CN108359670B - 提高砷胁迫水稻耐受性的microRNA基因及其应用 - Google Patents
提高砷胁迫水稻耐受性的microRNA基因及其应用 Download PDFInfo
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Abstract
本发明公开了提高砷胁迫水稻耐受性的microRNA基因及其应用,主要涉及植物功能基因组学领域。包括提高水稻对砷胁迫耐受性的微小RNA即osa‑miR812q,其核苷酸序列如SEQ ID NO.1所示。该microRNA的前体基因在水稻中过量表达后,转基因株系在砷胁迫下的耐受能力明显高于野生型水稻。本发明的有益效果在于:在水稻体内过表达本发明的microRNA,能够显著提高水稻对砷胁迫的耐受性,缓解砷对水稻的毒害,降低砷在水稻中的积累,对提高水稻品质具有重大的意义,为培育耐受砷胁迫的水稻新品种提供了新的基因资源。
Description
技术领域
本发明涉及植物功能基因组学领域,具体是提高砷胁迫水稻耐受性的microRNA基因及其应用。
背景技术
砷(Arsonism,As)是表土污染五大有害元素之一。近年来,采矿冶炼和含砷除草剂的使用加剧了土壤中砷的污染程度,中国土壤砷污染形势已相当严峻。
砷不仅影响土壤生态结构和功能,而且能抑制作物的生长发育,降低产量和品质。砷可通过食物链进入人体,作用于半胱氨酸残基导致酶和蛋白质构象畸变,并阻止二硫键生成,对人体具有强烈毒害作用,被列为I级致癌物。
水稻(Oryza sativa)对砷的吸收和积累能力远高于其他谷类作物,在缺氧的水稻土壤中,砷的主要存在形式为三价亚砷酸盐As(III),其毒性比五价砷酸盐As(V)更强,水稻砷污染已成为人类砷暴露危害的主要途径之一。中国是世界水稻种植和消费第一大国,因此,提出避免水稻砷伤害的有效措施和提高砷胁迫下的抗性的有效方法,将对保障农作物生态安全和人民健康、促进农业可持续发展具有重要意义。
microRNA(miRNA)是一种内源性非编码小分子RNA,通过序列配对调控转录后或翻译水平的靶基因表达,从而调控植物生长发育、激素分泌、信号转导、器官形态建成等过程。microRNA作为细胞逆境应答的激活因子,在重金属、干旱和高温等环境胁迫应答中发挥着重要作用。
近年来,随着高通量芯片和测序技术的广泛运用,miRNA的大规模高通量鉴定得以实现,重金属逆境应答相关miRNA的发掘取得了一定进展。Ding等人(Y.Ding,Z.Chen andC.Zhu,Microarray-based analysis of cadmium-responsive microRNAs in rice(Oryzasativa),J.Exp.Bot.,2011,62,3563–3573)利用miRNA微阵列芯片和荧光定量PCR技术从水稻中筛选到了19个与镉胁迫应答相关的miRNA。Yu等人(L.J.Yu,Y.F.Luo,B.Liao,L.J.Xie,L.Chen,S.Xiao,J.T.Li,S.N.Hu and W.S.Shu,Comparative transcriptome analysis oftransporters,phytohormone and lipid metabolism pathways in response toarsenic stress in rice(Oryza sativa),New Phytol.,2012,195,97–112.)利用高通量测序鉴定获得36个砷胁迫下差异表达的水稻miRNA。因此,利用miRNA的遗传操作改良作物对重金属胁迫的耐性已成为一个可行的技术手段。
