CN116004558B - 乙酰转移酶OsG2基因及其编码的蛋白质在调节水稻植株高度方面的应用 - Google Patents
乙酰转移酶OsG2基因及其编码的蛋白质在调节水稻植株高度方面的应用 Download PDFInfo
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Abstract
本发明公开了一种乙酰转移酶OsG2基因及其编码的蛋白质在调节水稻植株高度方面的应用,所述蛋白质的氨基酸序列如SEQ ID No:2所示。本发明通过超量表达OsG2基因来调控水稻等农作物的植株高度,本发明为培育植株高度可调控的水稻提供了一条重要途径,可应用在农业生产上栽培植株高度可调控的水稻,对节能节水、盐碱地的利用、增加粮食产量等。
Description
技术领域
本发明涉及一种乙酰转移酶OsG2基因及其编码的蛋白质在调节水稻植株高度方面的应用,属于基因工程领域。
背景技术
植物固着生长的生活方式,决定了其在生长发育的整个生命周期中,在遇到外界不利环境时,不能像动物一样选择通过移动来躲避。对植物生长、发育和繁殖产生不利影响的外界条件统称为逆境,植物主要受到两大逆境:生物和非生物逆境的影响。生物逆境胁迫是指植物、动物及微生物对植物形成的伤害,如草害、虫害、病害;非生物逆境胁迫是指由于过度或不足的物理或化学条件引发的对植物生长、发育和繁殖产生的不利影响,主要包括盐碱害、干旱、高温、低温、重金属等。在逆境胁迫条件下,植物进化出一套自己的防御系统,在分子、生理、发育和形态等多个水平上形成了适应机制,不同的逆境胁迫会引起植物在基因表达、代谢和生理性状等方面的特异的反应。
盐害是一种严重影响植物区域分布和生长发育的非生物限制性因子,是影响植物生长发育的主要非生物逆境胁迫之一,会同时对细胞造成渗透胁迫和离子毒害,并引发一系列的次级影响,包括导致氧化胁迫、破坏膜脂、蛋白质及核酸等细胞组分,并引起代谢紊乱。渗透胁迫是指土壤中高浓度的盐分降低了土壤水势,造成植物的吸水困难,甚至引起植物体内水分的外渗,从而造成植物的水分亏缺,产生生理干旱。在盐胁迫中,离子毒害指的是过量的Na+浓度抑制了植物对其他离子的吸收,从而产生了毒害作用。
水稻是单子叶植物,是世界上最重要的粮食作物之一,研究水稻各个时期生长发育的分子生物学机理以及对逆境胁迫的调控,不仅有助于了解水稻的生长发育机制,同时对提高水稻抗逆性,保证水稻的产量具有重要的意义。水稻属于盐敏感作物,特别是在幼苗期和发育期。在盐胁迫条件下,水稻会通过特异的表达大量的耐盐蛋白和若干网络信号分子来调控水稻体内的渗透调节方式,从而维持植株体内环境的稳定,抵抗盐胁迫对水稻造成的多种影响。通过研究水稻盐胁迫的响应机制,筛选水稻抗盐相关的基因,进一步解析其调控机理,培育耐盐胁迫的水稻品种,进而扩大水稻的种植面积,达到增加粮食产量的目的。
乙酰化是生物体内普遍存在的一种蛋白质共价修饰方式之一。N-末端乙酰化在蛋白质的稳定性和降解、蛋白质的亚细胞定位和蛋白-蛋白相互作用和复合体的形成以及细胞的生长、代谢和疾病的发生中均有重要的作用。N-末端乙酰化普遍存在于真核生物中,在酵母中大约占可溶蛋白的50%-70%,在哺乳动物中约占70%-90%,在古生菌和细菌中也发生N-末端乙酰化修饰,只是相对较少。目前已经发现的N-末端乙酰转移酶主要有7种,但研究主要集中在人类中,在植物中关于N-末端乙酰转移酶基因功能的研究还较少,尤其是乙酰转移酶在植物抗逆方面的研究。
发明内容
本发明要解决的技术问题是,寻找农作物抗盐胁迫的相关蛋白质和编码基因,以获得抗逆性改变的植株,使作物可适于在盐碱地上生长。
本发明提供一种乙酰转移酶OsG2基因编码的蛋白质在调节农作物抗盐胁迫性能的应用,所述蛋白质的氨基酸序列如SEQ ID No:2所示。
本发明进一步提供一种乙酰转移酶OsG2基因编码的蛋白质在调节农作物植株高度方面的应用,所述蛋白质的氨基酸序列如SEQ ID No:2所示。
