CN114807165A - 一种玉米ZmNAC78基因的应用 - Google Patents
一种玉米ZmNAC78基因的应用 Download PDFInfo
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- CN114807165A CN114807165A CN202210399568.XA CN202210399568A CN114807165A CN 114807165 A CN114807165 A CN 114807165A CN 202210399568 A CN202210399568 A CN 202210399568A CN 114807165 A CN114807165 A CN 114807165A
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- zmnac78
- gene
- maize
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Abstract
本发明提供了一种玉米ZmNAC78基因的应用，玉米ZmNAC78基因在玉米植株中过表达，用于提高玉米抗旱性；玉米ZmNAC78基因的核苷酸序列如SEQ ID NO:1所示，玉米ZmNAC78基因的氨基酸序列如SEQ IDNO:2所示。本发明的玉米ZmNAC78基因在玉米植株中过表达，用于提高玉米抗旱性。
Description
技术领域
本发明属于玉米抗旱技术领域，具体涉及一种玉米ZmNAC78基因的应用。
背景技术
玉米是重要的粮食、饲料和能源作物，随着人口的増加和工业的发展，对玉米的需求量不断增加。玉米产量的安全问题是国之大事,而干旱胁迫又频频严重发生,导致产量下降,要减少或解决这个问题,必须培育出抗旱型新品种。干旱胁迫已然成为影响玉米生长发育和产量最重要的非生物胁迫之一。挖掘优良抗旱基因,通过转基因或者分子辅助育种,将抗旱基因导入到骨干自交系中，从而有效提高玉米的抗旱性。
发明内容
本发明所要解决的技术问题在于针对上述现有技术的不足，提供一种玉米ZmNAC78基因的应用，该玉米ZmNAC78基因在玉米植株中过表达，用于提高玉米抗旱性。
为解决上述技术问题，本发明采用的技术方案是：一种玉米ZmNAC78基因的应用，所述玉米ZmNAC78基因在玉米植株中过表达，用于提高玉米抗旱性；所述玉米ZmNAC78基因的核苷酸序列如SEQ ID NO:1所示，所述玉米ZmNAC78基因的氨基酸序列如SEQ ID NO:2所示。
优选地，所述玉米ZmNAC78基因在玉米植株中过表达的方法为：
S1、ZmNAC78-pUC57质粒的合成：为方便载体构建，在ZmNAC78基因的核苷酸序列的两端添加Eco31I酶切位点序列，为方便后续蛋白互作研究，同时在3’端终止密码子前添加一段30个核苷酸的myc标签序列，合成带酶切位点和myc标签的ZmNAC78基因，连入pUC57载体中，得到ZmNAC78-pUC57质粒；
所述ZmNAC78基因的核苷酸序列如SEQ ID NO:1所示；
所述带酶切位点和myc标签的ZmNAC78基因的核苷酸序列如SEQ ID NO:3所示；
所述myc标签序列的核苷酸序列如SEQ ID NO:4所示；
所述ZmNAC78基因的核苷酸序列5’端添加的Eco31I酶切位点序列的核苷酸序列如SEQ ID NO:5所示；
所述ZmNAC78基因的核苷酸序列3’端添加的Eco31I酶切位点序列的核苷酸序列如SEQ ID NO:6所示；
S2、玉米ZmNAC78基因过表达载体的构建：
对S1中得到的基因质粒ZmNAC78-pUC57质粒和UBI启动子驱动的过表达载体质粒pCambia3300分别用Eco31I进行酶切，电泳验证片段大小后，分别得到ZmNAC78目的片段和pCambia3300载体大片段，用琼脂糖凝胶回收试剂盒将酶切电泳产物回收后，将所述pCambia3300载体大片段和所述ZmNAC78目的片段在T4连接酶的作用下，在温度为37℃的条件下连接反应1h，得到连接产物；
所述连接反应的体系为：pCambia3300载体大片段3μL、ZmNAC78目的片段5μL、T4DNA连接酶1μL、10×T4 DNA连接酶缓冲液1μL；
