CN117402877A - 长链非编码rna natal57在调节水稻产量相关性状中的应用 - Google Patents
长链非编码rna natal57在调节水稻产量相关性状中的应用 Download PDFInfo
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Abstract
本发明属于分子育种技术领域，具体涉及长链非编码RNA NATAL57在调节水稻产量相关性状中的应用。本发明从水稻中克隆得到长链非编码RNA NATAL57基因，其核苷酸序列如SEQ ID NO：1所示，利用基因编辑技术在水稻中抑制lncRNA NATAL57的表达能使水稻株高增高、稻穗变大、种子变大，说明NATAL57在调节水稻产量相关性状上具有重要的作用，将为水稻的分子育种提供新的思路和方向，在作物遗传改良上具有重要的意义。同时，RNA NATAL57将为水稻产量性状的遗传改良提供宝贵的资源，具有一定的研究价值和社会效益，并有望应用于实际生产中。
Description
技术领域
本发明属于分子育种技术领域，具体涉及长链非编码RNA NATAL57在调节水稻产量相关性状中的应用。
背景技术
水稻是最重要的粮食作物之一，其高产、稳产对粮食安全具有举足轻重的作用。水稻产量是一个复杂的农艺性状，主要由每株穗数、每穗粒数和粒重三个要素决定。这三个要素是水稻产量提高的关键调控因素，往往表现为多基因控制的复杂数量性状。因此，探索水稻高产的基因资源，对产量性状的优化和作物遗传改良具有重要意义。
长链非编码RNA(long noncoding RNAs，lncRNAs)是一类长度超过200nt，不具备编码蛋白功能的内源性RNA分子。研究发现，lncRNAs在调节水稻生长发育、应对生物胁迫及非生物胁迫过程中发挥着重要功能(Gao CX,et al.Roles of lncRNAs in Rice:Advancesand Challenges.Rice Science(2020):384-395.)。例如，lncRNA LAIR过表达可增加水稻籽粒产量，并上调多个LRK基因的表达(Wang Y,et al.Overexpressing lncRNA LAIRincreases grain yield and regulates neighbouring gene cluster expression inrice.Nat Commun(2018):1-9.)。Ef-cd是从开花激活因子OsSOC1基因座的反义链转录而来的lncRNA，可以促进氮素利用和提高光合作用速率，参与调控水稻成熟(Fang J,et al.Ef-cd locus shortens rice maturity duration without yield penalty.Proc Natl AcadSci USA(2019):18717-18722.)。lncRNA ALEX1在水稻中过表达，能够激活JA途径，对白叶枯病具有显著的抗性(Yu Y,et al.Transcriptional landscape of pathogen-responsive lncRNAs in rice unveils the role of ALEX1 in jasmonate pathway anddisease resistance.Plant Biotechnol J(2020):679-690.)。母本来源、调控胚乳发育的lncRNA MISSEN，MISSEN RNAi可以导致种子出现明显的“大肚子”表型(Zhou YF,et al.Theparent-of-origin lncRNA MISSEN regulates rice endosperm development.NatCommun(2021):6525.)。水稻ESP(Epigenetic short panicle,Os01g0356951)的功能获得性表观等位基因，编码一个假定的lncRNA，其表观遗传修饰与水稻穗结构的调节有关(LuanX,et al.Epigenetic modification of ESP,encoding a putative long noncodingRNA,affects panicle architecture in rice.Rice(2019):1-8.)。
然而，尽管水稻lncRNAs的研究取得了一定的进展，但对其研究作物产量的研究知之甚少。此外，lncRNAs数量众多，绝大部分lncRNAs确切的基因组注释和功能都还是未知的。因此，挖掘具有调控水稻产量性状的lncRNAs具有重要的意义。
发明内容
为了克服上述现有技术的不足，本发明提供了一种长链非编码RNA，命名为NATAL57，该RNA可以在水稻株型、穗型和粒型等目标性状上改良水稻的产量，为水稻遗传育种提供新的遗传资源。
为了实现上述目的，本发明所采用的技术方案是：
