CN117844860A - 油菜BnaREM1.3基因及其编码蛋白在调控作物抗旱性中的应用 - Google Patents
油菜BnaREM1.3基因及其编码蛋白在调控作物抗旱性中的应用 Download PDFInfo
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Abstract
本发明油菜BnaREM1.3基因及其编码蛋白在调控作物抗旱性中的应用。本发明获得了BnaREM1.3基因的过表达株系,发现其抗旱性相比于野生型显著提高,同时,利用CRISPR/Cas9技术得到了BnaREM1.3基因的突变体株系,发现其抗旱性相比于野生型显著降低。综上结果表明,利用油菜BnaREM1.3基因可以创制不同抗旱性且能够稳定遗传的油菜,为油菜的抗旱育种提供了重要的基因资源和种质资源,在油菜分子设计育种中具有广阔的应用前景。
Description
技术领域
本发明属于植物基因工程技术领域,涉及一种BnaREM1.3基因及其编码蛋白在调控油菜抗旱性中的应用。
背景技术
甘蓝型油菜(Brassica napus,AACC,2n=38,以下简称油菜)是我国第一大油料作物,菜籽油产量占油料作物的47%以上,是最重要的食用植物油来源之一。我国油菜种植面积约为1.1亿亩,其中长江流域冬油菜约为1亿亩,北方春油菜约为0.1亿亩。油菜主产区容易受到季节性干旱或持续性干旱的影响,严重威胁油菜生长发育和产量。2022年,我国平均气温偏高,长江流域发生历史罕见夏秋冬连旱,对油菜生产造成严重损失。因此,挖掘油菜抗旱基因、解析其抗旱调控机制对油菜高产稳产具有重要意义。
当植物在苗期受到干旱胁迫时,其根冠比提高,地上部形态和地下部形态都会发生改变。地上部改变主要包括叶片萎蔫、叶片水势和含水量降低、地上部生物量降低、株高降低等,地下部根系的改变包括根系膨大和分支数增多等。这些改变会严重抑制植物的正常生长和发育甚至影响产量。植物抵御干旱胁迫是一个复杂的生物学过程,与多种生物学途径密切相关。目前,在模式植物拟南芥中已报道大量与抗旱相关的基因,而油菜中抗旱相关基因研究还较少。利用基因工程创制具有不同抗旱性的油菜,可以为油菜抗性育种提供重要的基因资源和种质资源。
Remorin蛋白首次在番茄的叶片中发现,其大小约为34kD,最初被命名为pp34。后因其依靠coiled-coil结构结合于质膜的结构特点类似于吸附在其他大型海洋生物体表面的海鱼Remora,将其命名为Remorin。Remorin(REM)是一种植物特异性膜脂纳米结构域定位蛋白,具有一个包含coiled-coil结构域的高度保守C端和一个不保守的N端,N端的多样性决定了其功能的多样性。它与抗去垢剂膜相互作用构成了脂筏的主要成分,脂筏是质膜上富含胆固醇和鞘磷脂的微结构域,与膜的信号转导密切相关。随着对Remorin蛋白研究的不断深入,在越来越多的植物中鉴定到了Remorin蛋白并解析了其功能。与脂筏的功能多样性相同,Remorin蛋白在植物生物进化、生长发育、抗病抗逆信号转导等方面发挥重要作用。
Remorin蛋白具有功能多样性。首先,它参与植物的生物进化过程,研究人员在藻类植物中没有鉴定到Remorin蛋白而在苔藓类和蕨类植物中鉴定到Remorin蛋白,这意味着Remorin蛋白的出现是随着植物适应陆地环境进化而来。Remorin蛋白参与植物的生长发育,在短尾紫花苜蓿和日本莲花等豆科植物中,Remorin蛋白与共生受体样蛋白激酶相互作用,这是结瘤所必需的。Remorin蛋白还能诱导活性氧,上调烟草中与程序性细胞死亡有关蛋白的含量。Remorin蛋白还参与了植物的抗病抗逆和信号转导过程。目前,已经发现植物Remorin蛋白能够调控水稻条纹病毒、稻瘟病、番茄青枯病、早酥梨抗黑星病、马铃薯青枯病、辣椒青枯病、烟草晚疫病等在细胞间移动进而影响植物的抗病性,这是由于Remorin蛋白能够作用于植物和微生物的相互作用。此外,Remorin蛋白还能通过与植物激素协同调控植物的信号转导影响植物的抗逆性。水杨酸能够依赖Remorin蛋白调控脂质排布进而介导胞间连丝的关闭,阻碍病毒在细胞间的移动,这也是Remorin蛋白参与生物逆境调控的重要原因之一。水稻Remorin蛋白也能通过与体细胞胚胎发生受体激酶结合协调脱落酸和油菜素内酯的信号传导。Remorin蛋白也可以调控植物响应非生物逆境,如胡杨参与调控拟南芥镉耐受性和抗盐性等。
