CN112375763A - Ebs1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用 - Google Patents
Ebs1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用 Download PDFInfo
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- CN112375763A CN112375763A CN202011211307.8A CN202011211307A CN112375763A CN 112375763 A CN112375763 A CN 112375763A CN 202011211307 A CN202011211307 A CN 202011211307A CN 112375763 A CN112375763 A CN 112375763A
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Abstract
本发明公开了EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用,该EBS1基因的蛋白编码区的核苷酸序列如SEQ ID No.1所示。本发明鉴定并筛选到一种抗碳酸氢盐胁迫的基因EBS1,提供了EBS1作为正向调控因子在调控拟南芥碳酸氢盐抗性和改善盐碱地种植的新用途,为培育耐碳酸氢盐植株提供依据。
Description
技术领域
本发明涉及生物技术技术领域,主要涉及EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用。
背景技术
全球盐碱地面积达9.5亿公顷,约占陆地面积的三分之一,而我国盐碱地面积近1亿公顷,在碱性土壤中植物受到多重压力,如渗透胁迫,营养匮乏,离子毒害,高pH胁迫等。土壤营养缺乏、高酸碱度、含盐度高、富含碳酸氢根等特点,影响植物正常代谢,因此加深对植物耐盐碱机制的研究有助于土壤资源高效利用,发展农业生产。
目前发现,在碱性环境下植物会调节一系列生理活动如增强铁、氮和微量营养素的吸收或输送能力(Romera FJ,et al.1992.Effects of bicarbonate,phosphate andhigh pH on the reducing capacity of Fe-deficient sunflower and cucumberplants.Journal of Plant Nutrition 15:1519–1530;);增加活性氧清除剂的合成(GongB,et al.2013.Comparative effects of NaCl and NaHCO3 stress on photosyntheticparameters,nutrient metabolism,and the antioxidant system in tomatoleaves.Scientia Horticulturae 157:1–12;);盐的离子区域化,维持正常的光合作用等来应对盐碱胁迫。
我国碱化土壤的形成大部分与土壤中碳酸盐的累计有关,碳酸氢钠是碱性土壤中的主要成分之一,通常用于模拟实验室条件下的碱性胁迫。碳酸氢盐对植物的胁迫的具体机理尚不清楚,以前认为来自两个方面:盐胁迫和碱胁迫。有研究对碳酸氢钠、氯化钠和高碱度对植物生长损害程度进行比较,发现低浓度碳酸氢钠(小于10mM)处理就能使叶片失绿并最终出现致死症状,相当于10-20倍浓度的氯化钠(200mM)带来的影响(Guan Q,etal.2017.Tolerance analysis of chloroplast OsCu/Zn-SOD overexp ressing riceunder NaCl and NaHCO3 stress.PLOS ONE 12:e0186052.;Ye X,etal.2019.Transcriptome profiling of Puccinellia tenuiflora during seedgermination unde r a long-term saline-alkali stress.BMC Genomics 20:589.)。