将得到的连接的产物转化大肠杆菌感受态细胞，将转化菌液涂布于含卡那霉素的LB固体培养基上，在温度为37℃的条件下培养12h后，挑选阳性克隆进行培养，用引物ZmNAC78-F和ZmNAC78-R进行菌斑PCR鉴定，鉴定到1080bp条带即为阳性克隆，得到玉米ZmNAC78基因过表达载体，即pCambia3300-ZmNAC78质粒；
所述菌斑PCR鉴定的PCR扩增的反应体系为：Taq酶1μL、引物ZmNAC78-F 2μL、引物ZmNAC78-R 2μL、10x buffer 2.5μL、dNTP mix5μL、无菌水补足至25μL；
所述菌斑PCR鉴定的PCR扩增的反应程序为：94℃预变性5min；94℃变性30s，58℃退火30s，72℃延伸1min，共35个循环后，72℃延伸10min；
所述引物ZmNAC78-F的核苷酸序列如SEQ ID NO:7所示；
所述引物ZmNAC78-R的核苷酸序列如SEQ ID NO:8所示；
S3、ZmNAC78基因的玉米遗传转化：
用S2中得到的pCambia3300-ZmNAC78质粒转化农杆菌，侵染玉米自交系B104的幼胚，经过诱导培养、共培养培养、筛选培养、分化培养、生根培养和移栽成苗，得到ZmNAC78转基因的阳性植株；
S4、ZmNAC78转基因的阳性植株的鉴定：S3中得到的ZmNAC78转基因的阳性植株通过bar试纸检测阳性植株，然后通过PCR扩增检测带酶切位点和myc标签序列的ZmNAC78基因，然后实时定量PCR检测，与玉米自交系B104相比，ZmNAC78基因表达水平升高，则为玉米ZmNAC78基因过表达植株。
本发明与现有技术相比具有以下优点：
本发明的玉米ZmNAC78基因在玉米植株中过表达，用于提高玉米抗旱性。
下面结合附图和实施例对本发明作进一步详细说明。
附图说明
图1是本发明的实施例2的ZmNAC78基因在干旱胁迫下表达水平检测图。
图2是本发明的实施例2的ZmNAC78-OE过表达植株中ZmNAC78表达水平检测图。
图3本发明的实施例2的ZmNAC78-OE过表达转基因植株抗旱性分析图。
图4本发明的实施例2的ZmNAC78-OE过表达转基因植株干旱胁迫下生存率图。
图5本发明的实施例2的ZmNAC78-OE过表达转基因植株叶片失水率图。
图6本发明的实施例2的ZmNAC78-OE过表达转基因植株叶片气孔形态图。
图7本发明的实施例2的ZmNAC78-OE过表达转基因植株叶片气孔大小统计图。
具体实施方式
实施例1
本实施例的玉米ZmNAC78基因的应用，所述玉米ZmNAC78基因在玉米植株中过表达，用于提高玉米抗旱性；所述玉米ZmNAC78基因的核苷酸序列如SEQ ID NO:1所示，所述玉米ZmNAC78基因的氨基酸序列如SEQ ID NO:2所示。
所述玉米ZmNAC78基因在玉米植株中过表达的方法为：
S1、ZmNAC78-pUC57质粒的合成：为方便载体构建，在ZmNAC78基因的核苷酸序列的两端添加Eco31I酶切位点序列，为方便后续蛋白互作研究，同时在3’端终止密码子前添加一段30个核苷酸的myc标签序列，合成带酶切位点和myc标签的ZmNAC78基因，连入pUC57载体中，得到ZmNAC78-pUC57质粒；
所述ZmNAC78基因的核苷酸序列如SEQ ID NO:1所示；
所述带酶切位点和myc标签的ZmNAC78基因的核苷酸序列如SEQ ID NO:3所示；
所述myc标签序列的核苷酸序列如SEQ ID NO:4所示；
所述ZmNAC78基因的核苷酸序列5’端添加的Eco31I酶切位点序列的核苷酸序列如SEQ ID NO:5所示；
所述ZmNAC78基因的核苷酸序列3’端添加的Eco31I酶切位点序列的核苷酸序列如SEQ ID NO:6所示；
S2、玉米ZmNAC78基因过表达载体的构建：
对S1中得到的基因质粒ZmNAC78-pUC57质粒和UBI启动子驱动的过表达载体质粒pCambia3300用Eco31I进行酶切，分别得到ZmNAC78目的片段(1000bp左右)和pCambia3300载体大片段(10000bp左右)，用琼脂糖凝胶回收试剂盒将酶切电泳产物回收后，将所述pCambia3300载体大片段和所述ZmNAC78目的片段在T4连接酶的作用下，在温度为37℃的条件下连接反应1h，得到连接产物；
