CN116606882B - OsbZIP79基因在增强水稻缺氮胁迫抗性中的应用 - Google Patents
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Abstract
本发明公开了OsbZIP79基因在增强水稻缺氮胁迫抗性中的应用。所述OsbZIP79基因的蛋白编码区的核苷酸序列如SEQ ID NO.1所示。本发明通过在普通水稻中过表达和敲除OsbZIP79基因,发现其能增强水稻缺氮胁迫抗性的新用途,并为培育具有缺氮胁迫抗性的水稻品种提供了重要的基因资源。
Description
技术领域
本发明涉及植物基因工程技术领域,尤其涉及OsbZIP79基因在增强水稻缺氮胁迫抗性中的应用。
背景技术
作为一种重要的大量营养元素,氮素(N)是植物生长发育的主要因素,而其缺乏对多种关键化合物的生物合成造成严重影响,例如,植物中的核苷酸、氨基酸、蛋白质、叶绿素和激素(Yang et al.,2015)。由于氮成为农业系统的一个重要限制因素,植物的当前生产力在很大程度上取决于施氮肥(Kaur et al.,2017)。因此,在农业实践中过度使用氮肥会产生无法估量的问题,导致稻田硝酸盐过盛不断增加,造成严重的经济问题和环境污染(Liet al.,2017)。在保持作物产量的同时,减少施肥是作物育种的主要目标(MasclauxDaubresse和Chardon,2011)。提高氮素利用效率(NUE)是实现可持续农业的一个有前景的解决方案,其目标是保护环境和满足人口增长。NUE是一个受环境和遗传因素控制的多方面特征,这在很大程度上取决于作物土壤中每单位有效氮的种子氮浓度和种子产量(Perchlik和Tegeder,2017)。因此,了解作物生命周期中N吸收、迁移和同化的众多设备对于提高NUE至关重要。
由于其在土壤中的浓度通常不足以实现作物的最佳生产量,缺氮胁迫是一个常见的问题,用于诱导氧化应激(Zhang et al.,2013;Lee et al.,2018;Safi et al.,2021)。例如,缺氮胁迫不仅增加了氧化应激(Zhang et al.,2013),还促进了植物激素和活性氧清除抗氧化剂的细胞串扰(Liu et al.,2021)。TaNBP1(鸟嘌呤核苷酸结合蛋白1)通过调节小麦中的ROS稳态和N获取参与缺氮胁迫相应及调节(Liu et al.,2018)。TaMPK14(丝裂原活化蛋白激酶14)在通过调节小麦中的ROS稳态和氮吸收来响应缺氮胁迫中至关重要(Shi etal.,2020)。GARP转录因子通过拟南芥中的ROS独立或依赖信号抑制缺氮胁迫响应(Safi etal.,2021)。GCN4通过在缺氮胁迫下启动灵芝中的抗氧化基因表达来控制二次代谢(Lianet al.,2021)。总之,调节氧化还原系统可能是缓解缺氮胁迫应激的潜在途径。
bZIP转录因子家族都具有一个高度保守的bZIP结构域,为60~80个氨基酸。它由两部分组成:高度保守的结合DNA的碱性区和多变的亮氨酸拉链区(Wang et al.,2008)。bZIP蛋白是一类重要的转录因子,它们可以识别包含ACGT基序的A盒、C盒和G盒等顺式作用元件,bZIP类转录因子通常经翻译后修饰或形成二聚体而得以发挥功能(Schütze et al.,2008)。许多逆境相关基因的启动子序列中包含ABA响应元件(ABRE),被激活的bZIP类转录因子能够结合在ABRE上,从而调节下游基因的表达。水稻(Oryza Sativa L.)是重要的粮食作物,研究发现,水稻OsbZIP23基因受干旱、盐碱、ABA 和聚乙二醇等多种胁迫诱导表达,OsbZIP23通过ABA依赖的信号通路调节非生物胁迫下许多胁迫相关基因的表达,在增加对ABA敏感性的同时增强了水稻的抗旱耐盐性(Xiang et al.,2008);OsbZIP72被证明是ABA响应和水稻抗旱的正调节因子,有望应用于培育水稻抗旱品种(Lu et al.,2009);OsbZIP46是ABA信号和水稻耐旱性的一个正向调节子,OsbZIP46含有的D结构域对激活活性具有负效应,因此过量表达OsbZIP46对耐旱性无正效应(Tang et al.,2012)。而水稻的bZIP转录因子家族在水稻缺氮胁迫方面尚未见相关的研究报导。
发明内容
本发明提供了OsbZIP79基因在增强水稻缺氮胁迫抗性中的新用途,该OsbZIP79基因为培育抗缺氮胁迫的水稻种质提供依据。
具体技术方案如下:
本发明提供了基因在增强水稻缺氮胁迫抗性中的应用,所述基因的编号为Os11g0152700,该基因的蛋白编码区的核苷酸序列如SEQ ID NO.1所示,其蛋白质编码区长度为1455bp,全基因序列如SEQ ID NO.2所示。
OsbZIP79基因编码的蛋白为bZIP家族转录因子,由484个氨基酸组成,其氨基酸序列如SEQ ID NO.3所示。
本发明将OsbZIP79基因作为目的基因导入水稻粳稻品种中花11(Oryza.SativaL.spp.japonica,var.Zhonghua11)中得到OsbZIP79基因的T0代过表达植株,经过连续自交,获得纯合高表达的T2代株系,命名为OE:OsbZIP79-1和OE:OsbZIP79-2。
