CN115838746A - 拟南芥bdr3基因在调控植物耐盐性中的应用 - Google Patents
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Abstract
本发明公开了拟南芥BDR3基因在调控植物耐盐性中的应用,所述拟南芥BDR3基因的编码区核苷酸序列如SEQ ID No.1所示。本发明通过对拟南芥BDR3基因缺失突变、过表达植株盐胁迫研究发现,BDR3基因缺失突变相对于野生型能显著提高其耐盐性,而过表达植物相对于野生型对盐更加敏感。说明BDR3基因对植物耐盐性具有重要的调控作用。
Description
技术领域
本发明属于基因工程领域,具体涉及一种拟南芥BDR3基因在调控植物耐盐性中的应用。
背景技术
盐胁迫是制约植物生长分布和农作物产量的重要环境胁迫因素,已成为世界性生态和农业问题。以Na+为主的盐离子在土壤中过度沉积后,在细胞生理水平上引起离子毒害、渗透胁迫和氧化损伤,严重破坏植物营养代谢和光合效率等生理活动,进而影响植物生长发育,降低农业生产(Deinlein et al.,2014;Morton et al.,2019)。因此,探究植物盐胁迫耐受性调控机制,对于通过基因工程改良和提高农作物耐盐能力,具有重要意义和应用价值。
植物响应盐胁迫是涉及多信号通路级联交叉的复杂生理过程(Yang&Guo,2018)。在植物耐盐性机制研究中,人们陆续发现植物通过维持Na+/K+平衡、渗透调节和氧化损伤控制与修复等核心途径来应对高盐环境(Zhu,2002;2016)。以模式植物拟南芥为例,当植物处于高盐生长环境中,位于细胞膜上的HKT(High Affinity K+Transporter)、AKT1(Arabidopsis K Transporter 1)、NSCC(Nonselective Cation Channel)和NORC(Nonselective Outward-Rectifying Conductance)等通道蛋白介导Na+进入胞内(Apse&Blumwald et al.,2002),经由Ca2+和ROS等第二信使感知盐信号,植物通过依赖于Ca2+的SOS(Salt Overly Sensitive)途径外排Na+(Zhu,2000;Ma et al.,2019;Yang et al.,2019)、NHX1反向转运体以及AVP1和V-ATPase蛋白液泡区域化Na+(Apse et al.,1999)来维持盐胁迫下胞内Na+/K+平衡,另有HKT1参与Na+由根部向地上部分的运输(Sunarpi et al.,2005)。另一方面,盐胁迫诱导植物体内带电荷代谢物、多元醇、可溶性糖和复合糖等渗透调节物质积累来降低胞内渗透势,稳定细胞结构(Zhu,2016;Yang&Guo,2018)。此外,植物能够通过调动抗氧化系统及时有效地清除过量ROS避免氧化损伤(Miller et al.,2010)。基于植物抵御逆境和维持生长发育双重需求,植物激素信号通路如ABA、JA、GA和BR等也在植物应答盐胁迫中发挥重要作用(Park et al.,2016)。在拟南芥基因组范围内鉴定盐胁迫应答基因,显示30%的基因转录水平受盐胁迫影响(Kreps et al.,2002)。
综上,多个胁迫诱导途径彼此交叉,构成了共同调控植物耐盐生理响应的复杂网络。目前为止,植物耐盐性调控网络仍存在许多未解问题,大量未知功能基因参与其中。BDR3基因编码脂肪代谢酶,其在植物生理上的作用尚不清楚。目前并没有BDR3基因参与调控植物抗逆的报道。因此,研究BDR3基因在植物逆境调控网络中的作用具有重要意义。
发明内容
本发明所要解决的技术问题为:提供了BDR3基因在调控植物耐盐性中的用途。
本发明的技术方案为:拟南芥BDR3基因在调控植物耐盐性中的应用,所述拟南芥BDR3基因的编码区核苷酸序列如SEQ ID No.1所示。
进一步地,所述应用为通过抑制植物中BDR3基因的表达来提高植物耐盐性。
进一步地,所述抑制植物中BDR3基因的方式为基因沉默、基因突变或基因敲除。