在拟南芥中,共有16个Remorin蛋白。其中只有4个已经验证了功能,包括AtREM1.2,AtREM1.3,AtREM4.1,AtREM4.2,其余12个基因尚未完成验证。已验证功能的4个基因中,都集中于拟南芥的病毒防御、植物激素信号转导、种子萌发和磷酸化方面。根据Remorin蛋白端序列不同可以将其分为六类:Group 1,Group 2,Group 3,Group 4,Group5,Group 6。其命名规则为:第一亚组(Group 1)排序为第1的蛋白为AtREM1.1,并以此类推。Group 1组Remorin蛋白根据N端脯氨酸含量高低分为Group 1a(8.9%)和Group 1b(14.4%)两个亚组。其中,Group 1b亚组的转录本能够被低温、脱落酸处理、微生物丁香假单胞菌等多种逆境诱导表达。Group 2组Remorin蛋白目前只在苜蓿属和欧洲大叶杨中被鉴定到,研究表明该亚组的Remorin蛋白可能与豆科植物结瘤密切相关。沉默蒺藜状苜蓿中的MtREM2.2基因能够减少根瘤的数目和生长畸形。Group 3组Remorin蛋白不含有N端结构域,其在植物中的研究还不清楚。Group 4组Remorin蛋白具有较长的N端,研究表明该组Remorin蛋白能够参与植物激素间的信号转导。水稻OsREM4.1蛋白能够与油菜素内酯受体结合共同协调水稻脱落酸和油菜素内酯信号的相互联系。Group 5组Remorin蛋白与Group3组相似,研究较少,只有苜蓿属植物的Remorin蛋白能够在种子中表达。Group 6组Remorin蛋白主要在分生组织中表达,可能参与了植物的生长发育。也有研究表明,玉米中的ZmREM6.3基因能够赋予玉米对玉米叶烧病、细菌性枯萎病和常锈病的数量抗性,证明该组Remorin蛋白也可能参与了植物抗病性的调控。
目前在油菜中,暂时没有关于Remorin蛋白及其基因的家族成员分类和功能研究。在番茄中过表达油菜BnaREM1.3的同源基因SIREM1.3能够抑制马铃薯X病毒的移动,同样地,水杨酸也能够通过依赖于水稻OsREM1.3蛋白的脂质重塑介导胞间连丝的通透性,减少病毒在植物细胞间的移动。BnaREM1.3的同源蛋白AtREM1.3蛋白还能够在膜微结构域促进拟南芥超敏诱导反应相关免疫复合物的形成,参与植物的免疫过程。此外,AtREM1.3蛋白也能够诱导拟南芥蛋白磷酸化,调节蛋白质间的相互作用。值得注意的是,油菜BnaREM1.3的同源基因AtREM1.3能够在拟南芥种子萌发和抵御低温过程中诱导表达,暗示其可能参与植物生长发育和耐受性调控。
本发明克隆了油菜BnaREM1.3基因并将其在油菜中高表达,获得的油菜苗期抗旱性显著升高。同时,利用CRISPR Cas9技术创建BnaREM1.3基因的突变体,获得的油菜苗期抗旱性显著降低。该结果表明BnaREM1.3基因在调控油菜苗期抗旱性中起着重要作用,这使它在创建油菜抗旱新种质方面具有重要的应用前景。
发明内容
本发明的目的在于提供BnaREM1.3基因在培育不同抗旱性转基因植物中的应用。
本发明中所涉及的油菜BnaREM1.3基因,编码一种Remorin蛋白,在油菜A4、A5染色体上各有一个拷贝,在C04染色体上有两个拷贝。它们分别是BnaA04.REM1.3(BnaA04g26460D)、BnaA05.REM1.3(BnaA05g05140D)、BnaC04.REM1.3a(BnaC04g04520D)、BnaC04.REM1.3b(BnaC04g50490D)。其中,BnaC04.REM1.3a的核苷酸序列和氨基酸序列分别如序列表SEQ ID NO:1和SEQ ID NO:2所示。其核苷酸序列长度大小为579bp,该基因编码192个氨基酸。
