CN117264956A - 甘薯IbSAP15基因启动子、表达盒、重组表达载体及其应用 - Google Patents
甘薯IbSAP15基因启动子、表达盒、重组表达载体及其应用 Download PDFInfo
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Abstract
本发明公开了一种甘薯IbSAP15基因启动子、表达盒、重组表达载体及其应用。所述的甘薯IbSAP15基因启动子的核苷酸序列为SEQ ID NO.1。本发明通过构建含有启动子IbSAP15pro和目的基因的重组表达载体,并将其转化到目标植物中,启动子IbSAP15pro能够驱动目的基因在目标植物中表达,同时目的基因的表达在盐胁迫、干旱胁迫和低温胁迫下明显上调,启动子IbSAP15pro可作为胁迫诱导启动子应用于植物的遗传转化过程中,在植物基因功能研究和耐逆育种中有着广阔的应用前景。
Description
技术领域
本发明属于基因工程技术领域,涉及一种甘薯IbSAP15基因启动子、表达盒、重组表达载体及其应用。
背景技术
启动子是RNA聚合酶和转录因子等相关蛋白识别并结合的DNA序列,包含多种反式作用因子,是调控基因转录水平的重要元件。植物遗传转化中常用的启动子有花椰菜病毒(Cauliflower mosaic virus,CaMV)35S启动子、E12Ω启动子(Wakita等,2001)、烟草泛素启动子(Nicotiana tabacum ubiquitin 4promoter,NtU4)(Yu等,2020)等组成型启动子。
甘薯是重要的粮食与经济作物,其产量高、耐贫瘠,因此提高甘薯对逆境的耐受能力、保证甘薯产量,对于保障粮食安全具有重要意义。目前,基因工程已广泛应用于甘薯品质与抗性改良中,在甘薯中同源或异源表达一些基因均能够提高甘薯对胁迫的耐受能力。使用组成型启动子能够使得目的基因在甘薯中过表达。然而,过表达某些基因会抑制甘薯块根的发育,通过特定的胁迫诱导启动子驱动目的基因能较好的解决这一问题。目前,甘薯中已有报道的胁迫诱导启动子有甘薯过氧化物酶A2(Sweetpotato peroxidase A2,SWPA2)启动子(Kim等,2003)和GIGANTEA(GI)启动子(Tang等,2021)等。这些启动子可以仅在胁迫条件下上调目的基因的表达,从而改良甘薯对非生物胁迫的耐受性。
植物胁迫相关蛋白(Stress-associatedprotein,SAP)家族成员基因编码具有A20/AN1锌指结构域的蛋白,具有转录激活/抑制活性或E3泛素连接酶活性,参与植物生长发育和胁迫应答的调控,是提高植物非生物胁迫耐性的优良候选基因(Giri等,2013)。初步的研究表明,甘薯IbSAP15能够响应盐胁迫和干旱胁迫,过表达IbSAP15能提高拟南芥对盐胁迫的耐受性(杨强强,2019)。
发明内容
本发明的目的之一在于提供一种甘薯IbSAP15基因启动子,命名为启动子IbSAP15pro,其核苷酸序列如SEQ ID NO.1所示。
本发明的目的之二在于提供一种表达盒,其包含上述甘薯IbSAP15基因启动子和目的基因,且甘薯IbSAP15基因启动子连接在目的基因CDS的5’端。
本发明的目的之三在于提供一种重组表达载体,其含有上述甘薯IbSAP15基因启动子。
进一步地,所述的重组表达载体,通过将上述甘薯IbSAP15基因启动子插入植物双元表达载体中,并连接在目的基因CDS的5’端制得。
本发明所述的植物双元表达载体为本领域常见的植物双元表达载体,在本发明具体实施方式中,采用的植物双元表达载体为pCAMBIA1300。
本发明的目的之四在于提供上述甘薯IbSAP15基因启动子在调控下游目的基因在非生物胁迫条件下上调表达中的应用。
本发明所述的目的基因为外源基因,可根据使用需求相应调整。在本发明具体实施方式中,采用的目的基因为GUS报告基因。
在本发明具体实施方式中,所述的非生物胁迫为盐胁迫、干旱胁迫和低温胁迫中的一种或一种以上。