本发明利用CRISPR/Cas9基因编辑技术,在水稻粳稻品种中花11(Oryza.SativaL.spp.japonica,var.Zhonghua11)中敲除OsbZIP79基因,制备得到OsbZIP79基因敲除植株osbzip79-1和osbzip79-2。
通过将T2代过表达纯合株系OE:OsbZIP79-1和OE:OsbZIP79-2,OsbZIP79基因敲除植株osbzip79-1和osbzip79-2和其对照普通水稻中花11植株进行低氮处理培养14天后统计株高、干重、氮含量和叶绿素含量,发现OsbZIP79过表达水稻经过低氮处理后株高、干重、氮含量和叶绿素含量均显著高于对照,而OsbZIP79基因敲除植株osbzip79-1和osbzip79-2水稻经过低氮处理后株高、干重、氮含量和叶绿素含量均显著低于对照。
通过将低氮处理培养14天后的水稻叶片取样,检测谷氨酸合成酶基因(OsGOGAT1)、过氧化物酶基因(OsPRX47)基因表达量,过氧化物酶(POD)酶活和双氧水含量。结果表明OsbZIP79过表达植株比普通水稻植株的谷氨酸合成酶基因(OsGOGAT1)、过氧化物酶基因(OsPRX47)基因表达量,过氧化物酶(POD)酶活显著升高,激发双氧水含量显著降低;而OsbZIP79敲除植株OsbZIP79谷氨酸合成酶基因(OsGOGAT1)、过氧化物酶基因(OsPRX47)基因表达量,过氧化物酶(POD)酶活显著降低,激发的双氧水含量则显著升高。
综合上述实验证明:过表达OsbZIP79基因能够提高水稻对缺氮胁迫的抗性,基因编辑OsbZIP79基因则降低了水稻对缺氮胁迫的抗性。OsbZIP79基因编码的bZIP类转录因子能够提高谷氨酸合成效率和清除活性氧的爆发,表明OsbZIP79基因在植物抗缺氮胁迫基因工程中具有十分重要的应用价值。
本发明还提供了过表达水稻基因OsbZIP79的相关生物材料在增强水稻缺氮胁迫抗性中的应用,其特征在于,所述相关生物材料包括:过表达载体或基因工程菌。
本发明还提供了一种培育抗缺氮胁迫的水稻种质的方法,包括以下步骤:
(1)构建含OsbZIP79基因的过表达载体;所述OsbZIP79基因的蛋白编码区的核苷酸序列如SEQ ID NO.1所示;
(2)将所述过表达载体转入农杆菌感受态细胞中,构建含OsbZIP79基因过表达载体的农杆菌工程菌A;
(3)将所述农杆菌工程菌A介导转化水稻愈伤组织,培育得到过表达纯合株系。
进一步地,在步骤(1)过表达载体的制备过程中,采用的上游引物如SEQ ID NO.4所示,下游引物如SEQ ID NO.5所示。
进一步地,所述农杆菌为根癌农杆菌菌株GV3101。
与现有技术相比,本发明具有以下有益效果:
(1)本发明通过在普通水稻中花11植株中过表达和沉默OsbZIP79基因,发现其增强水稻缺氮胁迫抗性的新用途,并为培育抗缺氮胁迫的水稻品种提供了重要的基因资源。
(2)本发明利用转基因技术提供一种抗缺氮胁迫的水稻种质的选育方法,并获得了抗缺氮胁迫的OsbZIP79基因过表达纯合植株。
附图说明
图1为OsbZIP79基因的组织表达(a)和亚细胞定位示意图(b)。
图2为实施例3中水稻OsbZIP79基因过表达植株与普通水稻植株缺氮胁迫14天后之后的植株株高、干重、氮含量和叶绿素含量;
其中,a为植株生长的照片,N-S表示氮充足;N-L表示缺氮胁迫;b为实施例2中构建的OsbZIP79基因T2代过表达纯合株系:OsbZIP79-OE-1和OsbZIP79-OE-2的基因表达含量验证;c为株高的柱状图;d为干重的柱状图;e为氮含量的柱状图;f为叶绿素的柱状图。
图3为实施例2中水稻OsbZIP79基因编辑靶标位点和突变类型(a)、突变后蛋白结构(b)以及蛋白序列(c)示意图。
图4为实施例3中水稻OsbZIP79基因编辑植株osbzip79-1和osbzip79-2与普通水稻植株缺氮胁迫14天后之后的植株株高、干重、氮含量和叶绿素含量变化情况;
其中,a为植株生长的照片,N-S表示氮充足;N-L表示缺氮胁迫;b为株高的柱状图;c为干重的柱状图;d为氮含量的柱状图;e为叶绿素的柱状图。
图5为实施例3中构建的OsbZIP79基因T2代过表达纯合株系OE:OsbZIP79-1和OE:OsbZIP79-2,和实施例3中水稻OsbZIP79基因编辑植株osbzip79-1和osbzip79-2缺氮胁迫14天后之后的谷氨酸合成酶基因(OsGOGAT1)、过氧化物酶基因(OsPRX47)表达量、过氧化物酶(POD)酶活和双氧水含量;
其中,a和b为OsGOGAT1表达量,c和d为OsPRX47表达量,e和f为POD酶活性,g和h为双氧水含量。