本发明第一方面提供了长链非编码RNA NATAL57在调节水稻产量相关性状中的应用，所述长链非编码RNA NATAL57的核苷酸序列如SEQ ID No.1所示。
应当理解，考虑到密码子的简并性，在不改变氨基酸序列的前提下，对上述编码基因的核苷酸序列进行修改，也属于本发明的保护范围内。
优选地，所述水稻产量相关性状包括株型、穗型和粒型。
优选地，所述调节水稻产量相关性状为通过抑制或降低NATAL57在水稻中的表达，进而改善水稻产量相关性状。所述改善水稻产量性相关性状包括使稻株增高、使稻穗增大，使稻粒长度增加。
本发明前期从水稻中克隆得到一个长链非编码RNA NATAL57，其核苷酸序列如SEQID No.1所示，SEQ ID No.1所示的核苷酸序列由2368个脱氧核糖核苷酸组成。经研究发现，通过抑制该基因后，会导致水稻出现株高增高、稻穗变大、种子变大的表型，有望为水稻株型、穗型和粒型的调节提供一种新的方法，对水稻遗传改良具有重要的理论意义和应用价值。
优选地，所述长链非编码RNA NATAL57还包括与其相关的生物材料，所述相关的生物材料包括编码NATAL57的蛋白，含有NATAL57的重组载体、表达盒、转基因细胞系或重组菌。
优选地，所述水稻的品种包括中花11号。
本发明第二方面提供了一种培育高产水稻的方法，具体为：利用基因编辑技术抑制长链非编码RNA NATAL57在水稻中的表达，或者降低长链非编码RNA NATAL57在水稻中的表达量，进而培育得到水稻产量性状改善的水稻植株，所述长链非编码RNA NATAL57的核苷酸序列如SEQ ID No.1所示。
优选地，利用RNAi技术，通过构建NATAL57 RNAi载体并转化水稻植株，进而抑制长链非编码RNA NATAL57在水稻中的表达，或者降低长链非编码RNA NATAL57在水稻中的表达量。
更优选地，用于构建NATAL57 RNAi载体的引物包括如SEQ ID No.4、5所示的正义链上下游引物和如SEQ ID No.6、7所示的反义链上下游引物。
与现有技术相比，本发明的有益效果是：
本发明首次公开了NATAL57的生物学功能，经研究发现，利用基因编辑技术在水稻中抑制NATAL57的表达能使水稻株高增高、稻穗变大、种子变大，说明NATAL57在调节水稻产量相关性状上具有重要的作用，将为水稻的分子育种提供新的思路和方向，在作物遗传改良上具有重要的意义。同时，lncRNA NATAL57将为水稻产量性状的遗传改良提供宝贵的资源，具有一定的研究价值和社会效益，并有望应用于实际生产中。
附图说明
图1为NATAL57 RNAi载体的构建示意图；
图2为NATAL57 RNAi植株的基因表达抑制效果；
图3为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的整体株型，比例尺为20厘米；
图4为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的穗型和粒型示意图；其中，A为穗型示意图，比例尺为10厘米；B为粒型示意图，比例尺为1厘米；
图5为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的粒长和粒宽示意图，比例尺为1厘米；其中，A为粒长示意图；B为粒宽示意图；
图6为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的产量相关性状统计，数据所示为平均值±标准偏差，数据分析采用t检验确定，星号表示与野生型相比在统计学上有显著差异(*P<0.05，**P<0.01；n＝3)；其中，A为一级分枝；B为穗粒数；C为千粒重。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法，如无特殊说明，均为常规方法，下述实施例中所用的试验材料，如无特殊说明，均为可通过常规的商业途径购买得到。
长链非编码RNA NATAL57调控水稻产量相关性状的作用研究
1、NATAL57 RNAi载体的构建
根据NATAL57的核苷酸序列(SEQ ID No.1所示的核苷酸序列)，设计589bp的正义链(SEQ ID No.2)和反义链(SEQ ID No.3)。同时，根据正义链和反义链的序列设计相应的引物用于构建NATAL57的RNAi载体(图1)，具体引物序列如下：
RNAi-sense589-BamH1-F：agaggatccGGTTTGCTATGTATCCTTGTTGTTC(SEQ IDNo.4)；
RNAi-sense589-Sac1-R：aggtgagctcGCTTGCAGAGTATTGTATAGGCAGG(SEQ IDNo.5)；
RNAi-antisense589-Kpn1-F：gctcggtaccGCTTGCAGAGTATTGTATAGGCAGG(SEQ IDNo.6)；