所述连接反应的体系为：pCambia3300载体大片段3μL、ZmNAC78目的片段5μL、T4DNA连接酶1μL、10×T4 DNA连接酶缓冲液1μL；
将得到的连接的产物转化大肠杆菌感受态细胞，将转化菌液涂布于含卡那霉素的LB固体培养基上，在温度为37℃的条件下培养12h后，挑选阳性克隆进行培养，用引物ZmNAC78-F和ZmNAC78-R进行菌斑PCR鉴定，鉴定到1080bp条带即为阳性克隆，得到玉米ZmNAC78基因过表达载体，即pCambia3300-ZmNAC78质粒；
所述菌斑PCR鉴定的PCR扩增的反应体系为：Taq酶1μL、引物ZmNAC78-F 2μL、引物ZmNAC78-R 2μL、10x buffer 2.5μL、dNTP mix5μL、无菌水补足至25μL；
所述菌斑PCR鉴定的PCR扩增的反应程序为：94℃预变性5min；94℃变性30s，58℃退火30s，72℃延伸1min，共35个循环后，72℃延伸10min；
所述引物ZmNAC78-F的核苷酸序列如SEQ ID NO:7所示；
所述引物ZmNAC78-R的核苷酸序列如SEQ ID NO:8所示；
S3、ZmNAC78基因的玉米遗传转化：
用S2中得到的pCambia3300-ZmNAC78质粒转化农杆菌，侵染玉米自交系B104幼胚的幼胚，经过诱导培养、共培养培养、筛选培养、分化培养、生根培养和移栽成苗，得到ZmNAC78转基因的阳性植株；
具体的方法为：首先，挑选大小均一的玉米自交系B104幼胚，在无菌操作台中剥取幼胚，34℃高温诱导暗培养3d；再放在培养箱25℃，暗培养7d。将诱导产生的愈伤切去内生芽，接种至继代培养基中25℃暗培养7d，继代两次，备用于农杆菌转化。愈伤组织置于无菌的侵染培养基，洗两次；最后一次洗完倒掉液体加入菌液(OD600＝0.4)，侵染30min；农杆菌侵染后，胚在无菌滤纸上吸干农杆菌，转移至共培养基表面，胚轴端与培养基接触(盾片朝上)，23℃暗培养3d。筛选2次，待长出完整的再生苗后转入生根培养基中，28℃，16h光照，进行生根壮苗，根长至2cm左右，进行炼苗移栽；
S4、ZmNAC78转基因的阳性植株的鉴定：S3中得到的ZmNAC78转基因的阳性植株通过bar试纸检测阳性植株，然后通过PCR扩增检测带酶切位点和myc标签序列的ZmNAC78基因，然后实时定量PCR检测，与玉米自交系B104相比，ZmNAC78基因表达水平升高，则为玉米ZmNAC78基因过表达植株(记为ZmNAC78-OE转基因植株)。
实施例2
本实施例为实施例1中的玉米ZmNAC78基因过表达植株(ZmNAC78转基因植株)的耐旱性分析：
通过qRT-PCR实验检测，发现ZmNAC78受干旱胁迫诱导上调表达(图1)。ZmNAC78-OE转基因植株中ZmNAC78基因表达水平显著高于对照玉米自交系B104(图2)。为了确认过表达ZmNAC78-OE玉米是否比玉米自交系B104的玉米抗旱，对两种玉米进行了干旱胁迫处理。正常生长时，土壤水分保持在90-100％，待玉米生长到三叶期时停止浇水。在干旱胁迫之前，B104和过表达ZmNAC78-OE玉米的长势差别不大，经13天的干旱胁迫后，两者在表型上出现显著差异，玉米自交系B104(图中Control)较过表达ZmNAC78-OE玉米出现严重的叶片打卷萎蔫和逐渐变黄现象。复水后，过表达ZmNAC78-OE玉米能够恢复到原来的生长状态(图3)，且其存活率显著高于玉米自交系B104(Control)(图4)，过表达玉米ZmNAC78-OE较玉米自交系B104具有更强的抗旱性，说明过表达ZmNAC78基因增强玉米抗旱性。通过进一步分析，发现ZmNAC78-OE转基因植株在干旱胁迫下，失水率显著低于对照玉米自交系B104(Control)(图5)。通过显微镜观察干旱胁迫下气孔关闭速率，发现ZmNAC78-OE转基因植株气孔关闭更迅速(图6)。在干旱胁迫处理5min和10min时，ZmNAC78-OE转基因植株气孔孔径显著小于对照玉米自交系B104(Control)(图7)。以上结果说明ZmNAC78可通过调节气孔运动增强抗旱性。
