基序列。该序列编码的蛋白ZmbHLH167通过利用CRISPR‑Cas9技术将ZmbHLH167的基因片段SEQ ID NO：2作为guide RNA进行转化玉米幼胚，并获得基因缺失突变体的植株。相对野生型籽粒而言，转基因突变体玉米籽粒显著变小但发芽率并未受到影响。生化分析表明，转基因突变体玉米籽粒的淀粉含量明显下降，而蛋白和总油脂含量显著上升，为创造高品质玉米提供遗传资源。

Description

玉米转录因子ZmbHLH167及其应用
技术领域
本发明涉及一种玉米转录因子ZmbHLH167及其应用。
技术背景
玉米（Zea mays）是世界上最重要的粮食作物之一，同时也是重要的禽畜饲料以及重要的工业原料。随着世界人口的不断增加，提高玉米的产量和品质对于人类来说具有重要的意义。玉米胚乳是玉米籽粒主要的营养储存场所，胚乳中含有大量的淀粉（占胚乳干重约80%）和蛋白（占胚乳干重约10%），玉米的胚中含有大量的油脂。
研究者通过对一系列玉米籽粒突变体的研究，希望能够提高玉米的产量和品质。例如， opaque2突变体使得人类必需氨基酸赖氨酸含量增加接近2倍。研究表明，此突变体蛋白品质的改善得益于非醇溶蛋白和醇溶蛋白比例的增加。因此，玉米籽粒突变体的产生对于研究和改造玉米具有极其重要的研究意义。
CRISPR-Cas9 技术是继锌指核酸酶（ZFN）、ES 细胞打靶和TALEN 等技术后第四种基因敲除的新技术，此种基因敲除的方法具有效率高、速度快、生殖系转移能力强及简单经济的特点，被广泛应用于敲除各个物种并且效率很高。其作用机制是利用靶点特异性的RNA 将 Cas9 核酸酶带到基因组上的具体靶点，从而对特定基因位点进行切割导致突变。
之前的研究报道，在模式植物拟南芥中有一个名为ZHOUPI (ZOU)的bHLH转录因子，ZOU的突变导师拟南芥种子胚乳滞留且导致胚的发育受阻，故认为ZOU是一个调控胚乳发育的重要转录因子。随后有文献报道在玉米中利用RNA干扰技术将ZOU在玉米中的的直系同源进行基因沉默（也就是ZmbHLH167），但由于RNA干扰后的籽粒仍有45%的残余表达，故并未观察到明显的突变表型。为了对ZmbHLH167进行更为透彻的研究，我们利用CRISPR-Cas9技术将ZmbHLH167进行基因敲除。敲除后发现籽粒明显变小，但籽粒的发芽率并未发生显著变化；生化成分分析发现，胚乳三大营养物质的含量的比例发生显著变化，使得玉米胚乳中较为匮乏的蛋白和油脂的含量均发生显著升高，故为高品质玉米的创造做出重要贡献。
发明内容
本发明的目的之一在于提供一种转录因子ZmbHLH167。
本发明的目的之二在于提供该转录因子ZmbHLH167的构建方法。
本发明的目的之三在于提供该转录因子的应用。
为达到上述目的，本发明采用如下技术方案：
一种玉米转录因子ZmbHLH167，其特征在于该转录因子具有SEQ ID NO:1所示的碱基序列。
一种载体，其特征在于该载体含有上述的转录因子ZmbHLH167。
一种构建上述的玉米转录因子ZmbHLH167的方法，其特征在于该方法的具体步骤为：采用pCAMBIA3301 为转基因载体，将SEQ ID NO:2所示序列作为gRNA spacer和scaffold连入pCAMBIA3301转基因载体中获得，其中pCAMBIA3301 载体经过改造，以玉米U6启动子和终止子表达gRNA，同时以玉米泛素启动子和NOS终止子表达玉米密码子优化的Cas9蛋白。
一种上述的转录因子ZmbHLH167在控制玉米籽粒大小的应用。
一种上述的转录因子ZmbHLH167在玉米胚乳中调控淀粉、蛋白和油脂成分含量中的应用。
本发明通过生物信息学分析找到胚乳特异高表达的转录因子ZmbHLH167，并通过CRISPR-Cas9基因编辑的方法获得了该转录因子的突变体。
通过ZmbHLH167蛋白含量的检测，发现ZmbHLH167突变体中ZmbHLH167蛋白完全缺失。
通过表型观察发现ZmbHLH167突变体籽粒显著变小且出现粉质表型。