RNAi-antisense589-Not1-R：actagtgcggccgcGGTTTGCTATGTATCCTTGTTGTTC(SEQID No.7)。
首先，以水稻cDNA为模板，将引物对RNAi-sense589-BamH1-F/RNAi-sense589-Sac1-R加入KOD FX Neo DNA聚合酶(TOYOBO BIOTECH，Shanghai)反应体系中，扩增获得片段，相应的PCR程序为：98℃2min；98℃15s、65℃45s，35个循环；68℃5min延伸。将获得的片段纯化后进行BamH1和Sac1双酶切反应，并用T4 DNA连接酶连接到经过BamH1和Sac1双酶切的载体(pRNAi43)中，获得插入正义链的重组载体。
通过相同方法，以水稻cDNA为模板，将引物对RNAi-antisense589-Kpn1-F/RNAi-antisense589-Not1-R加入KOD FX Neo DNA聚合酶扩增获得片段。将获得的片段纯化后进行Kpn1和Not1双酶切反应，并用T4 DNA连接酶连接到经过Kpn1和Not1双酶切的含正义链的重组载体中，得到NATAL57 RNAi载体(NATAL57-pRNAi43)(图1)。
NATAL57的核苷酸序列(lncRNA sequence)(2368bp，SEQ ID NO：1)：
ATCGCTCGCGGTCGCAGTCGCAGTCGCCCACACCCTTGCCGCACCATGGCTGGAACCCGCGGCGGATTCGAGTTATTGCACATGGTGGCACCAAGCATGAGGCAACAACTGCATTACTTGCAAGAAAGGCACAAGTCTCTAACACCTAGGCCAGAAAGCTCCTCACCCATCAGTTGCCTACAACCATGCAAAAGAGTAATCTCAGCAGTTCAAGAAGTCCCAATCTATATGCCACTACAGCTCTCTTCTGTCGACATGGTTTGAGACAGGCCAAACTTATTTGTTAAAGAAACTTAGGAATACTATTAAAATAAATCTGACAGTGAATATTCTAATTATATTCCTAAAATTTTCCTTCAAGTATTTAAGCATCTTTTGGTGGTTCTTATACAATTCCATGTGACATTTAAATTGTTGATAAACTACTTTCTTGATAAATCTGACAGTGAATATTCTGATTATATTCCTAAAGTTTTCCTTCAAGTATTTAAGCATCTTTTGGTGGTTCTTATACAATTCCATGTGGCATTTAAATTGTTGATAAACTACTTTCTTGATGAATTTTATATTAATGTATGATCAATTTCCATTGCAGAATCATTAGATGATTACTTGTGGCTTTCTTGCAAGTTGTGCAGTTGCTGCCTCAAGCTTGCTGCCTCCCTCTGCCAAATCTATCAGCAAAAAAAGAAAATCATGTCAGTTTCCTTGAACTATATACATATGATTAATCTAGAAACGCAGTATGTTGCCAAAAATCACACACAACTCAATCACATATGCATTTGAGATGAGGTAAAATGCTATCTGATTCACATAGTTCCAATATCTCAAGATGTGGGATATGCGTAGCACAAGTGCATCAGAGTGTTATAGTTGTTCAGTCTTCAGTACTTATTATTCTAGATTAATTTGGACCAACTTGTCAATTCAGCTGACACTTGTTTTTGTCTAGTGCTCCAGTAGCTTGGTTGTTTCATCATCATTAGACTTTTAAACCTTTGGTCCTAAACTCCTAGCTGAAAAATTTGCACACTGAACTTCCCTGTGTTCTAAGGAAAAAAAAAATCTAGACATAACCTTGCTGATCTGTATTATGGACTGGCCATTGGAGTGCATAATGCATAACTGCTGATCACTTTGTTGGCACAAAACTTACGCATAGACATAGGAGGTACTTATGTATAATTGTATCTTTAATCAGGACTTTGTTTTCAACCATTTTGTTGATATAACAGTTATACTGTAGTAATGATGAGTACAGTCTCCAATGTAAACTCAGGGCAAATCAGATTTATCCCTTTTTGGTGCTTGGTATCTTTTTGTATTTTTCATGAGCGATGGCATTTTTGAACCCAAAATAGAGGGAAAGAGAAGGATCTCTGTGGGGGCATGCTATGTACTACAGTTCTTTGAGACCATGTAGGTGTGGATGCTTGTTTTGACCTTTTCCTTAACATATAGAAAGAATAAACATTAAATAAATGATGACATGGAAATGAAATTCATAAAGGAGTACATAAGCTAGTACTCCTGAAAATATCACTTCATATTCCCCCATGATTCAGTAACATGAATATCATACCATCAGCAGCAGTGAAATTTTTATGCGTAGACGGTTTGTTTCTTAAGGTATATATTTTTTATGTTTACAATTGTCTGTCGAGTTTACTGGTCAATTGAGCAACAACGGTACTTTTAACTGTTAATATCACTTGGTTTGCTATGTATCCTTGTTGTTCTGGCTTTGTTGCTTTACAGCCATTCTGCTTACGCTATTGGCATCTGTTATTTAGAAAACCATTTTCTAACAGTTTTTAGATTTCCTTTCGAGTCTCCAACTGTTTTTAACCTCCAAGTTGCCACATAATGCATAAATATACCGAGTAAGTTTGCCCCCCTCTGAAATATGGATGTTAGATACTCAGACAGGTTCAGTATGTGTTGGGTGTTGTTTTCTGAGTTTGTGGTTGTCATAAGATGCAGCGAAATTACTGCGCTGTATCTTGGACCAATGCAATCAGCGAAGTCCTTATCGTGCCAAAAGGATGTGCATGGATTTTCCATCATTTGCAAACTTGAGGATGGCCTGCCCTGATGCCCTCAGATGCTGATGAAGTGTGTGGTTCATGAAAACGTGAGAAGCTAAGTAGTGGAGTAACGGGAGTGAGTACCAGTGTGGCCACTATGAAGAAAGTTAATTACTGAGAAATTAAGACTTGAGCATGTTATTTTCAAGCAACTTGTACAGCATTATATTGCACTACCATCAAATGCTTCTCCTGCCTATACAATACTCTGCAAGCTCTTGTATTGTACACTGTTTTATCTTGTTTAAAGTATGCTTGTTTTTGCTTATGTTGAGATT。
正义链核苷酸序列(589bp，SEQ ID No.2)
GGTTTGCTATGTATCCTTGTTGTTCTGGCTTTGTTGCTTTACAGCCATTCTGCTTACGCTATTGGCATCTGTTATTTAGAAAACCATTTTCTAACAGTTTTTAGATTTCCTTTCGAGTCTCCAACTGTTTTTAACCTCCAAGTTGCCACATAATGCATAAATATACCGAGTAAGTTTGCCCCCCTCTGAAATATGGATGTTAGATACTCAGACAGGTTCAGTATGTGTTGGGTGTTGTTTTCTGAGTTTGTGGTTGTCATAAGATGCAGCGAAATTACTGCGCTGTATCTTGGACCAATGCAATCAGCGAAGTCCTTATCGTGCCAAAAGGATGTGCATGGATTTTCCATCATTTGCAAACTTGAGGATGGCCTGCCCTGATGCCCTCAGATGCTGATGAAGTGTGTGGTTCATGAAAACGTGAGAAGCTAAGTAGTGGAGTAACGGGAGTGAGTACCAGTGTGGCCACTATGAAGAAAGTTAATTACTGAGAAATTAAGACTTGAGCATGTTATTTTCAAGCAACTTGTACAGCATTATATTGCACTACCATCAAATGCTTCTCCTGCCTATACAATACTCTGCAAGC。
反义链核苷酸序列(589bp，SEQ ID No.3)
GCTTGCAGAGTATTGTATAGGCAGGAGAAGCATTTGATGGTAGTGCAATATAATGCTGTACAAGTTGCTTGAAAATAACATGCTCAAGTCTTAATTTCTCAGTAATTAACTTTCTTCATAGTGGCCACACTGGTACTCACTCCCGTTACTCCACTACTTAGCTTCTCACGTTTTCATGAACCACACACTTCATCAGCATCTGAGGGCATCAGGGCAGGCCATCCTCAAGTTTGCAAATGATGGAAAATCCATGCACATCCTTTTGGCACGATAAGGACTTCGCTGATTGCATTGGTCCAAGATACAGCGCAGTAATTTCGCTGCATCTTATGACAACCACAAACTCAGAAAACAACACCCAACACATACTGAACCTGTCTGAGTATCTAACATCCATATTTCAGAGGGGGGCAAACTTACTCGGTATATTTATGCATTATGTGGCAACTTGGAGGTTAAAAACAGTTGGAGACTCGAAAGGAAATCTAAAAACTGTTAGAAAATGGTTTTCTAAATAACAGATGCCAATAGCGTAAGCAGAATGGCTGTAAAGCAACAAAGCCAGAACAACAAGGATACATAGCAAACC。
2、NATAL57 RNAi载体的遗传转化和转基因植株表达量检测