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。下述实施例中的实验方法,若未特别指明,实施例中的培养基及实验条件均为常规培养基和实验条件,如分子克隆实验手册(Green M.R.,Sambrook J.Molecular Cloning:A Laboratory Manual:Three-Volume Set.Cold SpringHarbor Laboratory Pr,2012)或按照相应实验试剂和仪器说明书建议的条件进行。实施例中所用的试验材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1水稻OsbZIP79基因的克隆,基因过表达工程菌和基因编辑工程菌的构建1、水稻总RNA提取
采用Tiangen Plant total RNA extraction kit提取水稻幼嫩叶片的总RNA,将所提取的RNA用Thermo Fisher公司的反转录试剂盒按说明书进行反转录得到cDNA(SEQ IDNO.1),存于-20℃保存备用。
2、OsbZIP79基因过表达工程菌A的构建
设计水稻OsbZIP79基因编码区序列的特异性扩增引物,引物序列及所携带的酶切位点如下:
OsbZIP79-OE-F:5’-ACTAGGGTCTCGCACCATGGGAGAAGCTAGCAGTAG-3’;(SEQ IDNO.4)
OsbZIP79-OE-R:5’-ACTAGGGTCTCTCGCCGAAGGCTGAATATTGGCTCT-3’;(SEQ IDNO.5)
以上述从普通水稻中花11植株叶片总RNA反转录得到的cDNA(ComplementaryDNA)为模板,通过聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增OsbZIP79基因外显子的核苷酸序列,经BsaI酶切后,连接至载体pCAMBIA1301,42℃热击转化涂板。
挑单克隆菌落,将摇菌后的菌液送至测序公司测序,测序结果显示载体包含OsbZIP79基因外显子的核苷酸序列,用全式金公司质粒提取试剂盒提取质粒,将质粒电击入GV3101农杆菌感受态,28℃培养两天后挑斑进行PCR验证,即得到含有过表达载体pCAMBIA1301-Ubi::OsbZIP79::GFP的农杆菌工程菌A。
3、OsbZIP79基因编辑工程菌B的构建
基于基因组(SEQ ID NO.1)序列,在线(http://crispr.hzau.edu.cn/CRISPR/)查找特异的靶标序列,选定靶标序列“GGCACCCTTGCAACCTCCGGTGG”,根据该靶标序列合成互补引物:
OsbZIP79-CRISPR-F:5’-GGCAGCACCCTTGCAACCTCCGG-3’;(SEQ ID NO.6)
OsbZIP79-CRISPR-R:5’-AAACCCGGAGGTTGCAAGGGTGC-3’;(SEQ ID NO.7)
SEQ ID NO.1:ATGGGAGAAGCTAGCAGTAGTTCAGGACATCCAAGGCAGAATCCT CATGTTCTTGGCTATGGCTTCCATGGAGCTATGCCCAACTCCTTGCCTTCTGCAAACTTGTTCGAGCAGCAAGGAGGTGCTAACTATTTTGGAGAACTAGAAGAGGCTCTGATGCAACAGGTGGCCACACTCAGGAGGACTCAACAAACTGCAACCACCACCTCCACCCTTCATCATGGAGACACCACCCCCTTCTCCACCACTGCTACTGCTGCGGCTACAGCTAGGCCTCCTCCTACACTGGACATCTTCCCATCCTGGCCTATGAGGTCTCTCCACACACCCAAGGAGGGTTCAAATGTGACAGCAGACACAACAGACTCGGAGAGCAGCAGCAAGAACAACAGCAACCAAAATGCCTCATCAGACCAGCATGTGCTAGTAGGAGACATGGCAGGCCAGTTCGACCAAATTCCACAGCAGGAACAACACAAGAAGATGGCAACTAACAGTCCCACTCATAGCAGCAAGACTGGCAAGGCGCTTGATCCAAAGACGATGAGAAGGCTCGCCCAGAACCGAGAAGCAGCACGGAAAAGCCGACTGCGAAAGAAGGCTTACATCCAGCAGCTTGAAAGCAGCAAGCTGAAGCTTGCTCAGATGGAACAGGATATCCACAGAGCTCGTTCCCAGGGCTTACTGCTTGGAGCTCCAGGTGGAAACACCAGCTCAGGTGCCGCAATGTTCGACGTCGACTACGCCCGGTGGCTGGAGGAAGACAGCCAGCGCATGGCTGAACTGCACGGAGGGCTGCACGCACATCTGCCGGACAGCGACCTCAGAGCCATCGTCGACGACACGCTAACCCACTACGACCACCTCTTCAACCTCAAGGGCATGGCGGCCAAGGCCGACGTGTTCCACCTCATCACCGGGATGTGGGCGACCCCAGCTGAGCGGTGCTTCCTCTGGATGGGCGGATTCCGGCCCTCCGAGCTGCTCAAGACATTGACACCCCAGCTGGATCCCCTGACTGAGCAGCAAGTGGTTGGTATCTGCAATCTTCAACAGTCATCGCAACAGGCAGAGGAAGCTCTCTCCCAGGGCTTGGATCAGCTGCATCAGTCATTAGCAGAAACCGTGGCTGGAGGGTCCCCGTTGGATGACCCCAACGTCGGGAGTTTCATGGGTCACATGGCTATTGCCCTCGGCCAGCTATCAAATCTTGAAGGTTTTGTCATACAGGCTGATAATTTGAGGCAGCAGACTATCCATCAGATGCACAGGATTCTGACAGTCAGGCAGGCAGCTCGATGTTTCTTGGCCATTGGAGAGTACCATAATCGCCTCCGTGCCCTGAGCTCACTTTGGGCTTCTCGCCCTCGAGAGATACTGGTGGCAGATGAAGGCAATTGTGGAGAGCTAAGCATTGCAGCACATCCATCTGAGAGCCAATATTCAGCCTTCTGA
全基因组序列如SEQ ID NO.2所示。
SEQ ID NO.2:TCATGACAAAAACCTTCGAGTGAAGACAAGACCCTAGACCGTCTC TCTCTCTCTCTGACCAAGGCTAGCTAGGGAAGGAAGAACAAAAACAGCATAGGAAGGACAACATCTTTCTCTGAACTGTGGGATTAGTACGCCCAAGAACGTGAAGCTATTACACCGTCAGATATATCAAGAAGATGCAGGCGGAGCAAGAGCAACTGCGCACAGAAACGGTCAGGCATGCCTGCTCTCCCTCCTGCTTGTCATAAAAGGGAAGCCACAAGCTGGTATGTTTTCTTGACTCAAATCTTCTTCGTTCAGATGTATGCTTGCTGAGAGCCTGAGATAGGTGTATTTGTGTAGTATCGATCTTTGATGTTTTTCCTTCTTTTTTTGTGCGTTTTGAGCGCAGAAGC
CAGATTGCTAGAAGGTAGTTCTTGAGGACTGAAGCAAGGTAGAAGAACTTGAGCTTCT
GGTTCGTGTGGAGCTACTGGGATAAGTTGGCTGCTGATTGCTGAAGATGGGAGAAGCT
AGCAGTAGTTCAGGACATCCAAGGCAGAATCCTCATGTTCTTGGCTATGGCTTCCATGG
AGCTATGCCCAACTCCTTGCCTTCTGCAAACTTGTTGTAAGAGCTCTCTCTCTCTCTCTC
TCTCTCTCTCTCTCTCCATGAGTCTTCTTTTCTCTTTTTGTGATTCAATTTATGCAGGTGC
ACCAAGAAACATGATCTGCACCATGCACTTTCTTTGCAAGTTTTGCCTAGCTAACCATA
CATATTTACTGGCTAAATCAAATTTCTTTTACTTGTTTTGACGTATGCTTGTCAACTTGAC
TTCTTTTTTTTTTACTATATACTGTTCTTCATGCTTCGTAGTACGTACACAAGAATCTTTCT
GTAGACCAATTTTGCACAATTTTCCCCCAATAAGCTGTTGGTCTAATGCTAATACAAATT
AATTACCCCTATGCATCCCTGACAGCGAGCAGCAAGGAGGTGCTAACTATTTTGGAGAA
CTAGAAGAGGCTCTGATGCAACAGGTGGCCACACTCAGGAGGACTCAACAAACTGCA
ACCACCACCTCCACCCTTCATCATGGAGACACCACCCGTACGTACATCTCTCTCTCTCTC
ATGTGTGTGTGTGAGACCAGTTGAACAAGACCAAAAACACACAACCTTGTGAGGCAC
TTTCTGGAGAGGTTAGGGCATTGTAGTCAAAAGCAGCCATGGGAGAGCAGTAGAAGTG
GTGGCAGTTGTCCTGCTTGTGCTGTGTGCAGTACTACCTTGGCATGGCCATGGCATGAG
AGTGTGACTACCTTGTGACTCCTCTCCATCTCTCTAGTCTCTTGCAGTACGAGTACCAG
AGAGAGTGTCTACCAACTTTCTGGCTCCCTGACTGCAAGCACAGCTCAAAGAGAATAA
ATTCCACTGACTTTCACTGTGACAGGGACCCCACACCTTGTGATCCCAATGCAGCCTTT
TCACCTCTGGTGTATTGCCATGCATTTCTTGATCTCCTTCTCCCCTATGATATCTTCTTGC
AGCCTTCTCCACCACTGCTACTGCTGCGGCTACAGCTAGGCCTCCTCCTACACTGGACA
TCTTCCCATCCTGGCCTATGAGGTCTCTCCACACACCCAAGGTATGCTTTGCATCTACTG
TCTACTGTACTGCCTATTGGTTGTATCAACGACATGTGGTTTTACACAAGTATTAGCTCT
GGTAAATATTCATGCTGCATATAAAGTTTTTTACAGCTGTGGTCTGAGCCCTGGCTTTGC