本发明还提供一种乙酰转移酶OsG2基因编码的蛋白质在调节农作物籽粒大小方面的应用,所述蛋白质的氨基酸序列如SEQ ID No:2所示。
作为本发明的优选方案,通过超量表达乙酰转移酶OsG2基因来提高农作物的抗盐胁迫性能。
作为本发明的优选方案,所述乙酰转移酶OsG2基因的核苷酸序列如SEQ ID No:1所示。
作为本发明的优选方案,所述核苷酸序列还包括在SEQ ID No:1所示的核苷酸序列中添加、取代,插入或缺失一个或多个核苷酸而生成的突变体、等位基因或衍生物。
作为本发明的优选方案,所述农作物为禾本科植物。
作为本发明的优选方案,所述农作物包括但不仅限于水稻、玉米、小麦等禾本科植物。
乙酰转移酶OsG2基因是水稻乙酰转移酶家族GNAT(GCN5-related N-acetyltransferase,putative,expressed)亚家族成员之一,来源于水稻(Oryza Sativa),NCBI网站(http://www.ncbi.nlm.nih.gov)基因编号为LOC9269142,RICE网站(http://rice.plantbiology.msu.edu/)基因编号为LOC_Os02g46700;是编码序列表中SEQ ID NO.2所示氨基酸序列的蛋白质。
为描述方便,本发明将SEQ ID No:1所示的核苷酸序列的基因称为OsG2基因。本发明将SEQ ID NO.2所示氨基酸序列的蛋白质称为OsG2蛋白。
本发明提供一种乙酰转移酶OsG2基因及该OsG2基因编码的蛋白质在农作物盐胁迫中的应用,为研究农作物(特别是水稻)的耐盐性调控机制提供了一个候选基因。缺失该基因时,水稻对盐胁迫的敏感度增加,并表现出不耐受的表型。而OsG2基因超量表达的转基因水稻植株则表现出增强的盐胁迫抗性。此外,对突变体植株进行进一步形态学观察发现,突变体水稻株高变矮,籽粒变小,千粒重减小,OsG2基因可能通过直接或者间接修饰水稻种子发育中的某些关键基因,进而影响最终籽粒大小变化。
本发明将包括所述OsG2基因的表达载体导入农作物中,筛选获得抗逆性(抗盐胁迫性)改变的农作物植株。
本发明的OsG2基因在植物整个生长周期持续表达,在植物的不同组织均能检测到OsG2基因的存在,盐胁迫处理发现OsG2基因在根和叶中的表达量在150mM NaCl的诱导下均呈现出一定程度的上升。
本发明所述转基因敲除突变体,在150mM NaCl处理之后,经检测盐胁迫相关基因(如OsSOS1/OsSOS2/OsSalT/OsABI5,均为水稻已知基因)的表达量均有不同程度的显著升高。植物在受到盐胁迫时,Ca2+依赖的抗盐途径(Salt Overly Sensitive pathway),简称SOS途径,被迅速激活,OsSOS1和OsSOS2是SOS途径的关键基因。而OsSalT是一种类似于黄芩素的凝集素蛋白,是水稻根中被高盐诱导的最显著的蛋白之一。OsABI5是一种bZIP类转录因子,在盐胁迫条件下被诱导表达。突变体中OsSOS1/OsSOS2/OsSalT/OsABI5的表达量均上升,表明OsG2基因可能通过负调控这些基因的表达,达到抗盐胁迫的目的。
本发明通过转基因技术将含上述SEQ ID NO.1所示核苷酸序列的质粒或植物表达载体转染宿主细胞,所述宿主细胞包括但不限于大肠杆菌细胞、农杆菌细胞、植物细胞;再将宿主细胞导入农作物(如水稻)中。
综上所述,本发明研究表明乙酰转移酶OsG2基因编码的蛋白质在水稻等农作物应答盐碱逆境胁迫反应中及调控水稻籽粒大小上发挥重要的作用,进而可以利用该基因进行水稻等农作物的重要农艺性状和胁迫耐性的遗传改良。本发明通过超量表达OsG2基因来提高水稻等农作物的盐胁迫抗性,本发明为培育盐胁迫抗性增强的水稻提供了一条重要途径,可应用在农业生产上栽培盐胁迫抗性增强的水稻,对节能节水、盐碱地的利用、增加粮食产量等具有重要意义。
附图说明
图1是水稻乙酰转移酶OsG2基因的基因结构示意图及OsG2基因两等位突变体的突变位点示意图;