本发明通过对拟南芥BDR3基因缺失突变、过表达植株盐胁迫研究发现,BDR3基因缺失突变相对于野生型能显著提高其耐盐性,而过表达植物相对于野生型对盐更加敏感。说明BDR3基因对植物耐盐性具有重要的调控作用。
与现有技术相比,本发明具有以下有益效果:
利用正向遗传学技术,筛选到在植物耐盐性中起重要调控作用的BDR3基因,通过植物生理生化手段验证了BDR3参与调节植物耐盐适应过程。将BDR3基因在拟南芥中过表达,显著改变了转基因拟南芥抗盐能力,对植物耐盐性的研究提供了基因资源,具有重要的理论意义和应用价值。
附图说明
图1:左侧为野生型,右侧为突变体。通过正向遗传学手段筛选获得具有盐耐受表型的突变体,突变基因为BDR3。
图2:在300mM NaCl盐处理下3周幼苗的生长情况,Col-0代表拟南芥野生型,bdr3代表EMS诱变所得点突变体,bdr3-1和bdr3-2代表具有不同T-DNA插入位点的BDR3基因缺失突变体,如图显示突变体表现出盐耐受表型。
图3:在300mM NaCl盐处理下3周幼苗的生长情况,Col-0代表拟南芥野生型,bdr3代表基因缺失突变体,BDR3OE-1和OE-2代表BDR3过表达的两个独立转基因株系,转基因植物表现出盐敏感表型。
图4:以ACTIN为参照,在盐胁迫处理0-8h期间,BDR3基因表达呈下降趋势。
图5:在未处理和200mM NaCl盐处理下3周幼苗的叶片含水量情况,Col-0代表拟南芥野生型,bdr3代表EMS诱变所得点突变体,bdr3-1和bdr3-2代表具有不同T-DNA插入位点的BDR3基因缺失突变体。在未处理下,突变体与野生型叶片含水量无明显区别,在盐处理下,bdr3点突变体和T-DNA插入突变体的叶片含水量显著高于野生型。
图6:在200mM NaCl盐处理下3周幼苗的组织化学染色情况。上下两组分别代表针对活性氧物质超氧阴离子和过氧化氢积累,莲座叶的NBT染色和DAB染色图片。
具体实施方式
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为从商业渠道购买得到的。
一、实验方法
1.材料与种植方法
在本研究中植物材料以拟南芥(Arabidopsis thaliana)哥伦比亚生态型(Col-0)为野生型,拟南芥BDR3基因点突变体由本实验室构建,T-DNA插入突变体购买自ABRC。
将经过表面消毒的种子点播在含有土和蛭石(2:1)的混合土壤中,4℃冷室春化处理3d后置于培养室,培养条件为:温度25/20℃,光照周期(16h light/8dark),光照强度为150μmol/m2/s,湿度50%-70%。待种子萌发后幼苗生长3周,进行200mM NaCl的盐胁迫处理,正常培养的幼苗作为未处理对照。收取盐胁迫72h拟南芥叶片样品用于含水量测定及活性氧物质染色分析,其余样品均用液氮冷冻后放置于-80℃冰箱中备用。
2.叶片相对含水量测定
待拟南芥幼苗生长3周后,根据上述胁迫处理条件,分别从对照组和胁迫组中选择大小长势一致的野生型Col-0和突变体各剪取5片莲座叶称取鲜重(FW),浸没在培养皿中吸满水24h后,用滤纸擦干再测定其饱和鲜重(SW),在烘箱65℃烘干至恒重,称其干重(DW),根据公式:RWC(relative water content)=(FW-DW)/(SW-DW)计算相对含水量。
3.活性氧物质的组织化学染色
DAB染色检测H2O2积累,二氨基联苯胺(3.3’-Diaminobenzine,DAB)可与H2O2在产生处发生反应,形成红褐色斑点。首先配制DAB染色液:先称取0.3g固体粉末DAB(需4℃避光保存),溶解于300mL蒸馏水中,使溶液浓度为1mg/mL,再用浓醋酸调节pH至5.0,组织染液需新鲜配置。将盐胁迫处理后的拟南芥幼苗放有DAB染色液的小瓶中,置于28℃恒温培养箱避光孵育过夜,次日去除染色液后,加入80%乙醇,沸水浴中煮沸10min用于叶绿素脱色,待脱色完全后在日光灯下观察叶片的染色情况。