将第一部分获得的NATAL57 RNAi载体(NATAL57-pRNAi43)通过农杆菌EHA105介导的遗传转化方法侵染水稻中花11的胚性愈伤组织，经过共培养、阳性愈伤筛选、再分化、生根、移栽等步骤后得到潮霉素抗性的遗传转化植株(T0代转基因材料)。具体操作方法参照文献“Nishimura,A.,Aichi,I.,&Matsuoka,M.(2006).A protocol for Agrobacterium-mediated transformation in rice.Nature protocols,1(6),2796-2802.”。
将T0代转基因材料种于温室获得T1代种子。T1代种子消毒后于1/2MS培养基(含50mg/L Hyg)中培养7天，洗掉培养基后置于自来水中继续培养水稻幼苗(光暗周期12h/12h，温度28℃，相对湿度75％)。将3周龄秧苗移栽至大田(广东省农业科学院实验基地)，株距为20厘米×20厘米。待植株成熟后收取T2代种子，消毒后于1/2MS培养基(含50mg/L Hyg)中培养7天，洗掉培养基后置于自来水中继续培养7天，取叶片进行NATAL57表达量检测(图2)。具体检测方法为：用trizol提取RNA，反转录试剂使用PrimeScriptTMRT reagent Kitwith gDNA Eraser(Takara)，qPCR使用试剂为TBPremix Ex TaqTMII(Tli RNaseHPlus)(Takara)，所有操作均按照说明书操作规范进行。qPCR所用引物的序列为：NATAL57(F:TGGCTTTCTTGCAAGTTGTGC，SEQ ID No.8；R:GTGATTTTTGGCAACATACTGCG,SEQ ID No.9)；FhaB(F:GAGAAGCAGAAGCAGAAGGA，SEQ ID No.10；R:CTTGAATAGGACCATCAGGC,SEQ IDNo.11)。反应程序为95℃30s；95℃10s、60℃30s，40个循环。相对表达量用2-ΔΔct计算。以FhaB(NCBI Reference Sequence:XM_015788020.2)作为内参，以野生型中花11号水稻样品作为对照组，最终得到NATAL57表达明显受抑制的植株。
3、NATAL57 RNAi植株的产量相关性状观察
待第2部分中移栽至大田的植株成熟后进行产量相关性状的观察和统计。植株表型分析表明，NATAL57 RNAi植株表现出株高增加(图3)，稻穗增高增大(图4A)，籽粒增大(图4B)，籽粒长度明显增加(图5)的表型，从统计数据来看，NATAL57的表达抑制增加了水稻的千粒重(图6)。上述结果说明，NATAL57可以在水稻株型、穗型和粒型等目标性状上改良水稻的产量，为水稻遗传育种提供新的遗传资源，具有重要的潜在应用价值。
综上可见，通过抑制或降低NATAL57在水稻中的表达，可以使水稻表现出株高增加，稻穗增大，籽粒长度增加的表型，在调节水稻产量相关性状方面具有重要的前景。
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
Claims (8)
1.长链非编码RNA NATAL57在调节水稻产量相关性状中的应用，其特征在于，所述长链非编码RNA NATAL57的核苷酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的应用，其特征在于，所述水稻产量相关性状包括株型、穗型和粒型。
3.根据权利要求1所述的应用，其特征在于，所述调节水稻产量相关性状为通过抑制或降低NATAL57在水稻中的表达，进而改善水稻产量相关性状。
4.根据权利要求1所述的应用，其特征在于，所述长链非编码RNA NATAL57还包括与其相关的生物材料，所述相关的生物材料包括含有NATAL57的重组载体、表达盒、转基因细胞系或重组菌。
5.根据权利要求1所述的应用，其特征在于，所述水稻的品种包括中花11号。
6.一种培育高产水稻的方法，其特征在于，利用基因编辑技术抑制长链非编码RNANATAL57在水稻中的表达，或者降低长链非编码RNA NATAL57在水稻中的表达量，进而培育得到水稻产量性状改善的水稻植株，所述长链非编码RNA NATAL57的核苷酸序列如SEQ IDNo.1所示。
7.根据权利要求6所述的一种培育高产水稻的方法，其特征在于，利用RNAi技术，通过构建NATAL57 RNAi载体并转化水稻植株，进而抑制长链非编码RNA NATAL57在水稻中的表达，或者降低长链非编码RNA NATAL57在水稻中的表达量。
8.根据权利要求7所述的一种培育高产水稻的方法，其特征在于，用于构建NATAL57RNAi载体的引物包括如SEQ ID No.4、5所示的正义链上下游引物和如SEQ IDNo.6、7所示的反义链上下游引物。
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