CN114717236A - 一种辣椒受外源糖添加诱导型启动子的克隆及应用 - Google Patents
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Abstract
本发明公开了一种辣椒受外源糖添加诱导型启动子,所述启动子的核苷酸序列如SEQIDN0.1所示,所述启动子来源于辣椒CaRH57基因的上游启动子区。本发明同时公开了上述诱导型启动子的表达载体和转基因植物的构建方法。本发明克隆到了辣椒中与拟南芥AtRH57同源的基因CaRH57的启动子,该启动子为诱导型启动子,不仅能够启动外源基因(GUS基因)在转基因植物(拟南芥)中表达,并且经过外源糖培养处理,该启动子能够增强外源基因GUS的表达,可应用于开展辣椒抗糖胁迫育种。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种辣椒受外源糖添加诱导型启动子的克隆及应用。
背景技术
启动子是基因的一个组成部分,控制基因表达的起始时间和表达的程度。根据其作用方式可分为三类:组成型启动子、诱导型启动子和组织特异型启动子。诱导型启动子是指在某些特定的物理或化学信号的刺激下,该种类型的启动子可以大幅度地提高基因的转录水平。
植物为了生存,在遭遇非生物胁迫时,必须通过形态及生理生化代谢等方面的调整,以适应或抵抗环境胁迫。近年来的研究表明,许多植物基因的表达都受逆境胁迫的诱导,抗逆基因的表达又是依靠其上游启动子的调控来实现的。诱导性启动子通常包含比较保守的顺式作用元件,利用这些保守元件可以推测新基因的可能功能,也可用这些环境应答基因的启动子与抗逆基因融合,从而使转基因植物更好地适应逆境。
糖既是能量和碳骨架的来源,同时也是调控植物生长和发育的重要信号分子。糖响应基因对于植物逆境适应具有重要意义。
发明内容
本发明的目的在于解决现有技术中存在的受外源糖诱导的启动子在异源植物中对下游目的基因表达水平较低的问题,提供一种辣椒受外源糖添加诱导型启动子的克隆及应用。
为解决上述技术问题,本发明采用的技术方案如下:一种辣椒受外源糖添加诱导型启动子,所述启动子的核苷酸序列如SEQ ID N0.1所示,所述启动子来源于辣椒CaRH57基因的上游启动子区。
优选地,扩增所述启动子的引物序列如SEQ ID N0.2和SEQ ID N0.3所示。
本发明同时提供了上述诱导型启动子在植物抗糖胁迫育种中的应用。
本发明还提供含有上述诱导型启动子的表达载体。
优选地,上述表达载体还含有GUS基因,所述表达载体的核苷酸序列如SEQ IDN0.4所示。
本发明还提供了一种转基因植物的构建方法,包括以下步骤:将上述表达载体导入目的植物,构建GUS基因表达受到外源糖添加诱导的转基因植物,所述目的植物为拟南芥。
优选地,所述外源糖添加的条件为在1/2MS培养基中添加4.5%的Glc。
本发明所具有的有益效果:本发明克隆到了辣椒中与拟南芥AtRH57同源的基因CaRH57的启动子,该启动子为诱导型启动子,不仅能够启动外源基因(GUS基因)在转基因植物(拟南芥)中表达,并且经过外源糖(4.5%的Glc)培养处理,该启动子能够显著增强外源基因(GUS基因)的表达,对有效开展辣椒抗糖胁迫育种具有重要意义。
附图说明
图1为实施例1中CaRH57启动子片段电泳图;
图2为实施例1中CaRH57启动子的序列分析;
图3为实施例4中CaRH57-GUS转基因拟南芥植株;野生型(Col-0)与CaRH57-GUS转基因植株的表型;
图4为实施例3和实施例4中CaRH57-GUS转基因拟南芥植株的GUS染色分析图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步的说明。
T-Vector(TaKaRa);含有外源基因GUS的二元载体、农杆菌和野生型拟南芥(Col-0)由西南大学植物分子实验室提供;LEAGENE GUS染色试剂盒购自LEAGENE。
实施例1CaRH57启动子克隆及序列分析
1.CaRH57启动子克隆
从PepperHub(http://pepperhub.hzau.edu.cn/)数据库中下载辣椒CaRH57基因DNA序列,选择ATG上游约1.5kb长度作为CaRH57启动子区域,以序列SEQ ID N0.2和SEQ IDN0.3为引物,通过PCR扩增获得CaRH57启动子片段,连入T-vector,通过测序获得正确的CaRH57启动子片段,CaRH57启动子的核苷酸序列如SEQ ID N0.1所示;CaRH57启动子片段电泳图如附图1所示,片段长度为1437bp;本实施例中PCR扩增所用引物详见表1:
表1CaRH57启动子扩增所用的引物序列
Primer name | Primer sequences(5`-3`) |
CaRH57pro-F | <u>GGTACC</u>TGATGAGCACAGTTACATAG(SEQ ID No.2) |