通过发芽率测试，发现ZmbHLH167突变体并不影响籽粒的发芽率。
通过生化分析发现，ZmbHLH167突变体胚乳的总淀粉含量显著下降，但总蛋白和总油脂的含量显著升高，为创造高品质玉米提供重要的遗传资源。
附图说明
图1 是ZmbHLH167转基因CRISPR-Cas9载体构建示意图。
图2 是ZmbHLH167 CRISPR-Cas9基因编辑两个阳性事件籽粒（1号事件和4号事件）中ZmbHLH167在基因组水平的编辑情况。
图3 是ZmbHLH167 CRISPR-Cas9 4号事件和6号事件未成熟籽粒中ZmbHLH167在蛋白水平的表达量检测。
图4是ZmbHLH167 CRISPR-Cas9 4号事件和6号事件成熟籽粒表型观察。
图5是ZmbHLH167 CRISPR-Cas9 4号事件发芽率检测。
图6是ZmbHLH167 CRISPR-Cas9 4号事件的总淀粉含量的检测。
图7是ZmbHLH167 CRISPR-Cas9 4号事件的总蛋白含量的检测。
图8是ZmbHLH167 CRISPR-Cas9 4号事件的总油脂含量的检测。
具体实施方式
下面结合具体实施事例，进一步阐述本发明。应理解，这些实例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体实验条件的实验方法，通常按照常规条件，如分子克隆（Molecular Cloning: A Laboratory Manual,3rd ed.）或植物分子生物学－实验手册（Plant Molecular Biology－A Laboratory Manual, Melody S. Clark编， Springer-verlag Berlin Heidelberg, 1997）中所述条件，或按照制造厂商所建议的条件。
实施例一： ZmbHLH167的CRISPR-Cas9转基因载体构建，并用于转基因转化。
选取本实验室之前已经构建完毕的适用于玉米的CRISPR-Cas9载体作为农杆菌转化玉米幼胚的载体。用Pst I酶切载体，将合成的用于转基因的ZmbHLH167特异序列连接guide RNA序列连同玉米U6启动子和终止子插入到载体中（图1），测序正确后，电击转化至EHA105农杆菌菌株。选取PBPA玉米品系的授粉8-12天的幼胚，大小约1.5mm左右作为受体材料，进行幼胚转化，具体流程：
1.农杆菌侵染10min-共培养20℃ 3天。
2.恢复培养28℃ 7天-筛选培养（双丙氨磷 1.5mg/l）28℃ 14天。
3.筛选培养（双丙氨磷 3mg/l）28℃ 14天 3-5轮。
4.获得抗性愈伤组织-暗再生培养28℃ 14-21天。
5.光再生培养28℃ 14-21天-获得阳性苗。
6.移入盆中，授粉并获得后代。
结果：选取1000个幼胚作为受体材料，经过转化筛选后获得9个转基因阳性事件。获得后对各个事件进行鉴定，通过TPS法抽提各事件植株的基因组，并针对guide RNA序列所在基因组的位置，设计跨越guide RNA序列PCR引物，扩增得到目的片段后进行TA克隆，挑选阳性克隆进行测序，获得了具有移码突变形式的4号和6号事件（图2）。扩繁保种，用于获得纯合突变体并进行下游分析。证明转基因突变体材料成功获得。
实施例二：ZmbHLH167的转基因4号和6号事件移码突变材料中ZmbHLH167表达情况的检测
1.取未成熟籽粒8-10颗。
2. 分别抽提移码突变的ZmbHLH167突变体材料和野生型材料总蛋白。使用液氮研磨达到粉末级，装入EP管中，加入IP裂解液，冰上裂解20min。
3. 最高转速离心，取上清。两个样品各取4μl的蛋白，加入1μl混有1M DTT的5×SDS 蛋白上样缓冲液，99℃变性10分钟后，立即将蛋白样品插在冰上。
4. SDS-PAGE电泳，堆积胶为5%，80V电泳半小时后，分离胶为12.5%，电泳时间约为2小时。
5. 200mA转膜1h。用TBST配置5%牛奶室温封闭1h。
6. 用ZmbHLH167抗体和tubulin抗体（sigma）以1/1000比例稀释在5%牛奶中。室温杂交1h。