NBT染色检测O2 -积累,在超氧阴离子存在的情况下,氮蓝四唑(Nitrobuletetrazolium,NBT)被还原为蓝色沉淀。首先配制NBT染色液:先称取0.05g固体粉末NBT(需4℃避光保存),溶解在100mL 25mM磷酸钾缓冲溶液(pH 7.6)中,使其溶液浓度为0.5mg/mL。将盐胁迫处理后的拟南芥幼苗放有NBT染色液的小瓶中,置于28℃恒温培养箱避光孵育过夜,次日去除染色液后,加入80%乙醇,沸水浴中煮沸10min用于叶绿素脱色,待脱色完全后在日光灯下观察叶片的染色情况。
4.拟南芥RNA提取和cDNA合成
利用Trizol试剂(宝生物工程有限公司,TaKaRa)提取拟南芥总RNA。称取50-100mg生长周期为3周的拟南芥幼苗叶片,液氮速冻后,利用球磨仪(Retsch,MM400)充分研磨后,加入1mL预冷的Trizol试剂,颠倒混匀,室温放置5min。加入200μL氯仿,用旋涡振荡器剧烈震荡15s,室温放置2-3min,4℃12000g离心15min。缓慢吸取上清液(500μL)于新的DEPC水处理过的1.5mL离心管中,加入500μL异丙醇,混匀后室温静置10min,4℃12000g,离心10min。用移液器吸走上清液,加入500μL预冷的75%乙醇(DEPC水配制)到沉淀中,震荡洗涤沉淀后,4℃,7500g,离心5min。将乙醇弃去,用移液器吸走离心管中的残余液体,室温晾干(或者将其放入通风橱晾干)10-15min。切勿使RNA干燥时间过长,以免RNA不好溶解,加入20μLDEPC水溶解RNA。取1μL RNA进行琼脂糖凝胶电泳初步检测RNA提取质量,用微量紫外分光光度计(昊诺斯,NanoPro)检测OD260和OD280已确定样品浓度和RNA纯度(OD260/A280比率最好为1.8-2.1),将样品置于-80℃冰箱中保存。
取1μg RNA,采用反转录试剂盒(宝生物工程有限公司,TaKaRa)去除基因组DNA以及进行mRNA反转录。反应体系为:5×gDNA Eraser Buffer 2μL、gDNA Eraser 1μL、总RNA 2μg、RNAse Free ddH2O加到10μL,42℃反应2min后,进行mRNA的反转录。向10μL的DNA消化产物中加入以下试剂:RT primer mix 1μL、5×Prime script buffer 2 4μL、Prime scriptRT Enzyme Mix 1μL、RNase Free ddH2O 4μL。混合均匀后,在PCR扩增仪中37℃反应15min,85℃5s。PCR反应结束后,将cDNA样品置于-20℃冰箱保存。
5.半定量PCR
根据基因序列,用Primer Premier 5.0软件进行引物设计。取AtActin基因作为内参,检测BDR3基因在盐胁迫处理后2、4、6、8h的表达情况。PCR反应程序:95℃预变性5min,95℃变性30s,58℃退火30s,72℃延伸10min,一共循环25次。所用引物见表1。
表1半定量PCR引物
6.基因克隆及载体构建
在TAIR网站搜索BDR3序列,利用生物信息学软件Primer Premier 5.0设计该基因特异性克隆引物(不含终止密码子),并加入BP接头序列(表2)。利用Q5 High-Fidelity DNA聚合酶(NEB,纽英伦生物技术北京有限公司),以拟南芥cDNA为模板,进行该基因的开放阅读框(ORF)序列扩增。在BP克隆酶(Gateway BP Clonase II,ThermoFisher Scientific)的催化下,通过BP反应,利用Gateway体系将目的片段连接到中间载体pDONR222上,确定目的基因与入门载体连接成功后,利用LR克隆酶(Gateway LR Clonase II,ThermoFisherScientific)将重组质粒和pGWB405进行LR反应,室温连接过夜,然后将2.5μl连接产物转化于大肠杆菌,提取质粒进行酶切鉴定并测序正确后,保存构建成功的载体pGWB405-BDR3。
表2基因克隆引物