图2是盐胁迫条件下野生型日本晴地下部分和地上部分OsG2基因表达分析;
图3是在150mM NaCl处理下,突变体植株表型分析图;
图4是水稻OsG2基因突变体植株盐胁迫相关基因的相对表达量分析;
图5是突变体植株大田不同生长时期表型分析;
图6是突变体植株大田不同生长时期株高统计;
图7是去壳情况下,野生型日本晴和突变体的种子图片;
图8是带壳情况下,野生型日本晴和突变体的种子图片;
图9是在150mM NaCl处理下,OsG2基因超量表达植株表型分析图。
具体实施方式
以下的实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1:转基因水稻材料的获得
1、转基因突变体植株的获得
本研究中用到的植株材料包括野生型日本晴水稻和两种突变体植株,并将两种突变体植株分别命名为osg2-1和osg2-2,本发明所采用的水稻盐敏感突变体是利用CRISPRCas9技术进行基因编辑后得到,基因编辑的结果是产生了不同突变位点的两种等位突变体,编辑方式如图1所示。
OsG2基因全长为4256bp,具有如图1所示的基因结构图。其中,NIP表示野生型日本晴水稻,用于对照;为了构建OsG2基因的突变体植株,使用CRISPR-Cas9基因编辑技术来创建。主要方法如下:
水稻T0代转化植株经PCR测序检测,筛选出双链纯合编辑的突变体,分别命名为osg2-1以及osg2-2。其中osg2-1材料编辑类型为第16-311和第548-549位(以起始密码子ATG中的A为1)分别删除了两段核苷酸序列,共298bp核苷酸被删除,发生移码突变,造成OsG2基因结构被破坏,从而无法表达出正常大小有功能的OsG2蛋白;osg2-2材料编辑类型为第16-647位的核苷酸序列被切除,使OsG2基因损失了632bp的DNA片段,导致移码,其结果同样无法产生结构完整的OsG2蛋白,使OsG2蛋白无法正常发挥功能。综上,上述方法产生了两种不同的OsG2等位突变体:osg2-1和osg2-2。
潮霉素标记基因以及Cas9基因检测结果显示上述两种突变体类型已分离出载体。CRISPR Cas9创建突变体的载体为pRGEB32(https://www.addgene.org/63142/),在水稻OsG2基因的外显子上设计3个靶位点来敲除OsG2基因。
靶位点序列为:
靶位点1:TGGGGTAGATTTCCGATCTT;
靶位点2:TTGAACGTGGTCAACGGCAA;
靶位点3:TCTCCACTACACCGAGCGTC。
潮霉素基因鉴定引物为:
F:CTCCATACAAGCCAACCACG;
R:GGAAGTGCTTGACATTGGGG。
Cas9鉴定引物为:
F:CGATAAGAACCTGCCCAACG;
R:GCTCTTTGATGCCCTCTTCG。
CRISPR编辑检测测序引物为:
F:TACTGCCTCTCTTCCTCGCC;
R:GACACCCCTGCAATTTGAGA。
2、转基因超量表达植株的获得
本研究中用到的另一种转基因植株材料是两个OsG2超量表达株系,在本发明中统一命名为OsG2-OE,OsG2基因在两个超量表达株系中的表达量均达到了70倍以上。产生超量表达转基因材料的具体操作过程如下:
首先,设计带有Gateway反应体系BP接头的OsG2特异性引物,通过PCR的方法从水稻总RNA反转录得到的cDNA中扩增得到OsG2基因的全长编码序列,将该片段经BP重组反应构建到pDONR223载体上,操作按Invitrogen公司提供的重组克隆试剂盒说明书进行。再将BP反应产物与相应的表达载体进行LR重组反应,本发明中所用表达载体为pCAMBI1300-33,具有强启动子UBI10,最后将含有OsG2基因编码序列的表达载体通过农杆菌介导的水稻遗传转化体系将其导入至水稻品种日本晴中,并最终得到OsG2基因不同表达倍数的转基因超量表达材料。
用于从总cDNA中扩增OsG2基因编码序列的引物为:
F:
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTTGGACCCAAGATCG
R:
GGGGACCACTTTGTACAAGAAAGCTGGGTGTTAGACATGACATCTCGTATCATC
实施例2:盐胁迫处理分析OsG2的响应情况
首先利用野生型日本晴进行盐胁迫处理,分析OsG2的响应情况。具体过程如下:挑选发育状况良好的野生型日本晴水稻种子,在不去壳的情况下放入一个盛有少量无菌水的培养皿中,培养皿的底部铺有一层吸水纸。将盛有种子的培养皿放在30-35℃培养箱中两天,待种子露白破胸后再转移至温度为25℃左右的培养箱中,达到催芽促根的目的。需要注意的是,在浸种发芽的过程中需要勤于更换培养皿中的无菌水,以保证种子进行正常的呼吸和生长。
挑选萌发后生长状况一致水稻幼苗转移至盛有Hoagland营养液的培养盒中,待幼苗在培养盒中生长约15天左右时,挑选生长状态一致的幼苗进行盐胁迫处理,处理时采用Hoagland营养液(含有150mM NaCl)进行水培。在处理之前取样作为对照组,然后在3天后对幼苗的地上和地下部分分别取样,每个时间点的取样重复为至少三个生物学重复。取样后立即放入液氮中速冻。然后转移至-80℃冰箱备用。
提取总RNA后反转录为cDNA,以cDNA为模板进行qPCR分析OsG2基因的表达情况。结果如图2所示:随着盐胁迫处理时间的延长,OsG2基因的表达量呈现明显上升趋势。因此,乙酰转移酶OsG2对水稻抗盐胁迫起正向调控作用。
用于鉴定OsG2基因的qPCR引物如下:
F:GGCCGTTCGCCTTGCT
R:ACCTGGCAACCACCATCTTC
用于鉴定做对照的ACTIN引物如下:
F:TGGCATCTCTCAGCACATTCC
R:TGCACAATGGATGGGTCAGA
实施例3:转基因突变体植株盐敏感度检测
实验材料:野生型日本晴、OsG2基因等位突变体
处理方式:水稻种子萌发方式如实施例2所述,挑选生长状态一致的野生型日本晴和突变体幼苗,转移至含有150mM NaCl的Hoagland营养液中,分别处理0天、5天、7天,观察对照组和实验组的叶片形态变化,进行表型记录。如图3所示,相对于野生型日本晴,突变体植株对盐胁迫更加敏感。
实施例4:水稻OsG2基因突变体植株盐胁迫相关基因的相对表达量分析
实验材料:野生型日本晴、OsG2基因等位突变体
将实施例3中盐胁迫处理7天之后的植物的地上和地下部分分别进行取样,每次取样不少于3个生物学重复;提取总RNA,进行反转录后得到cDNA,将cDNA作为模板,通过实时荧光定量PCR鉴定各个相关基因的表达水平。以野生型日本晴未处理时的各个基因相对管家基因ACTIN表达量为1,计算样品中对应基因的相对表达水平。
用于鉴定OsSOS1基因的引物如下:
F:CGGATCGCTCGAATTTGG
R:GAATGCCGGCTCCGAGTT
用于鉴定OsSOS2基因的引物如下:
F:CGCCGTCGCCATGAAG
R:TGGAGCATGCGGTGGTT
用于鉴定OsABI5基因的引物如下:
F:GAAGAATTCCAGGCTACCACC
R:TTGATCTCAGTCCACACCTCC
用于鉴定OsSalT基因的引物如下:
F:TCCACTGCAAGACTCTGGCC
R:CCAATTGCGTCGATAAGCG
如图4所示,相对与处理前的野生型日本晴,盐胁迫之后,盐敏感突变体中盐胁迫相关基因的表达量上升。缺失乙酰转移酶OsG2基因之后,相关基因的表达状态受到一定程度的影响。间接证明乙酰转移酶OsG2蛋白参与盐胁迫相关的复杂信号通路的调控。
实施例5:乙酰转移酶OsG2参与调控植株生长
如图5和图6所示,我们对不同生长阶段的野生型日本晴和乙酰转移酶OsG2基因的等位突变体osg2-1和osg2-2进行了相应的田间农艺性状统计,统计结果发现,与野生型日本晴相比,乙酰转移酶OsG2基因的等位突变体osg2-1和osg2-2株高出现一定程度的降低,在水稻生长发育的不同阶段这种差异都是持续显著存在的。由此说明,乙酰转移酶OsG2基因参与调控植株的生长。