TTTGGCATGTTGTAGGAGGGTTCAAATGTGACAGCAGACACAACAGACTCGGAGAGCA
GCAGCAAGAACAACAGCAACCAAAATGCCTCATCAGACCAGCATGTGCTAGTAGGAG
ACATGGCAGGCCAGTTCGACCAAATTCCACAGCAGGAACAACACAAGGTATGCAATGC
ATTGCTGTGATCTCGTATCACTGTTGCTTGATGCATCGGTGGTCTGGTATTGTTTCTGTGC
TTCCCGTTTCTGTGCTTTGATTTGTCTTGGTCATCCTTATCATGCAAGCGCAATGGTATTG
TTCCCTAGCCTGGCGTCATGCATGTCCCTAGCTCCACAACTTCCAAGATGCTCATGTAGA
TACATGCTCCTATAGGTAGAACAACCATTGTTCAGGGGCATTTAAGATCTCGCTGCTCCC
AAGATCGCCAATCCCAACACGTTTGGTGAATCATTTGGCCCTATAGAAAGTTCCAACTA
ATTTCCCTGATGCTTCTTCCATGGGACCAGTGTTTTCTACCATCAGACGCTACTAATATAT
TACTACTAGATAGAGAATATTAGTCAGTCATCTCTTTGCATTCTTAAGAAACAAAGAAGT
CATCTTCAATTTCTATTGGGACGATGCAAATCTTTATTTGACTGCACTTATTATCAGGAGT
ATGGACAATCTCTTCAGCAAGAAACAGTCGTTCTAGTTGATCTTTTATTGTTGGAATGGA
TAAGTTTAGAGGGTTTGGATATGTTAGATTAGTGCATGCTTGTTCATGTGGTGGCTGCAT
TTCTGCCATTGGCCTGCTGCAATAAGTGACAAATGAGCTTTCTTACAATATTGTCTCTAT
AGCTTGCAAACCTTGTGCGCTAATAATAAAAATGAACTAATCGATAATACAAATGATACT
TTGGGTCAGTGGTGCTTTAGTTGTTTTCTGGTCAATTGATGAATTGCCAATATATAGGAT
GAATAAATAATCGCATGGACTATTTAAATTCGCTCATAACCGAAAGAGAGCTCTACATAT
TAGGCCTCGACAGCAACATATAAAAAAAAAGCAGAACCAGTTTTTGTCTGTTGTTAGA
GTGGGTGGCTTCCACCAAATGTACACGACTGTTTTTATTATTGTGCTTGATGTAACGTGC
TGTAGAAACCATACTATATTGCTTACACCATAAACGCATACACATAGCATTGCTCTACCAT
TTATCCCCTAGTTAGTGTGTAATTACAGATTAATTTGTGCACAGGTGTAGTAAATTCCATA
TTCTGATTTGTTCATGTCCACTTGGCATGCAGAAGATGGCAACTAACAGTCCCACTCAT
AGCAGCAAGACTGGCAAGGCGCTTGATCCAAAGGTCTCTTATCTTCTTATTAGTTTGAT
GCTTAGAGTGTGCTGACGTGTGCTGTGTGCATGCATGCATGCCAAGCAGATGCACAGA
GCCATGCAGTGCAAGCTAATGACCATTTCGAGGCAGTATTTGAGATTGAGAGTTTTTTT
TTTCTCTTGGTGTCATCAGACGATGAGAAGGCTCGCCCAGAACCGAGAAGCAGCACGG
AAAAGCCGACTGCGAAAGAAGGTGAATGTTGTGACCGATACTCGGCACGCAGCAGTA
AATTTTCCATGTTCTAACAGTTTCGTGTATGGATCATACAGGCTTACATCCAGCAGCTTG
AAAGCAGCAAGCTGAAGCTTGCTCAGATGGAACAGGATATCCACAGAGCTCGTTCCCA
GGTAACTTTCTTTCAACACATCCATAATTTTATAATCGACAAATTGACTATGTGTTTAGCT
TAGTTATACAGCGTAATCTGATTTTTTGAGCTATGTTTAGGGCTTACTGCTTGGAGCTCC
AGGTGGAAACACCAGCTCAGGTGAATATATTTGATATAAACTTATCTATGAACTTCAGCT
GGGATCTGCAGACGGGACTATGGTTCACTGAAACTGAGCTGTATGACTGACATTTATTT
CCAGGTGCCGCAATGTTCGACGTCGACTACGCCCGGTGGCTGGAGGAAGACAGCCAG
CGCATGGCTGAACTGCACGGAGGGCTGCACGCACATCTGCCGGACAGCGACCTCAGA
GCCATCGTCGACGACACGCTAACCCACTACGACCACCTCTTCAACCTCAAGGGCATGG