申请人通过使用农杆菌介导的遗传转化方法将BnaC04.REM1.3a(BnaC04g04520D)基因转入到由组成型启动子启动的过表达载体并转化到油菜的基因组中,获得油菜BnaC04.REM1.3a过表达的油菜种质资源。同时利用CRISPR/Cas9基因编辑技术获得基因BnaREM1.3功能缺失的油菜种质资源。
接着,测定了过表达和突变体转化材料的抗旱性,发现BnaREM1.3正调控油菜抗旱性。
本发明中所述的表达载体是指现有技术中已知的、能够在植物中进行表达的任何载体,适用于本发明所述遗传转化的表达载体包括但不限于,如:35S-pCAMBIA1300S、PKSE401(中国农业大学陈其军课题组提供)等。
本发明获得的高抗旱性油菜,在其营养生长与生殖生长时期与正常植株没有明显的差异,该种质资源在油菜抗旱育种中具有十分重要的应用前景。
本发明获得的低抗旱性油菜,也能够被用于植物抗旱技术或相关药物研发的试验对象。
附图说明
序列表SEQ ID NO:1是分离自甘蓝型油菜的基因BnaC04.REM1.3a的核苷酸序列,其序列长度为579bp;序列表SEQ ID NO:2是分离自甘蓝型油菜的基因BnaC04.REM1.3a的氨基酸序列,其编码192个氨基酸。
图1:过表达载体35S-pCAMBIA1300S的质粒图谱。
图2:BnaC04.REM1.3a基因的克隆与过表达植株检测。A为BnaC04.REM1.3a基因的克隆;B为转化单株的qRT-PCR检测;C为转化单株的Western blot检测。*表示在Student’st test中P<0.05。
图3:油菜BnaREM1.3突变体的获得和鉴定。sgRNA1和sgRNA2分别位于基因BnaREM1.3的第三和第四个外显子区,获得的2个突变体的靶位点的编辑情况:CR1和CR2均为BnaA04.REM1.3、BnaA05.REM1.3、BnaC04.REM1.3a被编辑的三突突变体。
图4:油菜BnaREM1.3过表达和突变体的抗旱表型鉴定。A为抗旱试验的表型图;B为三次独立重复试验后的存活率统计情况(n=20)。*表示在Student’s t test中P<0.05,**表示在Student’s t test中P<0.01,***表示在Student’s t test中P<0.001。
具体实施方式
以下结合具体实施例,进一步定义本发明。应理解,这些实施例仅用于说明本发明而不是用于限制本发明的范围。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工具书《分子克隆:实验室指南》(New York:Cold Spring Harbor Laboratory,1989)中所述的条件,或者按照生产商提供的操作手册中建议的方法。
实施例1BnaC04.REM1.3a基因的克隆
REM1.3编码一个Remorin蛋白,在油菜A4、A5染色体上各有一个拷贝,在C04染色体上有两个拷贝。它们分别是BnaA04.REM1.3(BnaA04g26460D)、BnaA05.REM1.3(BnaA05g05140D)、BnaC04.REM1.3a(BnaC04g04520D)、BnaC04.REM1.3b(BnaC04g50490D)。其中,BnaC04.REM1.3a的CDS和氨基酸序列分别为序列表SEQ ID NO:1和SEQ ID NO:2所示的序列,包括完整的ORF阅读框及起始密码子ATG,共编码192个氨基酸。这四个拷贝具有很高的同源性,BnaC04.REM1.3a与其他三个拷贝的同源性分别为:(BnaC04.REM1.3b)89.21%、(BnaA04.REM1.3)89.56%、(BnaA05.REM1.3)98.27%。
(1)RNA的提取