说明碳酸氢钠对植物的毒害不是由钠离子引起的。另外,碳酸氢钠对植物的毒害效果也高于高碱度胁迫。通过转录组学方法,发现植物在氯化钠、氢氧化钠和碳酸氢钠的处理后,基因表达模式不同(Zhang B,et al.2018a.Transcriptome analysis of gossypium hirsutumL.reveal s different mechanisms among NaCl,NaOH and Na2CO3 stresstolerance.Scientific Reports 8:1–14.),这表明植物对高盐、高碱、碳酸氢钠胁迫反应是不同的,存在碳酸氢钠特有的信号途径。大量的研究表明碳酸盐和碳酸氢盐是驱动植物适应碱性土壤的决定性选择因素(Terés J,et al.2019.Soil carbonate drives localadaptation in Arabi dopsis thaliana.Plant,Cell&Environment 42:2384–2398.)。分离植物中的碳酸氢钠特异性反应信号对理解碳酸氢钠的胁迫机制具有重要意义。
发明内容
本发明筛选并鉴定到一个抗碳酸氢盐胁迫的基因EBS1,提供了EBS1作为正向调控因子在调控拟南芥碳酸氢盐抗性和改善盐碱地种植的新用途,为培育耐碳酸氢盐植株提供依据。
具体技术方案如下:
本发明提供了EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用,所述EBS1基因的蛋白编码区的核苷酸序列如SEQ ID No.1所示,其蛋白编码区长度为945bp,EBS1基因编码的蛋白的氨基酸序列如SEQ ID No.2所示。
本发明通过拟南芥在不同介质提供的碱性环境处理下,利用RNA-Seq分析筛选到差异表达基因(DEGs),利用加权相关网络分析(WGCNA)、层级聚类树、韦恩图分析等数据处理手段,缩小基因筛选范围,最终在受NaHCO3、NaOAc处理后拟南芥根部基因表达量变化呈相反趋势的19个基因中,选择受NaHCO3诱导表达量上升的13个基因,我们用这13个基因的缺失突变体展开碳酸氢钠处理实验进一步将目标锁定在3种NaHCO3敏感的突变体,之后将这3种敏感突变体在多种弱酸钠盐下培养,最终筛选到对NaHCO3敏感,而对其他弱酸钠盐不敏感的突变体ebs1。为了进一步验证ebs1的功能,用正常的EBS1回补ebs1突变体,得到ebs1的回补株系。实验表明ebs1的回补系恢复了对NaHCO3的抗性。通过构建过表达EBS1株系,发现对碳酸氢盐的抗性进一步加强。因此我们筛选并证实了EBS1基因正向调控对碳酸氢盐的抗性。
进一步地,所述应用的途径为通过增强EBS1基因的表达水平,提高拟南芥在碳酸氢盐胁迫下的抗性。
进一步地,所述增强的方法包括:过表达EBS1基因或其同源基因。
过表达的方法可以是利用增强型启动子增强植物内源表达,也可以通过诱导EBS1基因高水平表达。本发明是将目的基因蛋白编码序列克隆转载到含35S启动子的过表达载体上。
进一步地,所述碳酸氢盐为可溶性碳酸氢盐。
本发明还提供了一种增强拟南芥抗碳酸氢盐胁迫能力的方法,包括以下步骤:
(1)设计克隆EBS1的引物:正向引物CGGGGTACCATGCCCTTATTATCTGATAATG,反向引物2TGCTCTAGATTAAAATAAAACTTCGACACCGG,构建到含35S启动子的过表达载体;
所述EBS1基因的蛋白编码区的核苷酸序列如SEQ ID No.1所示;
(2)构建含步骤(1)所述的35S启动子过表达载体的农杆菌基因工程菌;
(3)将步骤(2)所述基因工程菌转化拟南芥,获得过表达EBS1的抗碳酸氢盐株系。
进一步地,步骤(1)中,正向引物的序列如SEQ ID No.5所示,反向引物的序列如SEQ ID No.6所示。
进一步地,步骤(2)中,所述农杆菌为GV3101。
本发明中所述基因筛选过程中RNA-seq选用在普通1/2MS培养基培养10天后的拟南芥苗,在NaHCO3、NaOAc处理四天后苗的根部和叶片分别作为测定对象。本发明中突变体NaHCO3敏感性实验、EBS1回补实验及过表达实验中根长生长作为判定植物适应碱性环境的标准。