CGGCCAAGGCCGACGTGTTCCACCTCATCACCGGGATGTGGGCGACCCCAGCTGAGCG
GTGCTTCCTCTGGATGGGCGGATTCCGGCCCTCCGAGCTGCTCAAGGTATGGATGACCT
TATCTCTGAACAAAACATCAGTCATCAGATTACAGCGTCTCTTGTATTTGCTGAATTGAG
TCTATGGCTAATTTGATCAGAAACAAGACACTCACTTCAGAGTTGAAAATGAAAAATTT
TCTCCAACAAATTCTTCAGGAAGATGAAACCAAGCTAAAATCTGAGCTCATCTTTCAAG
TATACATATACAATTCTCCAATCTCACTGCATATTTTTTATGTTGATGCAGACATTGACAC
CCCAGCTGGATCCCCTGACTGAGCAGCAAGTGGTTGGTATCTGCAATCTTCAACAGTCA
TCGCAACAGGCAGAGGAAGCTCTCTCCCAGGGCTTGGATCAGCTGCATCAGTCATTAG
CAGAAACCGTGGCTGGAGGGTCCCCGTTGGATGACCCCAACGTCGGGAGTTTCATGGG
TCACATGGCTATTGCCCTCGGCCAGCTATCAAATCTTGAAGGTTTTGTCATACAGGTAAA
GATTTCCAGTTGTATCTGCTACTCTTCAATATTGATCATCTTGTCATTTTGTCAGATCAAG
ATAGGAGTTGCACTCTCTCTTTGTGTGACTCAACTTCATAAATGAGCTAGTAACTACGTA
AAATTGTTGAGTGTAAAATGTTGCTGTGTTCAGTAGAATGTTTATCTGTCTAGCCACTGC
CCAGTAGCCAAAGCTGTGTCCTAGTAAACCTTAACAGTGCTGCCAAAAAATGTATTAAA
AATAATTAACTAGTCCATATGTAAGAGAATATATCATGTTATGGCTCCATCATGTAGAGAT
GTTACTGTATCTAACATTGTAGAAAGAAAAGACTTTCCAACTAAAACAAGTAGCAAAA
CAAATATGTACTGCCGGCAACTTTTAGTTAGATTCCAGGAATGGGTTTTACTGGAACAC
ACATAAAACTAAGGCCGTGTTTAGTTCGTGTGCCAAATTTTTTTTGAAGTATACGGACA
CATATTTAAAGTATTAAACGTAGACTAATAACAAAACAAATTACAGATTCCGCCTGTAAA
CTGCGAGACGAATTTATTAAGCCTAATTAACCCGTCATTAGCAAATGTTTACTGTAGCAT
CACATTGTTAAATCATGGCGTAATTAGGCTCAAAAGATTCGTCTCGCAATTTACATGTAA
ACTGTGCAATTGGTTTTTTTTCGTCCACATTTAATGCTCTATGCATGTATCCAAACATTTG
ATGTGATGGAATTTTTGGAAGTTTGAAGGGAACTAAACACTGCCTAATGTGCACCACAT
TGGATTTCCAGGCTGATAATTTGAGGCAGCAGACTATCCATCAGATGCACAGGATTCTG
ACAGTCAGGCAGGCAGCTCGATGTTTCTTGGCCATTGGAGAGTACCATAATCGCCTCCG
TGCCCTGAGCTCACTTTGGGCTTCTCGCCCTCGAGAGTAAACCCCATGCCTGTAGTTGA
TTCTTTTTTTCCTATAAAAAAGGGATATTCTCCTGCTCATAGTTATTGTAGTTAATATTTTT
TACTTTTACTAGATAATTTTGAAGTATGATTGAGCCATGACTTAATATAGGCGGGAATCAT
CTAAATAACTTATCAGAGATTGAGCCGTGCACTACTAATCAGGTTGAAATGGACCTTCC
TCCTAACAGGAAACAAAAGTCAGAAAGCTTATTTTTTAGACCATGTCACTTTCTCATTC
TTAAGGTTAAGATGCCCCTAAAGGACTGCCCCAATTATATTAAATTTAAACGAGTATAAT
TTACACTTTAACATATTCAGCCTTGAAAGTATATGATTCTCTTACCCTTAAAAGAACATG
ACAAAACACAGTGCATGTCATGGTGTAAAATAAGGACACGGTTTTCTGTGGCTTCTCTG
TAGATTTTTCAACTATTTCAGCCAACTAACCAAATTCTATATGTGAAACAAGCCACAAA
GTCAGCAGCTGTGACAGTACGTCCTTTTTTATGCATTTCTCATCTTATTTGCTGGTGCCTT
TCATTGCTTGTAGGATACTGGTGGCAGATGAAGGCAATTGTGGAGAGCTAAGCATTGCA