miR812是一种广泛存在于单子叶植物和双子叶植物中的miRNA。miR812可靶向CIPK10,CIPK10进一步和CBL相互作用从而激活CBL-CIPK信号转导通路,该通路在调节逆境胁迫耐受性中具有重要作用。已有研究报道miR812在植物对环境胁迫的应答过程中发挥着一定作用。Kansal等人(Kansal S,Mutum R D,Devi R M,et al.Unique miRNome duringanthesis in drought-tolerant indica rice var.Nagina 22.[J].Planta,2015,241(6):1-1.)对比了干旱胁迫下粳稻品种Nagina 22抽穗至开花期的microRNA表达谱,发现水稻miR812在干旱胁迫下差异表达,可能对干旱和高温胁迫具有敏感性。然而,目前尚未见到关于miR812在重金属胁迫下调控功能的报道。
发明内容
本发明的目的在于提供一种microRNA基因以及其在在提高水稻砷胁迫耐受性方面的应用。通过在水稻种过表达前述基因,培育耐受砷胁迫的水稻新品种。
本发明为实现上述目的,通过以下技术方案实现:
一种提高水稻对砷胁迫耐受性的微小RNA即osa-miR812q,其核苷酸序列如SEQ IDNO.1所示。
所述microRNA的前体序列为序列表中的SEQ ID NO.2。
所述microRNA前体序列的编码序列为序列表中的SEQ ID NO.3。
此编码序列被导入水稻细胞后可被水稻细胞转录成前体microRNA,所述的前体microRNA可被剪切并加工为成熟的microRNA,进而影响到相关生物学功能的发挥;此osa-miR812q前体序列的基因定位在水稻10号染色体上,实时荧光定量PCR检测结果表明,osa-miR812q在亚砷酸钠胁迫后发育过程中受到显著诱导。
包含上述编码序列的重组表达载体,为在pCAMBIA1301空载体的限制性内切酶位点Pst I和Kpn I之间插microRNA前体序列的基因,得到重组表达载体。
如序列表SEQ ID NO.1所示的microRNA基因在提高水稻砷胁迫耐受性中的应用。培出具有砷胁迫耐受性的转基因水稻。
其方法优选为通过PCR方法扩增miR812q的前体基因,构建含有miR812q前体基因的重组表达载体并将载体导入农杆菌,通过农杆菌侵染水稻愈伤组织,筛选获得具有砷胁迫耐受性的水稻植株。
对比现有技术,本发明的有益效果在于:
在水稻体内过表达本发明的microRNA,能够显著提高水稻对砷胁迫的耐受性,缓解砷对水稻的毒害,降低砷在水稻中的积累,对提高水稻品质具有重大的意义,为培育耐受砷胁迫的水稻新品种提供了新的基因资源。
附图说明
附图1为osa-miR812q前体基因的PCR扩增凝胶图谱;
附图2为35S:miR812q重组质粒菌液PCR扩增凝胶图谱;
附图3为35S:miR812q重组质粒酶切鉴定图;
附图4为35S:MIR812q过表达载体T-DNA区的结构图;
附图5为三叶期水稻在10mM NaAsO2处理7天后表型(WT为野生型;35S:MIR812q为转基因水稻);
图6为砷胁迫7天后水稻叶绿素含量的变化情况;
图7为砷胁迫7天后水稻地上部和根中的砷含量。
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。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所限定的范围。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1:osa-miR812q前体基因的克隆
用于基因克隆与表达的水稻品种为苏香粳3号(Oryza sativaL.subsp.Japonica,cv.Suxiang3)。克隆的osa-miR812q前体序列如序列表SEQ ID NO.2;前体序列表达的成熟序列如SEQ ID NO.1所示。