进一步地,上述甘薯IbSAP15基因启动子在调控下游目的基因在非生物胁迫条件下上调表达中的应用,具体应用方法为:
(1)将甘薯IbSAP15基因启动子插入植物双元表达载体中,并连接于目的基因上游,得到重组表达载体;
(2)通过农杆菌介导的遗传转化方法,将重组表达载体转化到目标植物中,并得到转化后的目标植物种子;
(3)利用转化后的目标植物种子,筛选出纯合转基因植物株系,培育相应植株,得到在非生物胁迫条件下上调表达目的基因的植株。
优选地,所述的目标植物为拟南芥,更优选为拟南芥Col-0生态型。
在本发明具体实施方式,步骤(1)中,植物双元表达载体为pCAMBIA1300-35S-GUS载体,重组表达载体为pCAMBIA1300-IbSAP15pro-GUS;步骤(2)中,目标植物为拟南芥Col-0生态型。
与现有技术相比,本发明具有以下优点:
(1)本发明首次分离了甘薯IbSAP15基因的启动子序列IbSAP15pro,该启动子序列中包含多种非生物胁迫应答和激素响应元件;
(2)本发明通过构建含有启动子IbSAP15pro和目的基因的重组表达载体,并将其转化到目标植物中,发现启动子IbSAP15pro能够驱动目的基因在目标植物中表达,同时目的基因的表达在盐胁迫、干旱胁迫和低温胁迫下明显上调,启动子IbSAP15pro可作为胁迫诱导启动子应用于植物的遗传转化过程中,在植物基因功能研究和耐逆育种中有着广阔的应用前景。
附图说明
图1为本发明中分离的甘薯IbSAP15基因启动子IbSAP15pro的PCR扩增电泳图。
图2为本发明中构建的植物表达载体pCAMBIA1300-IbSAP15pro-GUS的示意图。
图3为正常生长条件下IbSAP15pro-GUS拟南芥报告株系的GUS组化染色,(A)全株;(B)茎生叶;(C)莲座叶;(D)根。
图4为胁迫条件下IbSAP15pro-GUS拟南芥报告株系幼苗的GUS组化染色,(A)对照条件;(B)低温胁迫;(C)盐胁迫;(D)干旱胁迫。
具体实施方式
下述实施例中所用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径获得。
实施例1、甘薯IbSAP15启动子IbSAP15pro的分离
(1)依据已发表的甘薯品种泰中6号的参考基因组设计IbSAP15启动子克隆引物。所用引物如下:
IbSAP15pro-F:5’-ACTTCTACCGCCGGAGCTGC-3’(SEQ ID NO.2);
IbSAP15pro-R:5’-CTCTCTGTCTCTTTCTAGCTTTGTTTG-3’(SEQ ID NO.3)。
(2)使用徐紫薯8号基因组DNA为模板,使用高保真酶PrimeSTAR Max(宝生物,R045),进行PCR扩增。PCR体系为:PrimeSTAR Max Premix(2×)25μL,基因组DNA100ng,正反向引物IbSAP15pro-F、IbSAP15pro-R终浓度0.45μmol·L-1,补水至50μL。PCR反应程序为:预变性:98℃,30s。变性:98℃,15s;复性60℃,15s;延伸:72℃,40s。35个循环。终延伸:72℃,2min。
(3)PCR产物以1%琼脂糖凝胶电泳进行检测,按试剂盒说明书,使用GelDNA Extraction Mini Kit胶回收/DNA纯化试剂盒(诺唯赞,DC301)对目的片段进行回收,并将所回收的片段连接至/>Simple Cloning Kit试剂盒中的平末端克隆载体pEASY-Blunt上(全式金,CB111)。使用冻融法将连接产物转入大肠杆菌感受态DH5α中,通过菌液PCR挑选阳性克隆,送至上海擎科生物公司进行测序,测序正确的即为pEASY-Blunt-IbSAP15pro载体。