7. TBST洗膜6次，每次5min。
8. 用相应二抗室温杂交1h。
2.TBST洗膜3次，每次5min。
3.加入显色底物，用TANON化学发光成像仪现象。
结果显示内参Tubulin在两种材料中的含量基本一致，而ZmbHLH167仅存在于野生型基因型的材料中（图3），而转基因4号和6号事件移码突变材料中无法检测到ZmbHLH167蛋白。说明ZmbHLH167蛋白缺失是ZmbHLH167移码突变体的突变本质。
实施例三：ZmbHLH167 CRISPR-Cas9 4号事件突变体籽粒的表型观察
分别在自然光下和灯箱上观察ZmbHLH167的转基因移码突变材料籽粒的表型（图4）。观察发现ZmbHLH167的转基因移码突变材料籽粒与同一果穗上的野生型籽粒相比，籽粒明显变小。灯箱上观察发现ZmbHLH167的转基因移码突变材料籽粒与野生型籽粒相比，透光度显著降低，呈现粉质胚乳表型。
实施例四：ZmbHLH167 CRISPR-Cas9 4号事件突变体发芽率测试
挑选同一果穗的野生型与ZmbHLH167的Cas9缺失突变体各20 颗籽粒进行发芽率测试，平行取三组进行统计分析，结果发现ZmbHLH167的Cas9缺失突变体籽粒的发芽率与野生型籽粒相比并无明显差异(图5)，故ZmbHLH167的突变并不影响籽粒的萌发。
实施例五： ZmbHLH167 CRISPR-Cas9 4号事件突变体的胚乳总淀粉含量检测
1. 研磨成熟的玉米籽粒；
2. 取 100mg 粉末到 15ml 离心管，加入 200μl 的 80%乙醇，混匀；
3. 加入 2ml 的 2M KOH，于 4℃环境下，摇床摇晃 20min 以上；
4. 加入 8ml 的 1.2M 醋酸钠缓冲液（ PH=3.8），混匀，然后加入 100μl 的
bottle 1 和 100μl 的 bottle 2,50℃水浴 30min，中间间歇混匀；
5. 把反应液加入 50ml 容量瓶中，加水调节到 50ml；
6. 取出 500μl 到 EP 管中， 1800g 离心 10min；
7. 取 50μl 到 10ml 离心管中，加入 3ml GOPOD Reagent，在 50℃放置 20min；
8. 对照为 D-Glucose： 100μ l D-Glucose 标准品溶液加入 3ml GOPODReagent；空白对照为 100μl 水加入 3ml GOPOD Reagent；
9. 510nm 波长测每个样品，计算总淀粉含量
结果显示，ZmbHLH167突变体胚乳的总淀粉含量与同一果穗的野生型籽粒相比，总淀粉含量显著下调(图6)。
实施例六：ZmbHLH167 CRISPR-Cas9 4号事件突变体胚乳总蛋白含量检测
1. 在同一个成熟的果穗上收取野生表型和突变表型籽粒，去皮去胚，在液氮中研磨成粉末，抽干机中抽干至恒重。
2. 50mg 为一份单位，转移至 2mL EP 管中。野生表型和突变表型各三份。加入1mL 石油醚， 4℃摇床 1h。
3. 12000rpm 离心 10min，去除石油醚，抽干机抽干。
4. 加入1mL 硼酸钠蛋白提取液和 20µL 巯基乙醇，37℃摇床过夜。
5. 12000rpm 离心 10min，吸取 300µL 作为籽粒提取的总蛋白。
6. 另吸取300µL，加入700µL无水乙醇，室温摇床 2h。
7. 12000rpm 离心 10min，吸取 300µL 上清抽干即醇溶蛋白，用 200µL IPG 溶液溶解。
8. 沉淀即非醇溶蛋白，用 70%乙醇洗两遍，风干，用 200µL IPG 溶液溶解。
结果显示，ZmbHLH167突变体胚乳的总蛋白含量与同一果穗的野生型籽粒相比，总蛋白含量显著升高(图7)。
实施例七：ZmbHLH167 CRISPR-Cas9 4号事件突变体胚乳总油脂含量检测
1. 标样的配置。内标十九烷酸甲酯（ Methyl nonadecanoate, C19:0）溶于适量的正己烷溶液中，混匀，最终形成 1mg/ml 的溶液；各类外标可以根据购买量配置成合适的母液（置-20OC），工作液为 1mg/ml。