GCACATCCATCTGAGAGCCAATATTCAGCCTTCTGAAATCCTGTTTCAAATGTATTTTGC
AACTAACCTTCAAAGTAGGGAGCAAAAGCTTGTTTTTTAAATGAAATCTAGCACTCTAG
CAGATATGTGAGAAACTCAGAAGAGAGTTGGGAAACAATGGAAGGCTCCAGCTGCACT
AGCAAAATTTTCAGATGGAATGAATGGGATTGCATGATTTTCAGTTACTGATCATAAAGC
AAGTAGTTCACGTCCTTTCCTGGATGATATTTTCAGCATCTCTCCAGTTTTCAGCAATGC
CCAGTTCTACTAGAACTGCTCACATGACACAGGCACTTGTTCTGTGACCTGATGTAATC
AATTAATCATGCACACAACATGATACCAAACATCACATGATGTTCACAAACAATTAGCA
AACACAAATACACAATTATGTGC
在PCR仪中将上述引物融合,获得含有靶标序列的融合片段。
用BsaI限制性内切酶酶切载体pHun4c12,通过胶回收获得线性化载体。随后,将上述融合片段连入线性化的pHun4c12载体上(Jiang等,2019,Mutation of Inositol1,3,4-trisphosphate 5/6-kinase6 Impairs Plant Growth and Phytic Acid Synthesis inRice.Plants8(5):114),酶切鉴定获得正确载体,并进一步测序证实靶标序列已导入载体,正确载体命名为pHun4c12-OsbZIP79。同时利用热激转化法将pHun4c12-OsbZIP79导入农杆菌菌株EHA105中,即得到含有基因编辑载体pHun4c12-OsbZIP79的农杆菌工程菌B,用于后续遗传转化。
OsbZIP79基因编码的蛋白为bZIP家族转录因子,由484个氨基酸组成,其氨基酸序列如SEQ ID NO.3所示。
SEQ ID NO.3:MGEASSSSGHPRQNPHVLGYGFHGAMPNSLPSANLFEQQGGANYFG ELEEALMQQVATLRRTQQTATTTSTLHHGDTTPFSTTATAAATARPPPTLDIFPSWPMRSLHTPKEGSNVTADTTDSESSSKNNSNQNASSDQHVLVGDMAGQFDQIPQQEQHKKMATNSPTHSSKTGKALDPKTMRRLAQNREAARKSRLRKKAYIQQLESSKLKLAQMEQDIHRARSQGLLLGAPGGNTSSGAAMFDVDYARWLEEDSQRMAELHGGLHAHLPDSDLRAIVDDTLTHYDHLFNLKGMAAKADVFHLITGMWATPAERCFLWMGGFRPSELLKTLTPQLDPLTEQQVVGICNLQQSSQQAEEALSQGLDQLHQSLAETVAGGSPLDDPNVGSFMGHMAIALGQLSNLEGFVIQADNLRQQTIHQMHRILTVRQAARCFLAIGEYHNRLRALSSLWASRPREILVADEGNCGELSIAAHPSESQYSAF
实施例2水稻OsbZIP79基因过表达纯合植株和基因编辑纯合植株的获得
1、农杆菌介导的水稻基因遗传转化
利用含有pCAMBIA1301-Ubi::OsbZIP79::GFP过表达载体的农杆菌工程菌A和含有pHun4c12-OsbZIP79的基因编辑农杆菌工程菌B侵染水稻愈伤组织并经过分化培养基和生根培养基的筛选后,获得相应的遗传转化植株。
2、过表达纯合株系的获得
取上述T0代过表达苗叶片少许,使用RNeasy Plant RNA Mini Kit(Qiagen,Hilden,Germany)从植株材料(根、叶)中提取总RNA,并使用1μg总RNA,oligo-dT18引物和GoScriptTM逆转录系统(Promega)逆转录cDNA。使用SYBR Green GoTaq qPCRMaster Mix(Promega,WI,USA)进行定量实时聚合酶链反应(qRT-PCR)。水稻ACTIN基因用作内部对照,并使用2-ΔΔCt法计算OsbZIP79相对表达水平。
表1定量实时聚合酶链反应(qRT-PCR)引物表
将T0代转基因过表达植株自交分别得到T1代种子,从每个T0代过表达植株所产生的T1代中取6-9个阳性株继续自交产生T2代并进行分离分析。当T1代阳性株产生的T2代幼苗经过检测全部为阳性株,则该T1代植株为过表达纯合株,即获得OsbZIP79基因过表达的转基因纯系,命名为OsbZIP79-OE-1和OsbZIP79-OE-2;反之,则为杂合株。
3、基因编辑纯合植株的获得
将T0代转基因水稻生长至3-4叶期选取幼叶,CTAB法提取基因组DNA,利用pHun4c12-OsbZIP79特异引物(上游引物:5’-GCAGGCGGAGCAAGAGCAAC-3’;下游引物:5’-GCTGCTCGCTGTCAGGGATG-3’)扩增转基因水稻内源OsbZIP79基因,测序验证PCR产物,结果显示,OsbZIP79基因已发生单碱基缺失(图3),即获得OsbZIP79基因功能缺失的转基因纯系,命名为osbzip79-1和osbzip79-2。