总RNA的提取采用全式金公司的TansZol(目录号ET101),取甘蓝型油菜中双11号叶片于液氮中研磨粉碎,取100mg研磨好的样品转移到1.5mL离心管中,加入1mL的TransZol,上下剧烈颠倒数次使之充分混匀,室温静置5分钟;加0.2mL氯仿,剧烈振荡15秒,室温孵育3分钟;10000xg 4℃离心15分钟,此时样品分为三层,无色的水相(上层),中间层,粉红色有机相(下层);转移无色的水相于新的离心管中,加入0.5mL异丙醇,颠倒混匀,室温孵育10分钟;10000xg 4℃离心10分钟,去上清,在管侧和管底形成胶状沉淀;加1mL 75%乙醇(DEPC处理的水配制),剧烈涡旋;7500xg4℃离心5分钟;弃上清,室温晾干沉淀;沉淀溶于50-100μL RNA溶解液中;55℃孵育10分钟。取lμL抽提的总RNA在Nanodrop下测定RNA浓度,依据1.8<OD260/OD280<2.0鉴定RNA纯度。同时取lμL进行1%agrose电泳,检测完整性。
(2)cDNA的合成
反转录使用的是全式金One-Step gDNA Removal and cDNASynthesis SuperMix(目录号AE311)。以1μg总RNA为模板,依次加入Anchored Oligo(dT)18Primer 1μL,2×ES Reaction Mix 10μL,/>RT/RI Enzyme Mix 1μL,gDNARemover 1μL,RNase-free Water补充至20μL。将以上体系轻轻混匀后置于42℃30min,此步是合成第一链cDNA和去除gDNA。85℃加热5秒钟失活/>RT/RI和gDNA Remover。加入180μL RNase-free Water溶解合成的cDNA,待用。
(3)BnaC04.REM1.3a基因的扩增
根据上述分析所得的CDS序列设计引物,以甘蓝型油菜中双11的cDNA为模板,正向引物BnaREM1.3-KpnⅠ-F序列为5’-GCGggtaccATGGCGGAGGAGCAAAAA-3’,反向引物BnaREM1.3-PstⅠ-R序列为5’-GCGctgcagTTATTTGTCGTCGTCGTCCTTGTAGTCCATGAAACATCCACAAGTCGC-3’,扩增得到含有flag标签的BnaC04.REM1.3a全长CDS的片段。采用I-5TM2×High-Fidelity Master Mix(TSINGKE Biologica technology)进行PCR扩增。PCR扩增体系如下:
PCR扩增程序:98℃总变性1min;98℃变性15sec,58℃退火15sec,72℃延伸30sec,34circles;72℃总延伸5min。
扩增后产物通过琼脂糖凝胶电泳检测,扩增获得579bp的BnaC04.REM1.3a全长,产物挖胶回收使用天根琼脂糖凝胶回收试剂盒(http://www.tiangen.com/)。
实施例2BnaC04.REM1.3a基因过表达转化载体的构建
(1)对上述获得的BnaC04.REM1.3a片段用快速限制性内切酶KpnI和PstI进行双酶切,双酶切体系如下:
酶切反应在37℃水浴锅进行,时间1小时。酶切产物用天根的DNA纯化试剂盒回收。
(2)酶切产物连接到植物表达载体35S-pCAMBIA1300S,该载体含有组成型表达启动子和抗生素标记物。
连接方法如下:
连接反应条件:4℃过夜。
(3)转化大肠杆菌DH5α,转化方法如下:
吸取5μL连接产物加入50μL DH5α感受态细胞中吸打混匀,冰上放置30min;42℃水浴1.5min,冰上放置3min;加入400μL无抗液体LB培养基,37℃,150r/min摇床中活化45-60min;吸取活化后的菌液200μL涂布与相应抗性的固体LB培养基上,37℃倒置培养12-16h。之后筛选阳性克隆后提质粒酶切鉴定,选取3个阳性克隆送样测序,分析结果显示,BnaC04.REM1.3a基因的CDS序列成功连接上载体,即成功构建了转化植株的植物表达载体35S-pCAMBIA1300S-BnaC04.REM1.3a,35S-pCAMBIA1300S载体的质粒图谱如图1所示。