挑选饱满的水稻种子,70%乙醇消毒,蒸馏水冲洗后转移至湿滤纸,5%霍格兰营养液催芽2天。露白后转移至光温培养箱(16h光照/8h黑暗,温度25℃,相对湿度70%),每3天更换1次营养液。将18天龄幼苗用10mM NaAsO2进行胁迫处理,未处理组作为对照,每组3次重复。处理48h后对叶片取样,液氮冷冻并-80℃储存。使用Trizol法分别提取对照组和NaAsO2胁迫组总RNA。取叶片0.1g,用液氮研磨并加入1ml Trizol(Invitrogen)继续研磨至匀浆,转入1.5ml离心管中,12000rpm离心10min,取上清,室温静置5min,加入0.2ml氯仿震荡15s。室温放置5min,12000rpm离心15min,取上清。加入400μl异丙醇,-20℃放置2h。12000rpm离心10min,去上清,沉淀用1ml 70%乙醇洗涤2次,室温干燥5min,沉淀重溶于50μl RNase-free水,置于-80℃保存。根据miRBase(http://www.mirbase.org/)数据库中miR812q前体的序列信息,采用miRprimer3.0软件分别在前体发夹结构两端约50bp处设计引物并在两端添加酶切位点Pst I和Kpn I。所设计引物如下:
F1 5’-CGGGGTACCGACGTTGGGTACGAATATCTAC-3’
(SEQ ID NO.4,下划线序列为Kpn I位点)
R1 5’-AACTGCAGCCAGTACAGAATTAATACTGCCGT-3’
(SEQ ID NO.5,下划线序列为Pst I位点)
接着利用PrimeScriptTM(Takara)试剂盒反转录获得cDNA作为PCR模板,扩增osa-miR812q前体的编码基因。PCR采用SYBR Premix Ex TaqTM(Takara)在ABI7500荧光定量PCR仪上进行。以水稻5S rRNA为内参基因,去离子水作为阴性对照。PCR反应体系为:SYBRPremixEx TaqTM(2×)10μl,ROXReferenceDyeII(50×)0.4μl,cDNA模板2.5μl,正向引物(10μM)0.4μl,反向引物(10μM)0.4μl,加ddH2O至终体积20μl。PCR程序为:95℃预变性5min,95℃变性30s,60℃退火30s,72℃延伸30s,40个循环后72℃继续合成7min。PCR扩增产物进行1%琼脂糖凝胶电泳,采用上海生工胶回收试剂盒回收,PCR产物凝胶图谱如图1所示。
实施例2:osa-miR812q超表达载体的构建
将扩增出的PCR产物连接克隆载体pMD19-T(TakaRa)。10μl体系中加入1μlpMD19-T载体,5μlsolutionI,4μl纯化的miR812q前体DNA,混匀混合液,16℃连接6h,经PCR反应验证及测序鉴定正确后,得载体pMD19-miR812q。热击法将载体pMD19-miR812q转化到大肠杆菌DH5α感受态细胞,过夜培养,经氨苄青霉素(Amp)筛选、挑阳性克隆,测序后选取正确的菌液利用质粒小提试剂盒提取序列完全正确的阳性质粒pMD19-miR812q。用限制性内切酶Pst I和Kpn I酶切,回收酶切产物,同时用限制性内切酶Pst I和Kpn I酶切空载体pCAMBIA1301,回收载体骨架。酶切体系50μl,包括5μl 10×M buffer,30μl质粒,1μl KpnI、1μl PstI和13μl ddH2O,37℃水浴过夜。用T4连接酶将酶切产物和pCAMBIA1301载体骨架连接,连接体系10μl,包括1μl载体,7.5μlmiR812q DNA,1μl 10×T4连接酶buffer和0.5μl T4连接酶,16℃连接过夜。将连接产物热激转化入大肠杆菌DH5α感受态细胞中,37℃过夜培养,挑取阳性克隆进行测序。测序结果表明,得到了重组质粒pCAMBIA1301-35S:miR812q。
实施例3:农杆菌介导的水稻转化
将载体pCAMBIA1301-35S:miR812q导入农杆菌感受态细胞EHA105,操作如下:
1、农杆菌感受态的制备与转化:取农杆菌单菌落,加入5ml含有20mg/ml利福平的YM液体培养中,28℃摇床250rpm振荡培养过夜。吸取2ml菌液加入50ml YM培养基中,28℃振荡培养至OD600=0.5。4000rpm离心10min收集菌体,YM培养基悬浮菌体备用。将1μgmiRNA过表达载体质粒加入200μl农杆菌感受态细胞中,用枪头吹打混匀,依次在冰浴、液氮和37℃水浴中放置5min。加入800μl YM培养基,28℃振荡培养2-4h。取200μl菌液,涂布于含50mg/L卡那霉素(Kan)和40mg/L利福平(Rif)的YM培养基上,28℃暗培养箱中培养2-3天,挑取农杆菌单菌落。摇菌,提取农杆菌质粒DNA重新转入大肠杆菌DH5α中,进行菌液PCR和质粒酶切鉴定,结果分别如图2和图3所示。
2、水稻成熟胚愈伤组织的诱导:选取饱满的水稻成熟种子,脱壳后用70%酒精消毒2min,25%NaClO溶液浸泡30min,无菌水清洗浸泡30min,移置粳稻成熟胚诱导培养基中,28℃光照培养2周,诱导愈伤组织。去掉芽和胚乳,留下胚性愈伤组织,移入粳稻继代培养基中,28℃光照培养一周后用于转化。
3、感染与共培养:经鉴定正确的阳性质粒,通过农杆菌介导法转化到水稻愈伤。将农杆菌菌体悬浮于AAM液体悬浮培养基中,调整浓度至OD600 0.8-1.0,浸没继代培养一周的水稻愈伤组织,侵染l0min。取出愈伤组织,置于无菌滤纸上沥干40min。将愈伤组织置于铺有一层无菌滤纸的共培养基上25℃暗培养3天,用含500mg/L头孢拉定的无菌水清洗,转入含500mg/L羧卞青霉素(Car)和50mg/L潮霉素(Hyg)的选择培养基进行第一轮选择,28℃暗培养14天。将长有抗性愈伤的初始愈伤转到新的含250mg/L羧卞青霉素(Car)和50mg/L潮霉素(Hyg)的选择培养基上,进行第二轮选择,28℃光照培养直到颗粒性的抗性愈伤组织长出。选择生长旺盛的抗性愈伤组织转移至含潮霉素(Hyg)的分化培养基培养一周。转移到生根培养基上培养一周左右,移栽到温室土钵中生长。挑选生长正常的植株,进行潮霉素PCR筛选和GUS染色,获得水稻转基因株系,移至盆栽培养直至收种。
实施例4:转基因水稻砷耐受性鉴定
将T2代转osa-miR812q水稻种子、野生型苏香粳3号水稻种子在水稻营养土培养14天至3叶期,再将幼苗放在10mM NaAsO2的水稻营养液中处理7天,没有进行As处理的幼苗作为对照组,其他生长条件一致。观察转基因株系与野生型幼苗在As胁迫下的表型,并测定生长指标、生物量、光合色素含量和As含量。每个株系16株,所有数据均为3次重复的平均值,采用主成分分析法处理数据。
生长指标测定:株高、根长采用毫米刻度尺测量。
生物量测定:从茎基部剪断收获植株地上部,75℃烘干至恒重,称取干重。
叶绿素含量测定:称取0.1g水稻新鲜叶片,剪碎后,黑暗条件下采用95%乙醇浸提36h至叶片漂白。以95%乙醇为空白对照,利用分光光度计测定样品在波长645nm和663nm下的吸光值。
As含量测定:水稻地上部干样粉碎后用HNO3-HClO4消化,用ICP-8000电感耦合等离子体发射光谱仪测定砷含量。
测定结果:
过表达osa-miR812q对As胁迫下水稻生长的影响:
在NaAsO2处理下,过表达miR812q的转基因幼苗株高和根长度分别比野生型幼苗高11.86%(P<0.05)和10.93%(P<0.05),说明在过表达miR812q缓解了As对水稻生长的抑制。
过表达osa-miR812q对As胁迫下水稻生物量的影响:
As胁迫下,转基因水稻幼苗地上部鲜重比野生型高15.33%(P<0.05),地上部干重比野生型高10.62%(P<0.05)。
过表达osa-miR812q对As胁迫下水稻叶绿素含量的影响:
由图5可以看出,在10mM NaAsO2处理7天后,miR812q过表达的转基因幼苗叶片比野生型幼苗偏绿。表明砷胁迫过表达osa-miR812q的转基因水稻同野生型相比,叶片黄化趋势显著降低。如图6所示,As胁迫显著降低了野生型水稻幼苗叶片的叶绿素总含量,降幅达51.6%;而转基因幼苗叶片叶绿素总量仅下降19.1%,与对照组相比差异不显著。可见As胁迫下,转基因植株的叶绿素破坏程度比野生型水稻低。
过表达osa-miR812q对As胁迫下水稻As含量的影响
如图7所示,在As胁迫下,转基因与野生型水稻根中的As含量没有明显差异,但是转基因水稻地上部积累的As含量相比野生型显著降低,降幅达63.6%,这暗示miR812q过表达可能阻断了As从根到地上部的运输。
本发明的有益效果:
过表达osa-miR812q的转基因水稻对As胁迫有显著的耐受性。本发明缓解了As胁迫对水稻生长的抑制,提高了As胁迫下水稻叶绿素含量,降低了水稻地上部As含量和转运系数。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110>苏州科技大学
<120>提高砷胁迫水稻耐受性的microRNA基因及其应用
<160>5
<210>1
<211>24
<212>RNA
<213>水稻miR812q
<400>1
ACGUUGGGUA CGAAUAUCUA CGGC 24
<210>2
<211>242
<212>RNA
<213>水稻miR812q
<400>2
UACUCCAUCC GUCUCAAAAU AAGUGCAGUU UUGCACUAUU CAUACUUAAC AUUUGAACGU 60
UCGUCUUAUU UGAAAAUUUU UUAUGAUUAG UAUUUUUAUU GCUAUUAGAU GUUAAAACAU 120
AAAUAGUACU UUAUGUGUGA CUAAAUAUUU UCAAUUUUUU CACAAAAUUU UCAAAUAAGA 180
CGGACAGUCA AACGUUGGGU ACGAAUAUCU ACGGCUGCAC UUAUUUUGGG ACGGAGGUAG 240
UA 242
<210>3
<211>242
<212>RNA
<213>水稻
<400>3
TACTCCATCC GTCTCAAAAT AAGTGCAGTT TTGCACTATT CATACTTAAC ATTTGAACGT 60
TCGTCTTATT TGAAAATTTT TTATGATTAG TATTTTTATT GCTATTAGAT GTTAAAACAT 120
AAATAGTACT TTATGTGTGA CTAAATATTT TCAATTTTTT CACAAAATTT TCAAATAAGA 180
CGGACAGTCA AACGTTGGGT ACGAATATCT ACGGCTGCAC TTATTTTGGG ACGGAGGTAG 240
TA 242
<210>4
<211>31
<212>DNA
<213>人工序列
<400>4
CGGGGTACCG ACGTTGGGTA CGAATATCTA C 31
<210>5
<211>32
<212>DNA
<213>人工序列
<400>5
AACTGCAGCC AGTTTTTTTT TTTTTTTGCC GT 32
Claims (2)
1.如SEQ ID NO.1所示的osa-miR812q在提高水稻砷胁迫耐受性中的应用,其特征在于:具体表现为过表达osa-miR812q,可缓解As胁迫对水稻生长的抑制,可提高As胁迫下水稻叶绿素含量,可降低水稻地上部As含量和转运系数。
2.根据权利要求1所述的应用,其特征在于:通过PCR方法扩增miR812q的前体基因,构建含有miR812q前体基因的重组表达载体并将载体导入农杆菌,通过农杆菌侵染水稻愈伤组织,筛选获得具有砷胁迫耐受性的水稻植株。
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