CN116144659A - 拟南芥β-酮脂酰-ACP还原酶基因启动子及其应用 - Google Patents

拟南芥β-酮脂酰-ACP还原酶基因启动子及其应用 Download PDF

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CN116144659A
CN116144659A CN202211695857.0A CN202211695857A CN116144659A CN 116144659 A CN116144659 A CN 116144659A CN 202211695857 A CN202211695857 A CN 202211695857A CN 116144659 A CN116144659 A CN 116144659A
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ketoacyl
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姜伊娜
王双双
龙思宇
张强
卢磊
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Abstract

本发明公开了拟南芥β‑酮脂酰‑ACP还原酶基因启动子及其应用。本发明启动子具有SEQ ID No.1所示的核苷酸序列，控制拟南芥β‑酮脂酰‑ACP还原酶基因在正常生长发育的拟南芥幼苗下胚轴及根、成苗的叶片及雌蕊柱头中，以及接种白粉菌（G.cichoracerum,UCSC1）后的成苗叶片真菌侵染部位特异表达。本发明提供了一个新的组织特异、且受白粉真菌侵染特异诱导表达的启动子，为提高植物对白粉真菌抗性的基因工程育种提供了新的调控元件。

Description

拟南芥β-酮脂酰-ACP还原酶基因启动子及其应用
技术领域
本发明涉及植物基因工程技术领域；具体涉及一种拟南芥响应白粉真菌侵染特异表达的KAR(Ketoacyl-ACP reductase，KAR)基因启动子及其应用。
背景技术
由子囊菌亚门(Ascomycotina)白粉病目(Erysiphales)真菌引发的白粉病(Powdery mildew)是最常见的植物真菌病害之一，可侵染近万种被子植物，在小麦、茄科、豆科等许多重要农作物上发生普遍，给农业生产造成了巨大的损失(Celio and Hausbeck,1998；Byrne et al.,2000)。筛选及鉴定植物的抗白粉病相关基因并应用于生产是领域内的研究热点。
脂肪酸及其衍生物脂类是细胞的关键组成部分，主要为细胞及亚细胞器提供生物膜骨架；是细胞生命活动所必需的能量来源之一，也是生物体最佳的能量储存方式。对于脂肪酸在植物-微生物互作过程中的作用已有大量报道。脂肪酸的从头生物合成过程(FAS)包括缩合、还原、脱水、再还原四个阶段，由七种酶介导完成，包括：丙酮酸激酶(Pyruvatekinase，PK)、丙二酰基CoA-ACP转酰酶(Malonyl CoA-ACP transacylase，MCAT)、酮脂酰-ACP合成酶(Ketoacyl-ACP synthase，KAS)、酮脂酰-ACP还原酶(Ketoacyl-ACP reductase，KAR)、烯酰-ACP还原酶(Enoyl-ACP reductase，ENR)、羟烷基-ACP脱水酶(Hydroxyacyl-ACPdehydratase，HAD)和酰基-ACP硫酯酶B(Acyl-ACP thioesterase B，FatB)(Gah-Hyun Limetal.2017)。
TAIR数据库中的芯片表达数据显示，编码β-酮脂酰-ACP还原酶的KAR基因受白粉菌诱导高度特异表达，已有研究表明拟南芥KAR(At1g24360)基因的T-DNA插入突变体kar-1(SALK_011081)在接种白粉菌后出现了较野生型Col-0显著增强的抗性表型，表明脂肪酸合成过程参与调控植物白粉病抗性(Yina Jiang et al.,2017)，KAR基因在植物抗白粉病过程中发挥重要功能。此外，在玉米瘤黑粉菌(U.maydis)侵染宿主的转录组数据中，玉米中的KAR同源基因也受病原菌特异诱导表达，暗示脂肪酸合成过程可能广泛参与调控植物的抗活体营养型真菌过程(Doehlemann et al.,2008)。
启动子是位于结构基因5'端的上游特定核苷酸序列，包含可被转录因子识别和结合的顺式作用元件，在基因的表达调控中发挥着关键作用。植物的启动子可分为三类：组成型启动子、组织特异型启动子、诱导型启动子。在植物的基因工程育种中，组成型启动子例如花椰菜病毒CaMV35S能驱动外源基因在所有的植物组织和器官中表达，但也会导致细胞内物质和能量的过度消耗，而对植物的正常生长发育造成负面影响。克隆并鉴定植物受白粉菌特异诱导表达的启动子，将其应用到植物的遗传操作中，能精准调控植物抗病基因在病原菌侵染响应过程中表达，且不影响植物的生长发育，为农作物的抗病转基因新品种的培育打下基础。
发明内容
为了弥补现有技术的缺点与不足，本发明的目的在于提供一种拟南芥β-酮脂酰-ACP还原酶基因启动子及其应用。
本发明的目的通过下述技术方案实现：
一种拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子，其为SEQ ID NO.1所示的核苷酸序列。
一种含有所述β-酮脂酰-ACP还原酶基因KAR的启动子构建的植物表达载体。