(4)将构建正确的重组质粒载体导入农杆菌菌株GV3101,挑选阳性单克隆于-80℃冰箱保存。导入方法如下:
a.清洗电转杯:先用纯水洗,再用超纯水洗,倒掉,再用无水乙醇清洗(用1mL枪头吹打),倒掉无水乙醇,置于超净台晾干;
b.取农杆菌感受态GV3101 20μL;
c.取构建正确的重组质粒0.8μL,加入到20μL感受态中,轻轻吸打混匀,避免产生气泡;
d.将洗好吹干的电转杯置于冰中预冷,再将上述混合液靠杯壁打入;
e.将电转仪调至1800V;
f.将电转杯从冰中取出,用吸水纸擦干净电转杯外壁;
g.将电转杯放入仪器,连续按两下“push”键,几秒钟之后听到“滴”的一声则成功;
h.电击成功后,向电转杯中加入400μL无抗LB,吸打混匀,转移到无菌离心管中;
i.28℃活化1h左右,取100μL涂到含有对应抗性的平板上,用封口膜封好,倒置于28度培养箱培养2天,挑斑检测。
(5)农杆菌菌落检测
挑选菌落于双抗LB中,28℃培养1小时,取适量菌液进行PCR检测,保存阳性农杆菌菌液。
实施例3BnaREM1.3-CRISPR载体的构建
利用中国农业大学生物学院陈其军团队的CRISPR-Cas9***创建油菜BnaREM1.3突变体。实验操作步骤如下:
(1)登录网站http://www.genome.arizona.edu/crispr/CRISPRsearch.html,筛选靶点GCAGAAGAGAAGAGAGCAATGG和GAAGAGAGTGAGAAGTCA AAGG,分别位于基因的第三和第四个外显子区,可以同时靶向所有BnaREM 1.3基因。
(2)设计引物
DT1-BsF:ATATATGGTCTCGATTG GCAGAAGAGAAGAGAGCAAGTT
DT1-F0:TGGCAGAAGAGAAGAGAGCAAGTTTTAGAGCTAGAAATAGCDT2-R0:AACTTGACTTCTCACTCTCTTCCAATCTCTTAGTCGACTCTACDT2-BsR:ATTATTGGTCTCGAAACTTGACTTCTCACTCTCTTCCAA
(3)PCR扩增:以稀释100倍的pCBC-DT1T2为模板进行四引物PCR扩增。DT1-BsF和DT2-BsR为正常引物浓度;DT1-F0和DT2-R0稀释20倍。扩增体系为:
PCR扩增程序:98℃总变性1min;98℃变性15sec,56℃退火25sec,72℃延伸25sec,34circles;72℃总延伸5min。
(4)纯化回收PCR产物,建立如下酶切-连接体系(restriction-ligation):
反应条件:5hours at 37℃,5min at 50℃,10min at 80℃
(5)转化转化大肠杆菌DH5α:取5μL转化大肠杆菌感受态,Kan板筛选。阳性克隆筛选,方法如下:
U626-IDF+U629-IDR=726bp菌落PCR鉴定,U626-IDF和U629-IDF测序。测序正确的载体即为BnaREM1.3的CRISPR载体。
菌落PCR及测序引物:
U626-IDF:TGTCCCAGGATTAGAATGATTAGGC
U629-IDF:TTAATCCAAACTACTGCAGCCTGAC
U629-IDR:AGCCCTCTTCTTTCGATCCATCAAC
(rc:GTTGATGGATCGAAAGAAGAGGGCT)
(6)将构建正确的重组质粒载体导入农杆菌菌株GV3101,挑选阳性单克隆于-80℃冰箱保存。转化方法同实施例2。
实施例4遗传转化实验
(1)油菜的遗传转化
对构建好的BnaC04.REM1.3a过表达载体和CRISPR载体进行油菜的遗传转化,使用农杆菌介导的遗传转化方式,本发明中用于油菜转化的受体为甘蓝型油菜Westar,具体操作流程详见参考文献:An efficient Agrobacterium-mediated transformation methodusing hypocotyl as explants for Brassica napus.