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制。凡是根据发明技术实质对以上实施例所作的任何简单修改、变更以及等效变化，均仍属于本发明技术方案的保护范围内。
序列表
<110> 河南农业大学
<120> 一种玉米ZmNAC78基因的应用
<130> 2022.3.20
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Claims (2)
1.一种玉米ZmNAC78基因的应用，其特征在于，所述玉米ZmNAC78基因在玉米植株中过表达，用于提高玉米抗旱性；所述玉米ZmNAC78基因的核苷酸序列如SEQ ID NO:1所示，所述玉米ZmNAC78基因的氨基酸序列如SEQ ID NO:2所示。
2.根据权利要求1所述的一种玉米ZmNAC78基因的应用，其特征在于，所述玉米ZmNAC78基因在玉米植株中过表达的方法为：
S1、ZmNAC78-pUC57质粒的合成：在ZmNAC78基因的核苷酸序列的两端分别添加Eco31I酶切位点序列，同时在3’端终止密码子前添加myc标签序列，合成带酶切位点和myc标签的ZmNAC78基因，连入pUC57载体中，得到ZmNAC78-pUC57质粒；
所述ZmNAC78基因的核苷酸序列如SEQ ID NO:1所示；
所述带酶切位点和myc标签的ZmNAC78基因的核苷酸序列如SEQ ID NO:3所示；
所述myc标签序列的核苷酸序列如SEQ ID NO:4所示；
所述ZmNAC78基因的核苷酸序列5’端添加的Eco31I酶切位点序列的核苷酸序列如SEQID NO:5所示；
所述ZmNAC78基因的核苷酸序列3’端添加的Eco31I酶切位点序列的核苷酸序列如SEQID NO:6所示；
S2、玉米ZmNAC78基因过表达载体的构建：
对S1中得到的基因质粒ZmNAC78-pUC57质粒和UBI启动子驱动的过表达载体质粒pCambia3300分别用Eco31I进行酶切，电泳验证片段大小后，分别得到ZmNAC78目的片段和pCambia3300载体大片段，用琼脂糖凝胶回收试剂盒将酶切电泳产物回收后，将所述pCambia3300载体大片段和所述ZmNAC78目的片段在T4连接酶的作用下，在温度为37℃的条件下连接反应1h，得到连接产物；
所述连接反应的体系为：pCambia3300载体大片段3μL、ZmNAC78目的片段5μL、T4 DNA连接酶1μL、10×T4 DNA连接酶缓冲液1μL；
将得到的连接的产物转化大肠杆菌感受态细胞，将转化菌液涂布于含卡那霉素的LB固体培养基上，在温度为37℃的条件下培养12h后，挑选阳性克隆进行培养，用引物ZmNAC78-F和ZmNAC78-R进行菌斑PCR鉴定，鉴定到1080bp条带即为阳性克隆，得到玉米ZmNAC78基因过表达载体，即pCambia3300-ZmNAC78质粒；
所述菌斑PCR鉴定的PCR扩增的反应体系为：Taq酶1μL、引物ZmNAC78-F 2μL、引物ZmNAC78-R 2μL、10x buffer 2.5μL、dNTP mix 5μL、无菌水补足至25μL；
所述菌斑PCR鉴定的PCR扩增的反应程序为：94℃预变性5min；94℃变性30s，58℃退火30s，72℃延伸1min，共35个循环后，72℃延伸10min；
所述引物ZmNAC78-F的核苷酸序列如SEQ ID NO:7所示；
所述引物ZmNAC78-R的核苷酸序列如SEQ ID NO:8所示；
S3、ZmNAC78基因的玉米遗传转化：
用S2中得到的pCambia3300-ZmNAC78质粒转化农杆菌，侵染玉米自交系B104的幼胚，经过诱导培养、共培养培养、筛选培养、分化培养、生根培养和移栽成苗，得到ZmNAC78转基因的阳性植株；
S4、ZmNAC78转基因的阳性植株的鉴定：S3中得到的ZmNAC78转基因的阳性植株通过bar试纸检测阳性植株，然后通过PCR扩增检测带酶切位点和myc标签的ZmNAC78基因，然后实时定量PCR检测，与玉米自交系B104相比，ZmNAC78基因表达水平升高，则为玉米ZmNAC78基因过表达植株。
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