本发明中选取植物中天然存在、功能研究较为清楚、具有缓冲能力的醋酸盐(NaOAc)和磷酸盐(Na2HPO4/NaH2PO4)作为碳酸氢盐(NaHCO3)的对照。醋酸盐和磷酸盐比NaOH具有更强的缓冲能力,可以提供稳定的高碱环境。这样克服了以往研究中的用NaOH做对照时无法维持稳定的pH值的缺陷。醋酸盐和磷酸盐也比其他人工合成的碱性缓冲液合适:比如Tris、HEPES(N-2-羟乙基哌嗪-N-2-乙磺酸)等,虽然能提供稳定的碱性环境,但其对植物的生长有副作用。因此我们选取醋酸盐和磷酸盐作为碱盐对照,实验结果更可靠。
本发明所述应用途径是通过增强EBS1基因的表达水平,使得拟南芥在NaHCO3胁迫下具有更强的生长生存能力。为利用EBS1基因应对碳酸氢盐所致的盐碱土提供了方法。
与现有技术相比,本发明具有以下有益效果:
(1)本发明发现了EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的新用途,为为培育耐碳酸氢盐植株提供依据。
(2)本发明利用转录组分析和反向遗传分析鉴定了调控碳酸氢钠胁迫的基因EBS1,相比以往通过植物表型性状或生理实验测定应激响应活动,而后着手基因水平的研究不同。本发明从全基因组基因差异表达入手,不断缩小目标基因范围,筛选全面且更具有靶向性。然后利用分子生物学手段进行缺失回补实验,充分验证了调控碳酸氢钠胁迫的基因EBS1。
(3)本发明突破以往高碱胁迫植物生长的观念,提出盐碱土对植物的胁迫主要来自于高碳酸氢盐而非碱胁迫,为寻找改良植物耐盐碱能力提供了新思路。
(4)本发明通过基因编辑在拟南芥植株内过表达EBS1基因,发现植株对碳酸氢盐的耐受性明显增强,根系生长比野生型健壮;通过在其他植物体内过表达拟南芥基因EBS1,或找到其他植物体内EBS1同源蛋白基因,有望起到与拟南芥类似的的抗碳酸氢盐的效果,为开发耐碳酸氢盐植物品种开发提供了新途径。
附图说明
图1为实施例1中的拟南芥对碳酸氢钠和醋酸钠的剂量反应;
其中,A为拟南芥在不同剂量的NaHCO3、NaOAc处理10天后的生长状况,B为三次重复实验后与A图对应的根长统计图。
图2为实施例2受碳酸氢钠和醋酸钠调节的拟南芥差异表达基因的加权相关网络分析;
A为6298个DEGs构成的层次聚类树。16个主枝代表16个模块,;B为模块和样本之间的皮尔逊相关系数图。模块颜色与A图对应负相关和正相关分别用比例尺中的不同灰度颜色表示。显著相关值显示在每个模块的相应单元格上(P值≤0.05;括号中显示的是P值)。黑体字表示每个样本的最高相关值每个模块的基因数量显示在左侧。
图3为实施例2中拟南芥根样本的差异表达基因受到NaHCO3、NaOAc调节后调整基因表达情况的韦恩图,有17个基因由碳酸氢钠诱导而受醋酸钠抑制,另2个基因表现出相反的表达。
图4为实施例2中根样品在受碳酸氢钠诱导受醋酸钠抑制基因与模块M基因统计的韦恩图,有13个基因同属两区域。
图5为实施例3中受NaHCO3特异调节的基因型筛选。
其中,A表示3种基因的T-DNA突变体在多种碱盐胁迫下,生长10天后的生长状况图;B为该实验三次重复后,与A图相对应的根长情况统计图。
图6为实施例5中ebs1的回补株系对碱盐胁迫的敏感性实验;
A为ebs1、ebs1的回补株系在碳酸氢钠处理下生长10天后的生长状况,B为该实验重复三次试验后与A图相对应的根长情况统计图
图7为实施例6中ebs1的过表达株系对碱盐胁迫的敏感性实验,A为过表达株系在三种碱盐胁迫下的生长状况图,B为该实验重复三次试验后与A图相对应的根长情况统计图,经过线性模型统计分析(N≥24)。星标表示显著性(P值≤0.05)。
图8为实施例4中野生型对Na2HPO4的剂量反应实验图。
图9为实施例4中为多种碱盐处理的培养基在拟南芥生长前后pH值变化统计图。
图10为实施例6中qPCR检测EBS1过表达株系在NaHCO3处理下,该基因相对表达量。
具体实施方式
下面结合具体实施例对本发明作进一步描述,以下列举的仅是本发明的具体实施例,但本发明的保护范围不仅限于此。
实施例1拟南芥对NaHCO3、NaOAc的剂量反应
本实施例分析拟南芥对NaHCO3、NaOAc的剂量反应。
具体方法如下:
(1)将灭菌的野生型拟南芥Col-0种子置于含有指定碱性盐的MS培养基(1%蔗糖)上,设浓度区间0-5μM的NaHCO3实验组,每1.5μM设一梯度和0-8μM浓度区间的NaOAc实验组,每2μM设一梯度,在4℃下的两天春化处理后,将拟南芥幼苗移至生长室置于培养箱中培养,拟南芥培养条件为光强150-200μmol·m-2s-2,60%湿度,12/12h(明/暗)光周期。