所述的植物表达载体，其为pBGWFS7。
一种所述拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子或所述的表达载体在改善植物白粉真菌抗性上的应用。
一种所述拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子或所述的表达载体在植物育种中的应用。
一种所述拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子或所述的表达载体在培育转基因植物中的应用。
所述的植物包括拟南芥、番茄、水稻、小麦和玉米。
本发明相对于现有技术具有如下的优点及效果：
本发明克隆了拟南芥β-酮脂酰-ACP还原酶KAR基因启动子序列，利用植物表达载体，获得了KAR基因启动子连接GUS报告基因的纯合转基因材料pKAR1-708bp::GUS，对其幼苗、成苗、生殖期等不同生长发育阶段的植物组织器官材料进行GUS染色试验，检测到KAR启动子驱动GUS报告基因在拟南芥的下胚轴、根、叶片及雌蕊柱头中特异地表达。对短日照下生长至四周大的转基因材料叶片接种白粉菌后进行GUS染色和WGA染色试验，发现KAR启动子驱动GUS报告基因在白粉菌侵染的部位被特异诱导表达。本发明提供的KAR基因启动子序列不仅可用于基因在植株幼苗、叶片和雌蕊生殖发育中的特异表达的研究，还与植物的白粉病抗性相关，对采用生物强化手段改善作物对白粉病的抗性提供了研究基础和科学依据。
附图说明
图1为KAR启动子中顺式作用元件的生物信息学分析示意图；
图2为KAR启动子表达载体Promoter-pBGWFS7的构建示意图；
图3为KAR启动子响应生长发育过程的GUS染色结果分析；
图4为KAR启动子响应白粉菌侵染的GUS染色及WGA染色结果分析。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。除非另行定义，下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室指南(New York：Cold Spring HarborLaboratory Press)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。所用引物合成及测序工作由生工(上海)生物技术有限公司完成。
文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1.拟南芥KAR基因启动子的克隆
根据NCBI(https://www.ncbi.nlm.nih.gov/)网站所提供的拟南芥基因组序列，选取KAR基因转录起始位点上游708bp的范围作为候选启动子区域。本发明通过CTAB法提取拟南芥野生型Col-0叶片的基因组DNA，以此gDNA为模板，使用翊圣生物科技公司的高保真DNA聚合酶Hieff
Figure BDA0004023443720000031
Gold High Fidelity DNA Polymerase，设计并合成PCR正向及反向引物进行扩增。克隆获得KAR启动子的gDNA序列PKAR1-708bp后使用Hieff/>
Figure BDA0004023443720000032
Plus OneStep Cloning Kit试剂盒，利用重组交换方法将KAR基因启动子序列插入pENTR中间载体上，转化大肠杆菌DH5α，筛选到阳性克隆后提取质粒并测序，结果如SEQ ID NO.1所示。
所用PCR引物序列如下：
PKAR1-708bp-F：5′-GGAGCCCTTCACCGATTGAGGAACCAGGCA-3′；
PKAR1-708bp-R：5′-CGCGCCCACCCTTTGCCTGGTTCCTCAATC-3′；
PCR反应体系为：
Figure BDA0004023443720000033
Figure BDA0004023443720000041
PCR扩增条件为：
Figure BDA0004023443720000042
实施例2.拟南芥KAR基因启动子的生物信息学分析
将KAR启动子序列输入PLACE数据库，对序列所包含的顺式作用元件(cis-element)进行了预测。如图1所示，KAR启动子区富集了多种类型的顺式调控元件，其中包括生长发育及激素信号响应过程相关的元件，如：557-570位碱基和658-671位碱基是与脂肪酸合成代谢相关的AW-box元件、在270-275位碱基有与生长素(Auxin)信号响应相关的TGA-motif、在545-550位碱基和674-679位碱基有与油菜素甾醇(Brassinosteroid)响应相关的E-box元件、在115-119位碱基与甲基茉莉酸(Methyl Jasmonate)响应相关的MeJARE元件、在697-703位碱基与赤霉素(Gibberellins)响应相关的GARE元件、在575-581位碱基的与乙烯(Ethylene)信号相关的ERE元件、在640-649位碱基与水杨酸(Salicylic acid)响应相关的TCA-motif元件、在367-372位碱基含有与防御及胁迫响应相关的W-box元件等。
上述相关顺式作用元件的序列如下表所示：
Figure BDA0004023443720000043
Figure BDA0004023443720000051
以上结果表明KAR基因启动子可能会受到环境胁迫和生长素、水杨酸、脱落酸及乙烯等植物激素的诱导。