测序后得到长度为1102bp的IbSAP15启动子(IbSAP15pro),其序列如SEQ ID NO.1所示。将序列提交至PlantCARE在线数据库(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)进行分析,发现该启动子中除真核生物保守的核心启动子元件TATA-box和CAAT-box外,还包含激素应答和胁迫响应元件(表1)。激素应答元件包含脱落酸应答元件AAGAA和ABRE motif、茉莉酸应答元件as-1motif、水杨酸应答元件水杨酸TCAmotif、以及乙烯应答元件ERE。胁迫响应元件中包含NAC家族转录因子识别位点NRS、MYB家族转录因子识别位点,AP2/ERF(APETALA2/ethylene-responsive factor)家族转录因子识别位点CRT、TTTGTT、效应物响应元件W-box等,在甘薯及其他植物的基因功能研究和耐逆育种中具有重要应用价值。
表1甘薯IbSAP15基因启动子区顺式作用元件
实施例2、植物重组表达载体pCAMBIA1300-IbSAP15pro-GUS的构建
使用HindIII和Xba I双酶切后的pCAMBIA1300-35S-GUS载体为骨架,利用同源重组的方法将IbSAP15pro连接到上述骨架上,构建pCAMBIA1300-IbSAP15pro-GUS载体。具体步骤如下:
(1)IbSAP15pro的扩增
以pEASY-Blunt-IbSAP15pro为模板,使用高保真酶PrimeSTAR Max(宝生物,R045),参照实施例1中的体系和程序进行PCR扩增,其中所用引物如下:
IbSAP15pro-GUS-F:
5’-ACGACGGCCAGTGCCAAGCTTAGCAATTAGGGTTTCCTTTTTGCAG-3’(SEQ ID NO.4);
IbSAP15pro-GUS-R:
5’-CATAGGCCTACTAGTTCTAGACTCTCTGTCTCTTTCTAGCTTTGTTTG-3’(SEQ ID NO.5)。
(2)pCAMBIA1300-35S-GUS载体的双酶切
使用HindIII和Xba I(宝生物)双酶切pCAMBIA1300-35S-GUS载体,酶切体系如下:5μL缓冲液(10×),限制性内切酶Hind III和Xba I各2.5μL,pCAMBIA1300-35S-GUS载体2.5μg,补水至50μL。将上述体系37℃孵育30min。
(3)pCAMBIA1300-IbSAP15pro-GUS载体的连接与转化
参照实施例1中所述方法回收用于构建pCAMBIA1300-IbSAP15pro-GUS载体的IbSAP15pro目的片段。使用同源重组试剂盒2×MultiF SeamlessAssembly Mix(爱博泰克,RK21020)进行载体连接,连接体系如下:线性化的pCAMBIA1300-35S-GUS载体50ng,IbSAP15pro目的片段20ng,2×MultiF SeamlessAssembly Mix同源重组试剂5μL,补水至10μL。将连接体系置于PCR仪中,50℃孵育15min,随后立即置于冰上2min,使用冻融法转入大肠杆菌感受态DH5α中,按下述体系进行菌液PCR,挑选阳性克隆,送至上海擎科生物有限公司进行测序。挑选测序正确的单克隆提取质粒,即为构建好的植物重组表达载体pCAMBIA1300-IbSAP15pro-GUS。
实施例3、植物重组表达载体pCAMBIA1300-IbSAP15pro-GUS的转化
(1)使用酒精和次氯酸钠消毒拟南芥的种子,播种于1/2MS培养基上,4℃静置3d后取出,转移至光照培养箱中,光周期为16h光照/8h黑暗,温度为22℃,相对湿度为50%。7d后将拟南芥幼苗移栽至花盆中,继续置于光照培养箱中培养约3周至抽薹。
(2)使用冻融法将实施例2中构建得到的重组表达载体pCAMBIA1300-IbSAP15pro-GUS转化至农杆菌菌株GV3101感受态中,通过菌落PCR鉴定阳性克隆,接种于5mL含有利福平和卡那霉素的液体YEB培养基中,28℃、220rpm条件下过夜培养,至菌液浑浊。按1:100的比例,将过夜培养的农杆菌接种于新鲜的含有利福平和卡那霉素的YEB培养基中,28℃、220rpm条件下培养至OD600≈0.8。离心收集菌体,以1/2MS液体培养基(pH=5.8)重悬,使重悬后菌体OD600在1.0左右,加入表面活性剂Silwet L-77至终浓度0.02%。