(2)过表达转化单株的鉴定
提取获得的油菜过表达转化单株的基因组DNA,通过PCR检测外源基因片段的***,本发明中过表达的骨架载体为35S-pCAMBIA1300S,在骨架载体上设计引物pCAMBIA1300S Primer-P1(5’-CCAGGCTTTACACTTTATGC-3’),通过载体骨架引物与外源片段引物搭配来进行PCR(pCAMBIA1300SPrimer-P1和BnaREM1.3-KpnⅠ-F,BnaREM1.3-PstⅠ-R序列如实施例1所示),在PCR水平检测转基因苗。PCR体系为:Taq polymerase Mix 5μL;pCAMBIA1300S Primer-P1(10μmol/L)0.5μL;BnaREM1.3-KpnⅠ-F(10μmol/L)0.5μL;gDNA 1μL;ddH2O 3μL。PCR条件:94℃总变性5min;94℃变性30sec,558℃退火30sec,72℃延伸30sec,34circles;72℃总延伸5min。
对PCR获得的油菜转基因阳性苗进行qRT-PCR,以检测基因表达量。提取转化单株种子的RNA并进行cDNA的合成(方法同实施例1),定量引物利用Primer 5软件设计得到,产物大小在100bp-200bp之间,设计好后用参考序列进行BLAST比对,确保引物qREM-F(5’-AAAACCCATTGAGGAGGCCA-3’)和qREM-R(5’-GTGCCTTGTTCTCAGCCTTTG-3’)的特异性。BnaACTIN7-L(5’-CGCGCCTAGCAGCATGAA-3’)和BnaACTIN7-R(5’-GTTGGAAAGTGCTGAGAGATGCA-3’)作为油菜qRT-PCR的内参引物(详见Zhou et al 2012:BnMs3 is required for tapetal differentiation and degradation,microsporeseparation,and pollen-wall biosynthesis in Brassica napus)。反应体系为:
反应程序:94℃30s;94℃10s,60℃15s,72℃30s,45个循环;绘制溶解曲线。qRT-PCR在Bio-Rad CFX96 Real-Time System中进行。
根据内参引物进行标准化,不同重复间定量变异用delta-deltathreshold cyclerelative quantification(2-ΔΔCT)的方法计算。最后分析获得油菜过表达转化单株OE3、OE6和OE8(图2)。
(3)CRISPR转化单株的鉴定
对获得的油菜CRISPR转化单株进行测序筛选油菜突变体。首先利用引物Cas9-F(5’-AGACCGTGAAGGTTGTGGAC-3’)和Cas9-R(5’-TAGTGATCTGCCGTGTCTC-G-3’)鉴定Cas9蛋白,对于Cas9蛋白阳性单株进行目的基因的特异扩增以及测序鉴定。目的基因的特异扩增的方法为:分别用引物C-REM1.3-A05-F(5’-GGCTTACTTATGTGAACCTCTCCAT-3’)和C-REM1.3-A05C04a-R(5’-TTAGAAACATCCACAAGTCGCC-3’)特异扩增BnaA05.REM1.3;分别用引物C-REM1.3-C04a-F(5’-CTGCTTTGCTTCTAAAGCAAAC-3’)和C-REM1.3-A05C04a-R(5’-TTAGAAACATCCACAAGTCGCC-3’)特异扩增BnaC04.REM1.3;分别用引物C-REM1.3-A04-F(5’-GCGGTAGTTATACGTTTTCAATG-3’)和C-REM1.3-A04C04b-R(5’-TTAGAAACATCCACAAGTCGCC-3’)特异扩增BnaA04.REM1.3;分别用引物C-REM1.3-C04b-F(5’-GCCTTCCTAATTGAAGCGG-3’)和C-REM1.3-A04C04b-R(5’-TTAGAAACATCCACAAGTCGCC-3’)特异扩增BnaC04.REM1.3b。扩增方法同实施例4(2)所示。