(2)培养10天后拍照记录,用ImageJ测定根长,数据通过学生t检验或线性混合模型进行统计分析。
结果发现,NaHCO3和NaOAc在一定浓度下都能减弱植物生长,碳酸氢钠在5μM时诱导叶片坏死,并最终导致植物死亡;而醋酸钠即使在8μM时也没有致死作用,反而促进叶片花青素的合成(图1A)。根长测量数据表明,碳酸氢钠对植物造成的胁迫明显比醋酸钠严重(图1B)。在碳酸氢钠和醋酸钠处理下,植物之间的不同形态表明,不同的信号通路在对在应对不同的盐胁迫中被激活。
实施例2 RNA-Seq分析拟南芥在NaHCO3、NaOAc处理后的差异基因表达
在NaHCO3、NaOAc处理下分别对根和叶样品进行RNA测序,再鉴定和分析差异表达基因。
具体方法如下:
(1)将生长10天的幼苗转移到含有或不含有碳酸氢钠(3mM)、醋酸钠(4mM)的1/2MS培养基中(处理浓度根据实施例1选择)。四天后,分别收集这些幼苗的根和叶。每个基因型总共收集120株幼苗(40株/每板,3板/每个处理)作为一次重复。
(3)illumina HiSeq 4000在150bp的配对末端进行测序;微调-0.38用于移除适配器和低质量读数(Bolger AM,Lohse M,Usadel B.2014.Trimmomatic:a flexible trimmerfor illumina sequence data.Bioinformatics 30:2114–2120.)。
(4)通过DESeq2鉴定(Love MI,Huber W,Anders S.2014.Moderated estimationof fold change and dispersion for RNA-seq data with DESeq2.Genome Biology 15:550.)差异表达基因(DEGs);选择DEGs(log2样本间差异表达倍数值≥1,P值≤0.05)进行WGCNA分析。
(5)根据根样品在NaHCO3、NaOAc处理下分离出的DEGs绘制Venn图。
结果如图2所示,与对照组相比,四组类型样品共鉴定出6298个差异表达基因(DEGs);(log2样本间差异表达倍数值≥1,P值≤0.05)。对这些基因进行加权相关网络分析(WGCNA),四种样本类型中表达模式相似的基因被归入一个模块,得到16个模块,聚类分析树呈现16个主要分支,模块颜色与A图对应负相关和正相关分别用比例尺中的不同灰度颜色表示(图2A)。与此同时,还分离出处理与样品类型之间显著相关的模块(图2B):碳酸氢钠处理的根样品特异性相关的模块M相关值有最高相关系数0.96,因此我们将搜索的范围集中在根样本中的DEGs上,缩小受碳酸氢钠胁迫特异性调节的候选基因。根样品在NaHCO3、NaOAc处理下分离出的DEGs绘制Venn图(图3),有95个上调基因和223个下调基因重叠(图3)。19个(17+2)基因受碳酸氢钠和氢氧化钠调节变化的方向相反(图3)。该19个基因中,有13个包含在模块M的基因中,这些基因被碳酸氢钠特异性上调(图2B;图4)。
实施例3受NaHCO3特异性上调的13个基因的缺失性突变体进行NaHCO3敏感性实验
该13个基因T-DNA突变体从NASC(http://arabidopsis.info/BasicForm)购置。
具体实验方法如下:
(1)将13种突变体及Col-0点种在含NaHCO3(3mM)的1/2MS培养皿上,培养方法与实施例1一致;
(2)10天后拍照记录植物生长状况,用ImageJ测定根长。
结果:在NaHCO3敏感性验证实验筛选出AT5G07330(Salk_104370C)、AT3G05950(Salk_038626C)和AT4G17680(Sail_420_E12)的突变系在碳酸氢钠处理环境中植株生长状态不良,根系弱小,对胁迫敏感。(图5A)。
实施例4受NaHCO3特异诱导的基因型株系筛选
设置3种碱性盐(碳酸氢钠、醋酸钠和磷酸氢二钠)处理,盐浓度参考剂量反应实验实施1及补充的硫酸氢二钠剂量实验(图1B、图8),综合考虑pH值与根系生长状况,设置NaHCO3(3mM)、NaOAc(4mM)、Na2HPO4(7.5mM)。
具体方法如下:
(1)设置3种碱性盐处理,将3种突变体分别点种NaHCO3(3mM)、NaOAc(4mM)、Na2HPO4(7.5mM)调节碱性胁迫的1/2MS培养皿中,培养方法与实施例1一致。
(2)10天后拍照记录植物生长状况,用ImageJ测定根长。
该浓度设置下野生型拟南芥在三种处理下生长状况相似且植株生长前后培养基pH值相近(图9)。实验发现,只有AT4G17680的突变体表现出碳酸氢钠特异性敏感性;另外两个突变体(Salk_104370C和Salk_038626C)对所有碱性盐敏感(图5A和B)。因此,我们将AT4G17680的突变体命名为ebs1。
实施例5 ebs1回补系对NaHCO3的敏感性实验
具体ebs1回补系的制备方法为:
(1)用引物CGGGGTACCATGCCCTTATTATCTGATAATG(正向)和TGCTCTTAGATAAAAAAATAAACTTCGACACGG(反向)克隆EBS1的CDS序列。
(2)构建载体:克隆EBS1的2kb启动子,取代pCAMBI1300中的35S启动子。
(3)EBS1的PCR产物经Kpnl和Xbal限制性酶处理,并将片段***载体pCAM BI1300。
(4)将构建好的质粒转化农杆菌GV3101。
(5)花粉管通道法侵染拟南芥获得回补型植株。
敏感性实验方法为:设置3种碱性盐处理,将ebs1回补系分别点种NaHCO3(3mM)、NaOAc(4mM)、Na2HPO4(7.5mM)调节碱性胁迫的1/2MS培养皿中,培养方法与实施例1一致;10天后拍照记录植物生长状况,用ImageJ测定根长。
结果表明,ebs1的回补表达将ebs1的碳酸氢钠敏感表型保存到野生型水平(图6A和B)。
实施例6 EBS1过表达植株对NaHCO3的敏感性实验
具体EBS1过表达植株的制备方法为:
(1)设计克隆EBS1的引物:正向引物CGGGGTACCATGCCCTTATTATCTGATAATG,反向引物TGCTCTAGATTAAAATAAAACTTCGACACCGG,构建到含35S启动子的过表达载体。所述序列如SEQ ID No.1所示。
(2)构建含步骤(1)所述的35S启动子过表达载体的农杆菌GV3101基因工程菌;
(3)将步骤(2)所述基因工程菌转化拟南芥,获得过表达EBS1的抗碳酸氢盐株系。
(4)q-PCR检测EBS1在拟南芥植株的表达量(结果如图10),引物正向引物:qEBS1-FCGGATAACAGAGAGAGACGCTACG及反向引物:qEBS1-R GCTATGGCTTGTTGGAGATGAG,TUB2作为内参基因,qbase+2.1用于产生相对表达式值。
敏感性实验方法为:设置3种碱性盐处理,将EBS1过表达植株分别点种NaHCO3(3mM)、NaOAc(4mM)、Na2HPO4(7.5mM)调节碱性胁迫的1/2MS培养皿中,培养方法与实施例1一致;10天后拍照记录植物生长状况,用ImageJ测定根长。
EBS1过表达植株仅表现出对碳酸氢钠的增强抗性,而其对氢氧化钠和磷酸氢二钠的敏感性没有变化(图7A和B)。q-PCR鉴定碳酸氢钠处理下的EBS1基因的表达量相较对照明显上升。
综合以上研究,本发明发现了EBS1基因正调控碳酸氢根离子的特异抗性,通过增强该基因的表达,能提高植物在碳酸氢钠胁迫下的生长状况和碳酸氢盐抗性。
序列表
<110> 浙江农林大学
<120> EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 945
<212> DNA
<213> 拟南芥 (Arabidopsis thaliana)
<400> 1
atgcccttat tatctgataa tgaacgcata ggtctcttct ctgatagctc aatggcgatt 60
caagctcagc atccttcacg tttcttcttc aacaacagta acggacaaga agctagtgat 120
tgttcgttgc agcctcaaga cactcctttc actaatttca ccaaagctgg ggttgattca 180
agaaaacgat caagagaagt ttattcatcg gctctgatga accctccacc tccaaaaccg 240
tcgcaagtta ttgatataac cgagttgttg cagaaaacgc ctaacgtggt ttccactggt 300