CaRH57pro-R | <u>GTCGAC</u>CTCTCAACTGGATATTTGG(SEQ ID No.3) |
表1中下划线所标注的序列(GGTACC、GTCGAC)为引入的限制性内切酶识别位点。
2.CaRH57启动子序列分析
利用PLACE数据库(http://www.dna.affrc.go.jp/PLACE/)进行启动子调控元件分析,结果见表2和附图2:
表2CaRH57启动子所含有的启动子必需元件及响应环境胁迫的元件汇总表
实施例2构建含有CaRH57启动子和报告基因GUS的重组载体
在实施例1的基础上,将实施例1获得的正确无突变的CaRH57启动子片段转接入含有报告基因GUS的双元表达载体中;获得含有CaRH57启动子和报告基因GUS的重组载体,即CaRH57-GUS表达载体,其核苷酸序列如SEQ ID N0.4所示。
实施例3构建转基因拟南芥植株
在实施例2的基础上,将实施例2中构建成功的CaRH57-GUS二元载体转入农杆菌中,再通过花絮浸染法转入野生型拟南芥(Col-0),收取T0代种子,通过潮霉素筛选,得到拥有潮霉素抗性的转基因拟南芥植株CaRH57-GUS。
对转基因拟南芥植株CaRH57-GUS进行GUS染色鉴定。GUS染色鉴定方法为:取待染转基因拟南芥植株CaRH57-GUS加入适量GUS染色液使其完全浸没组织,37℃孵育过夜,70%乙醇脱色,拍照观察。如附图4所示,附图4中左侧植株为在常规培养基1/2MS(sugar free)中培养的转基因拟南芥植株CaRH57-GUS的GUS化学染色图,可以看到植株有明显染色,因此确定GUS基因的表达,证明CaRH57启动子序列是具有启动子功能的,并能在异源植物中有效启动外源基因的表达。
实施例4糖诱导实验
以野生型拟南芥(Col-0)种子和实施例3中获得的转基因拟南芥植株CaRH57-GUS的T1代种子为糖诱导实验对象。将野生型拟南芥(Col-0)种子进行消毒,4℃纯化3天,在1/2MS(sugar free)培养基中培养5天后,分别转置1/2MS(sugar free)和1/2MS(sugar free)添加4.5%Glc的培养基中垂直培养2天。对实施例3中获得的转基因拟南芥植株CaRH57-GUS的T1代种子进行同样操作。实验结果如附图3所示,野生型拟南芥(Col-0)和转基因拟南芥植株CaRH57-GUS在1/2MS(sugar free)培养7天后,长势无明显差别。
对生长在1/2MS(sugar free)培养基和1/2MS(sugar free)添加4.5%Glc培养基中的转基因拟南芥植株CaRH57-GUS进行GUS染色。结果如附图4所示,1/2MS(sugar free)添加4.5%Glc的培养基培养后的转基因拟南芥植株的染色深度明显大于1/2MS(sugar free)培养基培养后的转基因拟南芥植株的染色深度,说明报告基因GUS在外添加4.5%Glc的培养中增强了表达。因此,在外源糖诱导的培养条件下,CaRH57启动子可以显著增强外源基因GUS的表达,致使转基因植株染色加深,证明CaRH57的启动子明显受到外源糖的诱导。
综上所述,本发明构建了含有诱导型启动子CaRH57和外源基因(GUS报告基因)的表达载体,并且转入拟南芥,获得转基因拟南芥植株CaRH57-GUS。实施例3和实施例4说明本发明构建的CaRH57启动子能有效启动外源基因(GUS报告基因)在拟南芥植株中的表达,并且该启动子在外源糖诱导条件下(4.5%Glc)能显著增强外源基因(GUS报告基因)的表达,是一种响应外源糖诱导表达的诱导型启动子,其在基因工程植物育种中有着非常重要的应用价值。
本发明的说明书和附图被认为是说明性的而非限制性的,在本发明基础上,本领域技术人员根据所公开的技术内容,不需要创造性的劳动就可以对其中一些技术特征做出一些替换和变形,均在本发明的保护范围内。
Claims (7)
1.一种辣椒受外源糖添加诱导型启动子,其特征在于,所述启动子的核苷酸序列如SEQID N0.1所示,所述启动子来源于辣椒CaRH57基因的上游启动子区。
2.根据权利要求1所述的诱导型启动子,其特征在于,扩增所述启动子的引物序列如SEQ ID N0.2和SEQ ID N0.3所示。
3.一种表达载体,其特征在于,含有权利要求1或2所述的诱导型启动子。
4.根据权利要求3所述的表达载体,其特征在于,还含有GUS基因,所述表达载体的核苷酸序列如SEQ ID N0.4所示。
5.一种转基因植物的构建方法,其特征在于,包括以下步骤:将权利要求4所述的表达载体导入目的植物,构建GUS基因表达受到外源糖添加诱导的转基因植物,所述目的植物为拟南芥。
6.根据权利要求5所述的构建方法,其特征在于,所述外源糖添加的条件为在1/2MS培养基中添加4.5%的Glc。
7.如权利要求1或2所述的诱导型启动子在植物抗糖胁迫育种中的应用。
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