使用化学转化法进行农杆菌转化,取-80℃保存的农杆菌感受态GV3101放置在冰上待融化,加入1.5μL重组质粒pGWB405-BDR3,轻轻拨动管底混匀,依次在冰上静置5min,放入液氮里5min,37℃水浴5min,冰浴5min;转化后加入600μL 无抗生素的LB培养基,28℃摇床摇晃1.5h使其扩繁,用无菌涂布棒将菌液涂布于含50μg/mL壮观霉素和25μg/mL利福平的LB固体培养基上,于28℃恒温培养箱中倒置培养48h。挑取菌落加入5mL含有壮观霉素和利福平的LB液体培养基中,于28℃100rpm,培养48h,吸取活化好的农杆菌菌液1mL加入到100mL LB液体培养基(含壮观霉素和利福平抗生素)中28℃100rpm扩大培养,培养至菌液浓度为OD600=0.5-0.6。
选择长势良好、处于盛花期的拟南芥进行侵染,转化方法为浸花法;将菌液3750rpm离心15min,收集菌体,将菌液重悬在含有5%蔗糖的1/2MS液体培养基中,加入10μL表面活性剂Silwet 77,将其倒入方形培养皿中,将花全部浸染在菌液中,然后将侵染后的拟南芥植株放入保鲜袋中,放在黑暗条件下24h。一周后重复侵染一次,持续培养至拟南芥成熟,收取种子,即为T1代。
二、试验结果
1.拟南芥盐耐受突变体的筛选与bdr3的获得
为挖掘植物耐盐性新的调控因子,我们使用化学诱变剂EMS(甲基磺酸乙酯)处理拟南芥野生型Col-0种子,称取大约10,000粒种子置于15mL离心管中。首先利用0.1%的Tween20对种子进行表面清洁,再用无菌水清洗5次后加入10mL无菌水及25μL EMS至终浓度为2.5%,孵育结束后用无菌水清洗种子五次,去除多余诱变剂。将处理后的种子播撒在含有150mM NaCl的土壤中,通过正向遗传学手段筛选EMS诱变后具有盐敏感或盐耐受生长表型的植株,如图1所示,左侧为野生型Col-0,右侧为诱变突变体,相对于野生型叶片褪绿、发黄萎蔫的盐敏感表型,突变体植株则显示健康生长状态,表现出盐耐受生长表型。
利用二代测序对盐耐受突变植株回交后的自交株系进行基因组测序,采用CTAB法对自交株系产生的两组盐敏感表型分离植物的叶片,分别进行全基因组DNA提取。测序平台为Illumina NextSeq500。利用PuTTY客户端运行Linux操作系统,分析比对背景样品和突变体样品的全基因组序列测序结果。结果显示,突变位点高频率出现在BDR3(BOUNDARY OFROP DOMAIN3)基因上,表明其功能的改变或者缺失导致植物出现盐耐受表型。
2.BDR3负调控植物耐盐性
为验证上述植株的盐耐受表型是否由于BDR3的突变导致,以及证明BDR3是否参与调控植物耐盐性。我们从拟南芥生物资源中心(Arabidopsis Biological ResourceCenter,US)订购了BDR3的T-DNA插入突变体bdr3-1、bdr3-2,并构建了BDR3过表达株系BDR3-OE。观察其在盐胁迫下的生长表型。结果显示在300mM NaCl盐处理后,bdr3突变体幼苗表现出了盐耐受生长表型(图2),BDR3过表达株系BDR3-OE则显示对盐敏感的生长状态,植株在盐处理后整体发黄萎蔫(图3)。此外,BDR3基因在盐处理2-8h后,其转录水平呈下降趋势,受到盐信号的抑制(图4)。综上表明BDR3参与盐胁迫应答且在拟南芥耐盐性中起负调控作用。
3.在盐胁迫下bdr3突变体的生理生化响应
对拟南芥野生型Col-0和bdr3突变体在盐胁迫下的生理应答进行观察,包括叶片含水量、活性氧物质过氧化氢和超氧阴离子的产生。如图5观察到盐胁迫处理72h后的bdr3、bdr3-1、bdr3-2突变体叶片的相对含水量均高于野生型。此外,化学组织染色显示bdr3相关突变体中活性氧物质的积累稍低于野生型但不显著(图6),以上结果均进一步说明BDR3参与调控植物耐盐性。
Claims (3)
1.拟南芥BDR3基因在调控植物耐盐性中的应用,所述拟南芥BDR3基因的编码区核苷酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的应用,其特征在于,所述应用为通过抑制植物中BDR3基因的表达来提高植物耐盐性。
3.根据权利要求2所述的应用,其特征在于,所述抑制植物中BDR3基因的方式为基因沉默、基因突变或基因敲除。
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