cttcgattat ttcatgatca gtcacagaat caacaacagt ttttttcttc tcttcccgga 360
gatgttaccg gaaagattaa acggcaaaga gatgaactag acagattcat tcagactcag 420
ggtgaagagt tgcggcgtac gttagcggat aacagagaga gacgctacgt agagttattg 480
tgcgctgcgg aggagatagt cggacggaaa ttgagaaaaa aagaagcgga gttggagaaa 540
gccacgcgcc gtcacgctga gctagaagcg cgtgtagctc acatcgtgga agaggcgcga 600
aactggcagt taagagcggc gacgcgggaa gctgaggtgt cctcgttaca tgctcatctc 660
caacaagcca tagctaaccg cctagatacg gcggcgaaac agagtacgtt cggagaagac 720
ggcggagacg cggaagaagc agaggacgct gaatcggttt acgtggatcc ggagcggatc 780
gaattgatcg gaccaagttg taggatttgc cggcggaaat ctgcgacggt gatggcattg 840
ccgtgtcaac atttgattct ttgtaatgga tgcgacgtcg gagcagtgcg agtctgtccg 900
atttgcctcg ccgtgaagac ttccggtgtc gaagttttat tttga 945
<210> 2
<211> 300
<212> PRT
<213> 拟南芥 (Arabidopsis thaliana)
<400> 2
Met Pro Leu Leu Ser Asp Asn Glu Arg Ile Gly Leu Phe Ser Asp Ser
1 5 10 15
Ser Met Ala Ile Gln Ala Gln His Pro Ser Arg Phe Phe Phe Asn Asn
20 25 30
Ser Asn Gly Gln Glu Ala Ser Asp Cys Ser Leu Gln Pro Gln Asp Thr
35 40 45
Pro Phe Thr Asn Phe Thr Lys Ala Gly Val Asp Ser Arg Lys Arg Ser
50 55 60
Arg Glu Val Tyr Ser Ser Ala Leu Met Asn Pro Pro Pro Pro Lys Pro
65 70 75 80
Ser Gln Val Ile Asp Ile Thr Glu Leu Leu Gln Lys Thr Pro Asn Val
85 90 95
Val Ser Thr Gly Leu Arg Leu Phe His Asp Gln Ser Gln Asn Gln Gln
100 105 110
Gln Phe Phe Ser Ser Leu Pro Gly Asp Val Thr Gly Lys Ile Lys Arg
115 120 125
Gln Arg Asp Glu Leu Asp Arg Phe Ile Gln Thr Gln Gly Glu Glu Leu
130 135 140
Arg Arg Thr Leu Ala Asp Asn Arg Glu Arg Arg Tyr Val Glu Leu Leu
145 150 155 160
Cys Ala Ala Glu Glu Ile Val Gly Arg Lys Leu Arg Lys Lys Glu Ala
165 170 175
Glu Leu Glu Lys Ala Thr Arg Arg His Ala Glu Leu Glu Ala Arg Val
180 185 190
Ala His Ile Val Glu Glu Ala Arg Asn Trp Gln Leu Arg Ala Ala Thr
195 200 205
Arg Glu Ala Glu Val Ser Ser Leu His Ala His Leu Gln Gln Ala Ile