对扩增的目的片段进行PCR产物测序,测序结果用DSDecode在线网站(http://skl.scau.edu.cn/dsdecode/)分析靶位点的编辑情况。测序结果显示,获得了BnaREM1.3被编辑的两个突变体独立株系(CR1和CR2)。两个突变体的BnaA04.REM1.3、BnaA05.REM1.3、BnaC04.REM1.3a均被编辑(图3)。
(4)转化所得植株的抗旱相关表型分析
同时在田间收获已纯合的突变体、过表达和野生型种子,将其种入温室中进行苗期干旱试验。温室温度恒定为25℃,光照/黑暗时间为16h/8h。实验设置对照组和处理组,每个株系种植20株以上幼苗以用于取样和存活率统计。两组幼苗均正常浇水生长至3周大,然后处理组停止浇水开始干旱处理,对照组保存充足的水分供应。干旱处理7天后,进行表型的观察检测、拍照和取样等。继续对处理组的幼苗进行干旱胁迫,待首先出现干旱敏感表型的株系出现大多数的死亡后,对所有干旱处理组复水,并统计存活率。三次重复试验验证表型和存活率。存活率=存活的幼苗数/总幼苗数。
室内干旱试验结果表明,正常水分供应条件下的3周大的幼苗在长势上无显著差异,干旱胁迫处理后,相比于野生型,两个突变体株系均出现了更加严重的萎蔫程度。相反,两个过表达株系则出现了更强的抗旱性。继续处理一段时间后进行复水,3天后统计幼苗存活情况。在复水3天后,相比于野生型,过表达株系表现出更多的单株存活情况而突变体株系则出现更多的植株死亡(图4A)。将干旱试验重复3次,每次均统计存活率,结果表明,相比于野生型植株,过表达植株的存活率显著增加而突变体植株的存活率显著降低(图4B)。
综上所述,基因BnaREM1.3在调控油菜抗旱性中发挥重要的作用。
在本发明中所提及的所有文献都在本申请中引用作为参考文献,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明所讲述内容后,本领域的技术人员可以对本发明做各种改动或者修改,这些等价形式同样属于本申请所附的权利要求书所限定的范围。
Claims (6)
1.油菜BnaREM1.3基因及其编码蛋白在调控作物抗旱性中的应用。
2.如权利要求1所述的应用,其特征在于:所述油菜BnaREM1.3基因的核苷酸序列如SEQID NO:1所示,或者是与SEQ ID NO:1所示序列具有80%以上同源性且编码同一功能蛋白的其它序列。
3.一种提高油菜抗旱性的方法,其特征在于:使用农杆菌介导的遗传转化方法,将含有油菜BnaREM1.3基因的植物表达载体转化到油菜的基因组中,获得BnaREM1.3基因过表达的油菜品种。
4.如权利要求3所述提高油菜抗旱性的方法,其特征在于:所述植物表达载体是35S-pCAMBIA1300S,该载体含有组成型表达启动子和抗生素标记物。
5.一种降低油菜抗旱性的方法,其特征在于:使用农杆菌介导的遗传转化方法,将含有油菜BnaREM1.3基因的CRISPR/Cas9植物基因编辑载体转化到油菜的基因组中,获得BnaREM1.3基因被编辑的油菜品种。
6.如权利要求5所述降低油菜抗旱性的方法,其特征在于:所述CRISPR/Cas9植物基因编辑载体是PKSE401。
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