2. 脂肪酸的提取。
a）混合的玉米籽粒中取 50 粒， 45 OC 烘干 60 小时，用高速万能粉碎机粉碎。
b)每个样品称取两份 0.2～0.3g 玉米粉末放入 15ml 的细胞培养管中，加4ml无水甲醇∶氯乙酰(10∶1）的混合液， 5ml 约 1mg/ml 内标，振荡混匀， 80OC 水浴 2 小时。
c)水浴结束后取出，冷却到室温，加 5ml7％K2CO3 溶液中和脂肪酸。
d）置 4度冰箱待于气相色谱上机。
e) 气相色谱操作条件：
柱温：采用程序升温方式，开始以 220OC 保持 13min，然后以 20OC/的速率上升至 240OC 并保持 5min，整个过程为 18min。氢火焰离子化监测器（ Flame IonizationDetector, FID）检测温度：2500C。
结果显示，ZmbHLH167突变体胚乳的总油脂含量与同一果穗的野生型籽粒相比，总油脂含量显著升高(图8)。
<110> 上海大学
<120> 玉米转录因子ZmbHLH167及其应用
<160> 2
<210> 1
<211> 1875
<212> DNA
<213> 基因序列
<400> 1
ATGTC TCAGG AAGGA GCCAA CCTGC CGCAA GAAGT GGTGG GGAGC CATGA TCAGG CCACC 60
GCCCC CCACG GCAGC ATCCC TGCAC CGGCT GATTC CAACC CCAGC TCTGT CAGCA ACCTG 120
GCCAG CGCCG TCAAC AACGG CGGGT CGTCT GAGTG TGCCA GCCCA GCGGT GCTCT CTGCC 180
GGCGA GGACA ACAAC GCGGC GTCGT CCAAG ACCGC CAGCC CGGCG GTGCT CTCTG CCGGC 240
GAGGA CAACA ACGCG GCGTC GTCCA AGACC GCCAG CCCGG CGGTG CTCCA TGCTG CCGAC 300
GACAA CAACG CGGCG TCCAA GATCG CAGGC CCGGC GGTGC TCCTT GCCGG CGAGG AAAAC 360
AAGGC CAAGC TCAAG CTCGC GAGCC CGGCA TCGCT CCTTG CCGGC GAGGA GAAGG GCAAC 420
AACGC TGATG AGTTC AAGCT CGCTA GCCCA AGGAT GCTCC ATGAC TGCGA CGACA ACAAT 480
GCCGG GTCCC ATGCT GGCGT GCGCA ACTGC AACGC CGCCC AGTCC AAGCT CGCCA ACCCG 540
GCGGT TCTCC ATGCC GGCGA TGACA AGAAG GGCGG ATCCA AGATC GCCGG CCCGG AAGTG 600
CCCCA CGCCA GTGTG ACCAG CACCA CCGGA TCCAA GCTCA ACGGC CCGGC GGCGC TCCAT 660
GCCGG CAATG ACAAC GACAG CGGCG GATCC AGGCT CCCAA ACCCC GCAGT GCTCC ATGCC 720
AGCAA AGACA TGGAC AACAA CGCCG GGCGG CCCAA GTTCG CCAGC CCGGT GTTGC TCCAT 780
GCTGG CGAGG ACAAC AAAGC CAGGA CCAAG CTCGC AATCC AGGCG GGCGG TGGCG GTAAC 840
GCCGG AGCCG GATCG TCCAG GCTCA CCAGA TCGGC GGCGC TCCAT GCCGG GAAGG ACAAC 900