实施例6:乙酰转移酶OsG2参与调控种子的发育
水稻籽粒大小与其产量和品质等性状密切相关。如图7和图8所示,在去壳和不去壳的情况下,相比于野生型日本晴,突变体的籽粒大小包括种子的长度和宽度都比野生型的有所降低。这些结果表明,该基因参与水稻籽粒大小的调控过程,缺失该基因后导致水稻种子变小,进而影响水稻的产量。
实施例7:转基因超量表达株系耐盐性表型分析
本实施例选择了两个OsG2基因表达量均增加70倍以上的转基因超量表达株系进行了150mM NaCl盐胁迫处理实验。处理方式:水稻种子萌发方式如实施例2所述,挑选生长状态一致的野生型日本晴和OsG2基因超量表达植株,转移至含有150mM NaCl的Hoagland营养液中,分别处理0天、2天、5天,观察对照组和实验组的叶片形态变化,进行表型记录。实验结果如图9所示,胁迫2天或者5天后,OsG2-OE植株的卷叶程度明显轻于对照日本晴,因此OsG2基因超量表达的转基因水稻株系具有较强的盐胁迫耐性。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
SEQUENCE LISTING
<110> 武汉大学
<120> 乙酰转移酶OsG2基因及其编码的蛋白质在调节水稻植株高度方面的应用
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Gln Asp Ser Glu Ile Glu Asp Leu Phe Arg Tyr Asn Ser Ser Arg Lys
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Asp Leu Thr Leu Leu Tyr Ile Leu Thr Leu Gly Val Val Glu Ser Tyr
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Arg Asn Leu Gly Ile Ala Ser Ser Leu Val Arg Glu Val Ile Lys Tyr
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Ala Ala Ser Ile Ser Asn Cys Arg Gly Val Tyr Leu His Val Ile Ser
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Tyr Asn Gln Pro Ala Ile Ser Phe Tyr Asn Lys MET Leu Phe Lys Leu
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Val Arg Arg Leu Pro His Phe Tyr Tyr Ile Arg Gly Gln His Tyr Asp
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Leu Val Ser Gln Asn Asn Lys Arg Ile Ile Gly Gly Asp Asp Thr Arg
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Cys His Val
Claims (3)
1.一种乙酰转移酶OsG2基因编码的蛋白质在调节水稻植株高度方面的应用,所述蛋白质的氨基酸序列如SEQ ID No:2所示。
2.根据权利要求1所述的应用,其特征在于,通过乙酰转移酶OsG2基因的超量表达来调节水稻植株的高度。
3.根据权利要求1所述的应用,其特征在于,所述乙酰转移酶OsG2基因的核苷酸序列如SEQ ID No:1所示。
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