实施例3.拟南芥KAR基因启动子融合GUS标签的转基因株系的获得
通过Gateway LR反应，将KAR基因启动子从pENTR中间载体上构建至pBGWFS7植物表达载体(如图2所示)，转化大肠杆菌DH5α，筛选到阳性克隆后提取质粒测序比对，用冻融法将测序验证正确的pBGWFS7-PKAR1-708bp载体质粒转入根癌农杆菌菌株GV3101中，用农杆菌介导的浸花法转化至拟南芥野生型Col-0(野生型拟南芥Col-0购自ArabidopsisBiological Resource Center)背景中。配制含转化辅助剂silwet-77(购自Biotopped公司，CAS.NO:27306-78-1，使用浓度为200μL/L)和高浓度蔗糖(50g/L)的1/2MS培养基溶液，将农杆菌稀释至OD600约为1.0，选择抽薹至10-15cm的拟南芥，将角果和已经完全开放的花剪去，仅留下刚刚露白及幼嫩的花蕾进行浸染，浸花后的拟南芥用黑色塑料袋包好，避光保湿22℃过夜。约4周后对转化植株收种子。将收集所获的T1代转基因种子撒于灭菌土上，待幼苗长出第一对真叶时，喷洒适量0.05％的Basta溶液，每隔三天喷一次，连续喷洒三次。选取生长健康、发育良好的幼苗移栽并单株收种，再通过Basta溶液对T2代种子进行筛选，全抗Basta的幼苗对应株系为纯合株系。其中，pBGWFS7空载体转化Col-0得到的纯合株系，记为Empty Vector，简称EV。
实施例4.KAR启动子响应生长发育过程的表达情况分析
收集T3代转pBGWFS7空载拟南芥和T3代转pBGWFS7-PKAR1-708bp拟南芥植株7天大的幼苗、四周大的成苗莲座叶和抽薹后的花序等组织，放入10ml离心管中，置于冰上。倒入适量GUS染液至没过材料，抽真空15分钟，用锡箔纸包裹10ml管，置于37℃摇床，220rpm摇5h至过夜。倒掉GUS染液，换95％乙醇脱色2-4h后，换75％乙醇脱色至阴性对照材料呈白色，在体式解剖镜下观察并且照相记录。
以转pBGWFS7空载体拟南芥为对照，T3代转pBGWFS7-PKAR1-708bp植株营养期和生殖期不同组织器官的GUS染色结果如图3所示。结果显示：在7天大的转基因拟南芥幼苗中，KAR启动子驱动报告基因在下胚轴及中柱鞘中特异表达；在短日照下生长至四周大的成苗莲座叶中，KAR启动子驱动报告基因仅在新叶叶缘的水孔位置及极少数叶脉中有极低表达；在开花后，KAR启动子驱动报告基因在成熟的雌蕊柱头中有特异表达。以上结果表明，KAR基因的启动子在拟南芥生长发育过程中呈现明显的组织特异表达特点。
实施例5.KAR启动子响应白粉菌侵染过程的情况分析
将白粉菌(G.cichoracerum,UCSC1)接种到短日照下四周大的T3代转pBGWFS7空载拟南芥和T3代转pBGWFS7-PKAR1-708bp拟南芥植株莲座叶上，分别取未接菌的对照材料和接菌后的转基因材料莲座叶先进行GUS染色，后将GUS染色结束的叶片用PBS溶液洗3次以上，加入提前配好的WGA染液(WGA：PBS＝1：500)，室温避光静置过夜，在体式解剖镜的白光通道及绿色荧光通道下分别观察GUS及WGA染色情况并且照相记录。
结果显示，在白粉菌侵染后1-4天的叶片上只能看到显示白粉菌侵染结构的WGA绿色荧光信号，而KAR启动子驱动的GUS报告基因一直保持仅在叶片边缘水孔位置表达的模式，未见其他GUS信号。而到了第5天时，在叶片上检测到与白粉菌绿色荧光染色高度重叠的GUS蓝色信号(图4)，表明此时KAR驱动报告基因在白粉菌侵染的位置显著表达。该结果证实KAR启动子在拟南芥响应白粉菌侵染的过程中受诱导表达的模式具有高度的时空特异性。综上，本发明中所举例说明的发明可适当地在不存在本文中未具体公开的任何要素、限制的情况下实施。因此，例如术语“包含/包括”等应理解为开放式的且没有限制。另外，本发明中采用的术语和表达用作描述而非限制的术语，并且并非旨在使用排除所示出且描述的特征或其部分的任何等同特征的此类术语和表达，但是应认识到，在本发明所要求保护的范围可进行多种修改。因此，应理解，尽管已通过优选实施方案和任选特征具体公开了本发明，但是本领域技术人员可采用本发明中公开的其中体现发明的修改方案和变化方案，并且这样的修改方案和变化方案被认为在本发明的范围之内。

Claims (7)

1.一种拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子，其为SEQ ID NO.1所示的核苷酸序列。
2.一种含有权利要求1所述β-酮脂酰-ACP还原酶基因KAR的启动子构建的植物表达载体。
3.根据权利要求2所述的植物表达载体，其为pBGWFS7。
4.一种权利要求1所述拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子或权利要求3所述的表达载体在改善植物白粉真菌抗性上的应用。
5.一种权利要求1所述拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子或权利要求3所述的表达载体在植物育种中的应用。
6.一种权利要求1所述拟南芥β-酮脂酰-ACP还原酶基因KAR的启动子或权利要求3所述的表达载体在培育转基因植物中的应用。
7.根据权利要求4、5或6所述的应用，其特征在于，所述的植物包括拟南芥、番茄、水稻、小麦和玉米。
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