GGCGC CGGAT CCAAG CCCGC CATCC AGGTG GTGCC ACGCC TCCAT GCCGG CGGCA AGGAC 960
AACAA CGCTG GGTCG TTCAA GGCTG CCAGA CCGTC GGCGG CCGAC TCCGG CGAGA GCAAC 1020
GCCAA GGAGG GAAAG AGCAA CGTAG CTGGA GAACA ACGTG CCCGT GAGGC CGGCG TGGGC 1080
TGTGG CGGCG GGAAG GGCAA CGCCG CCGCG GTGGA GGATG TGGAC CACGA CTTGC ACATC 1140
TTTAC GGAGA GGGAG CGGAG GAAGA AGATG AAGAA CATGT TCAGC ACCCT ACACG CGCTC 1200
CTCCC GCAGC TCCCC GACAA GGCTG ACAAG GCCAC CATAG TCGGG GAGGC TGTAA CCTAC 1260
ATCAA GACTC TGGAA GGCAC CGTCC AGAAG CTGGA GAAGC TGAAG CTGGA GCGCA AGCGC 1320
GCGCT GGCAG CGCAG CAGCA GCTGA TGGCT GGTGC CGGCA GCAAC CGCGC GTCGT CCGCG 1380
CGCCA TCCCG CACCA GCGCC GTCGT CACCG TCGTC GTCGT CGAGG GAGGC GAACG TGGCG 1440
GACAT GGTCC ACGGT TGGCA TGCGC AGCAG GCCGC CGCGA ACAAG GCCCT GGCAG CGGAG 1500
GCCGG GGCGG GCGGC TCCTC CTCTG CCGCC GCCTC GCTGC CCCGT GGAGC GGTGC CCTTC 1560
CCCGC GCCCG CGGCG GGGTT CCAGA CGTGG TCCGG GCAGA ACGTC GTGGT GAGCG TGGCC 1620
AGCAA CGAGG CGTAC ATCAA CCTGC ACTCC CCGCG GCAGC CGGCG GGCAC CCTGA CCAAG 1680
GCGCT GTTCG TGCTG GAGAG GCACC GCATC GACGT CGTCA CGACG ACCAT CTCCA CCCAG 1740
GACGG CTTCC ACATG TACGG CATCC ATGCA CGCGT TAATT CGGCT TCCGC TTCGG CTCGC 1800
TTTCC GGAGA ATCTG TGTGC TGAAG ACAGG TTCAA GCTGG CGGTG TCGGA GATGC TGCAG 1860
CTGAT CAACA TCTGA 1875
<210> 2
<211> 20
<212> DNA
<213> 基因序列
<400> 2
gggca acgcc gccgc ggtgg 20

Claims (2)

1.一种靶向玉米转录因子ZmbHLH167的CRISPR-Cas9表达载体的构建方法，所述玉米转录因子ZmbHLH167的序列如SEQ ID NO:1所示，其特征在于，所述构建方法的具体步骤为：采用pCAMBIA3301为转基因载体，将SEQ ID NO:2所示序列作为gRNAspacer和scaffold连入pCAMBIA3301转基因载体中获得，其中pCAMBIA3301载体经过改造，以玉米U6启动子和终止子表达gRNA，同时以玉米泛素启动子和NOS终止子表达玉米密码子优化的Cas9蛋白。
2.根据权利要求1所述方法构建的靶向玉米转录因子ZmbHLH167的表达载体在玉米胚乳中调控淀粉、蛋白和油脂成分含量中的应用，其特征在于：所述玉米胚乳的总淀粉含量下降，总蛋白和总油脂的含量升高。
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