210 215 220
Ala Asn Arg Leu Asp Thr Ala Ala Lys Gln Ser Thr Phe Gly Glu Asp
225 230 235 240
Gly Gly Asp Ala Glu Glu Ala Glu Asp Ala Glu Ser Val Tyr Val Asp
245 250 255
Pro Glu Arg Ile Glu Leu Ile Gly Pro Ser Cys Arg Ile Cys Arg Arg
260 265 270
Lys Ser Ala Thr Val Met Ala Leu Pro Cys Gln His Leu Ile Leu Cys
275 280 285
Asn Gly Cys Asp Val Gly Ala Val Arg Val Cys Pro
290 295 300
<210> 3
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cggggtacca tgcccttatt atctgataat g 31
<210> 4
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgctcttaga taaaaaaata aacttcgaca cgg 33
<210> 5
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cggggtacca tgcccttatt atctgataat g 31
<210> 6
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgctctagat taaaataaaa cttcgacacc gg 32
Claims (7)
1.EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用,其特性在于,所述EBS1基因的蛋白编码区的核苷酸序列如SEQ ID No.1所示。
2.如权利要求1所述的EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用,其特性在于,所述应用的途径为通过增强EBS1基因的表达水平,提高拟南芥在碳酸氢盐胁迫下的抗性。
3.如权利要求1所述的EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用,其特性在于,所述增强的方法包括:过表达EBS1基因或其同源基因。
4.如权利要求1~3任一项所述的EBS1基因在增强拟南芥抗碳酸氢盐胁迫能力中的应用,其特性在于,所述碳酸氢盐为可溶性碳酸氢盐。
5.一种增强拟南芥抗碳酸氢盐胁迫能力的方法,其特征在于,包括以下步骤:
(1)设计克隆EBS1的引物,构建到含35S启动子的过表达载体;所述EBS1基因的蛋白编码区的核苷酸序列如SEQ ID No.1所示;
(2)构建含步骤(1)所述的35S启动子过表达载体的农杆菌基因工程菌;
(3)将步骤(2)所述基因工程菌转化拟南芥,获得过表达EBS1的抗碳酸氢盐株系。
6.如权利要求5所述的增强拟南芥抗碳酸氢盐胁迫能力的方法,其特征在于,步骤(1)中,正向引物的序列如SEQ ID No.5所示,反向引物的序列如SEQ ID No.6所示。
7.如权利要求5所述的增强拟南芥抗碳酸氢盐胁迫能力的方法,其特征在于,步骤(2)中,所述农杆菌为GV3101。
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JIPENG CHEN ET AL: "An S-ribonuclease binding protein EBS1 and brassinolide signaling are specifically required for Arabidopsis tolerance to bicarbonate", 《JOURNAL OF EXPERIMENTAL BOTANY》 * |
李菲等: "碳酸氢盐胁迫对拟南芥根部基因表达的影响", 《分子植物育种》 * |
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