(3)修剪抽薹的拟南芥,剪掉已经开放的花和果荚,将剩余的花序浸泡在侵染液中30至60s。将拟南芥暗培养24h,随后置于光照培养箱中直至种子成熟。使用含有20mg·L-1潮霉素的1/2MS固体培养基筛选转基因拟南芥株系,最终得到具有IbSAP15pro-GUS表达盒的纯合拟南芥株系,命名为IbSAP15pro-GUS报告株系。
实施例4、甘薯IbSAP15启动子活性分析
(1)以45日龄的拟南芥IbSAP15pro报告株系检测IbSAP15pro在植物中的活性。
(2)用以下条件处理14日龄的拟南芥IbSAP15pro报告株系,检测IbSAP15pro在低温、盐胁迫和干旱胁迫下的活性。低温胁迫:将拟南芥幼苗转移至用1/2MS液体培养基浸润的滤纸上,置于4℃条件下培养12h;盐胁迫和干旱胁迫:将拟南芥幼苗转移至含有150mmol·L-1NaCl或300mmol·L-1甘露醇的1/2MS液体培养基浸润的滤纸上,22℃培养12h。对照:将拟南芥幼苗转移至1/2MS液体培养基浸润的滤纸上,22℃培养12h。
(3)将上述样品浸泡于GUS染色液中,37℃过夜染色,并以脱色液(乙醇:乙酸:丙三醇=3:1:1)脱色至无背景颜色。其中GUS染色液各组分母液及工作液配制方法如表2所示。
表2GUS染色液的配制
实验结果表明,45日龄的拟南芥IbSAP15pro报告株系叶片和根中均可见明显蓝色,表明甘薯IbSAP15基因启动子IbSAP15pro能够驱动GUS基因在拟南芥叶片和根中表达。在未经处理的14日龄拟南芥IbSAP15pro报告株系中有较浅的蓝色,而在经盐胁迫、干旱胁迫或低温胁迫处理,特别是低温胁迫处理后的报告株系中蓝色加深,表明甘薯IbSAP15基因启动子IbSAP15pro在盐胁迫、干旱胁迫,特别是低温胁迫下明显增加GUS基因的表达,使报告株系呈现更深的蓝色。
Claims (10)
1. 甘薯IbSAP15基因启动子,其特征在于,核苷酸序列如SEQ ID NO.1所示。
2.一种表达盒,其特征在于,包含权利要求1所述的甘薯IbSAP15基因启动子和目的基因,且甘薯IbSAP15基因启动子连接在目的基因CDS的5’端。
3.一种重组表达载体,其特征在于,含有权利要求1所述的甘薯IbSAP15基因启动子。
4.根据权利要求3所述的重组表达载体,其特征在于,通过将权利要求1所述的甘薯IbSAP15基因启动子插入植物双元表达载体中,并连接在目的基因CDS的5’端制得。
5.根据权利要求4所述的重组表达载体,其特征在于,所述的植物双元表达载体为pCAMBIA1300。
6.权利要求1所述的甘薯IbSAP15基因启动子在调控下游目的基因在非生物胁迫条件下上调表达中的应用。
7.根据权利要求6所述的应用,其特征在于,目的基因为GUS报告基因,非生物胁迫为盐胁迫、干旱胁迫和低温胁迫中的一种或一种以上。
8.根据权利要求6所述的应用,其特征在于,具体应用方法为:
(1)将甘薯IbSAP15基因启动子插入植物双元表达载体中,并连接于目的基因上游,得到重组表达载体;
(2)通过农杆菌介导的遗传转化方法,将重组表达载体转化到目标植物中,并得到转化后的目标植物种子;
(3)利用转化后的目标植物种子,筛选出纯合转基因植物株系,培育相应植株,得到在非生物胁迫条件下上调表达目的基因的植株。
9.根据权利要求8所述的应用,其特征在于,步骤(2)中,所述的目标植物为拟南芥。
10.根据权利要求8所述的应用,其特征在于,步骤(1)中,植物双元表达载体为pCAMBIA1300-35S-GUS载体,重组表达载体为pCAMBIA1300-IbSAP15pro-GUS;步骤(2)中,目标植物为拟南芥Col-0生态型。
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