实施例3OsbZIP79基因过表达水稻对缺氮胁迫抗性试验
对实施例2所得水稻OsbZIP79过表达T2代纯合株系:OsbZIP79-OE-1和OsbZIP79-OE-2,OsbZIP79基因功能缺失的转基因纯系:osbzip79-1和osbzip79-2与其对照普通水稻植株缺氮胁迫处理。
1、水稻缺氮胁迫
将灭菌的水稻种子在蒸馏水中浸泡,并发芽至7天大小。缺氮胁迫试验按照先前的报告进行(Lee S,Marmagne A,Park J,Fabien C,Yim Y,Kim SJ,Kim TH,Lim PO,Masclaux-Daubresse C,Nam HG(2020a)Concurrent activation of OsAMT1;2andOsGOGAT1 in rice leads to enhanced nitrogen use efficiency under nitrogenlimitation.Plant J 103(1):7–20)。将7天龄的幼苗转移到含有10mM(氮充足,N-S)或0.1mM NH4Cl(缺氮胁迫,N-L)作为唯一氮源的1/2MS液体培养基中,并再生长14天。所有幼苗生长在生长室(8小时黑暗/16小时光照,25℃/30℃,300μmol光子m-2s-1,相对湿度约70%)中。
2、株高、干重、氮含量和叶绿素含量测定
对水稻植株进行取样,记录表型株高数值。将植株在105℃下干燥2小时,然后在60℃下干燥2天,直至达到恒定重量,称取整个植株干重。
将约300mg水稻叶片在70℃下干燥70小时,然后研磨成粉末,通过Auto Kjeldahl分析设备(KJETEC 2300;FOSS,Hillerod,Denmark)测量N含量。
取100mg新鲜叶片,用液氮研磨后,加入5mL 80%丙酮。在黑暗中温育1小时后,将混合物以12,000×g离心3分钟。吸取上清液,用分光光度法在645和663nm处测量吸光度,用80%丙酮作为空白对照。总叶绿素含量(mg/g)=20.2(A645)+8.02(A663)。
结果如图2和图4所示,株高、干重、氮含量和叶绿素含量越高,缺氮胁迫耐受性越好;OsbZIP79过表达纯合株系缺氮胁迫耐受性显著高于普通水稻植株,OsbZIP79基因编辑植株缺氮胁迫耐受性显著低于普通水稻植株。
3、谷氨酸合成酶基因(OsGOGAT1)、过氧化物酶基因(OsPRX47)基因表达量,过氧化物酶(POD)酶活和双氧水含量测定
取上述水稻叶片少许,使用RNeasy Plant RNA Mini Kit(Qiagen,Hilden,Germany)从植株材料(根、叶)中提取总RNA,并使用1μg总RNA,oligo-dT18引物和GoScriptTM逆转录系统(Promega)逆转录cDNA。使用SYBR Green GoTaq qPCRMaster Mix(Promega,WI,USA)进行定量实时聚合酶链反应(qRT-PCR)。水稻ACTIN基因用作内部对照,并使用2-ΔΔCt法计算OsbZIP79、OsGOGAT1和OsPRX47相对表达水平。
过氧化物酶(POD)使用POD测定试剂盒(北京索莱宝科技有限公司)测量抗氧化酶的活性。过氧化氢使用过氧化氢测定试剂盒(北京索莱宝科技有限公司)进行双氧水(H2O2)测量。将约10mg植株材料(根、叶)在液氮中粉碎,并加入500μL裂解物,4℃条件下以8,000×g离心10分钟。吸取125μL上清液与等量的过氧化氢检测试剂混合,并在室温下温育5分钟,然后用分光光度法在415nm处测量吸光度,并从标准曲线计算过氧化氢浓度。
结果如图5所示,谷氨酸合成酶基因(OsGOGAT1)、过氧化物酶基因(OsPRX47)基因表达量,过氧化物酶(POD)酶活越高,缺氮胁迫耐受性越好;双氧水含量越低,缺氮胁迫耐受性越好;OsbZIP79基因编辑植株缺氮胁迫耐受性显著低于普通水稻植株。
Claims (4)
1. 水稻基因OsbZIP79在增强水稻缺氮胁迫抗性中的应用,其特征在于,所述OsbZIP79基因的蛋白编码区的核苷酸序列如SEQ ID NO.1所示。
2. 水稻基因OsbZIP79编码的bZIP家族转录因子在增强水稻缺氮胁迫抗性中的应用,其特征在于,所述该bZIP家族转录因子的氨基酸序列如SEQ ID NO.3所示。
3.一种培育抗缺氮胁迫的水稻种质的方法,其特征在于,包括以下步骤:
(1)构建含OsbZIP79基因的过表达载体;所述OsbZIP79基因的蛋白编码区的核苷酸序列如SEQ ID NO.1所示;
(2)将所述过表达载体转入农杆菌感受态细胞中,构建含OsbZIP79基因过表达载体的基因工程菌;
(3)将所述农杆菌工程菌介导转化水稻愈伤组织中,培育得到过表达OsbZIP79蛋白且稳定遗传的纯合株系。
4. 如权利要求3所述的方法,其特征在于,在步骤(1)过表达载体的制备过程中,采用的上游引物如SEQ ID NO.4所示,下游引物如SEQ ID NO.5所示。
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