CN116199757B - 转录因子OsNF-YC1在调控水稻籽粒大小中的应用及方法 - Google Patents
转录因子OsNF-YC1在调控水稻籽粒大小中的应用及方法 Download PDFInfo
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Abstract
本发明涉及转录因子OsNF‑YC1在调控水稻籽粒大小中的应用及方法，其中，转录因子OsNF‑YC1的CDS序列如SEQ ID No.1所示，编码蛋白的氨基酸序列如SEQ ID No.2所示。通过对水稻中的转录因子OsNF‑YC1进行敲除或下调其表达，可以使水稻籽粒的粒宽和粒厚降低，呈现出轻微的细长粒外形，千粒重降低，这为水稻的籽粒改良提供了一种新的基因和方法。
Description
技术领域
本发明涉及植物基因工程技术和水稻分子育种技术领域，具体涉及转录因子OsNF-YC1在调控水稻籽粒大小中的应用及方法。
背景技术
水稻作为世界上最重要的粮食作物之一，世界上一半的人以稻米为食，然而，近年来随着世界人口增加，全球气候变化，可用耕地面积减少，水稻产量更是关乎人类粮食安全问题，因此，提高水稻单产就成为了解决粮食安全的关键所在。在构成水稻产量的三个要素中包括单位面积有效穗数、穗粒数和千粒重。而水稻的千粒重在很大程度上被水稻粒形包括粒长、粒宽、粒厚、长宽比等影响着。
在近年的研究中，研究人员已经克隆了很多控制水稻籽粒大小的基因，并初步明确了其分子调控机理。主要包括转录调控途径、内源激素调控途径、泛素-蛋白酶体途径、G蛋白信号传导途径等多种调控途径(Li,N.,&Li,Y..(2016)Signaling pathways of seedsize control in plants.Current Opinion in Plant Biology,33,23-32.)。例如：在转录调控途径中，GS2编码一个生长调控因子，与转录共激活子OsGRFs互作，调控细胞伸长和细胞分裂，超表达可显著增加籽粒重量，进而提高水稻产量(Hu,Y.,Liang,W.,Yin,C.,etal.(2015)Interactions of OsMADS1 with Floral Homeotic Genes in Rice FlowerDevelopment.Molecular Plant,8(9):1366-1384)。在内源激素调控途径中，GW5编码一个钙调素结合蛋白,可与糖原合酶激酶GSK2互作并抑制其活性,从而调节油菜素内酯响应基因的表达水平和生长响应，进而调控粒宽和粒重(Liu,J.,Chen,J.,Zheng,X.,Wu,F.,Lin,Q.,Heng,Y.,et al.(2017)GW5 acts in the brassinosteroid signalling pathway toregulate grain width and weight in rice.Nature Plants,3:17043.)。在泛素-蛋白酶体途径中，GW2编码一个环型E3泛素连接酶，通过将其底物锚定到蛋白酶体进行降解，从而负调节细胞的分裂(Hao,J.,Wang,D.,Wu,Y.,et al.(2021)The GW2-WG1-OsbZIP47pathway controls grain size and weight in rice.Molecular Plant,14(8):1266-1280.)。水稻中3个G蛋白γ亚基GS3、DEP1和GGC2。在G蛋白信号传导途径中，DEP1和GGC2的C端结构域介导G蛋白信号传导，其功能发挥依赖G蛋白β亚基RGB1和α亚基RGA1，它们对籽长的调节作用是加性的。GS3本身对籽粒大小无影响，但它与DEP1或GGC2竞争性结合Gβ，缩短粒长(Yu,J.,Miao,J.,Zhang,Z.,Xiong,H.,Zhu,X.,Sun,X.,et al.(2018)Alternativesplicing of OsLG3b controls grain length and yield in japonica rice.PlantBiotechnology Journal,16,1667–1678.)。
核因子Y转录因子(NF-Y)又称CCAAT盒结合因子或血红素相关蛋白，是一种由NF-YA,NF-YB和NF-YC亚基组成的三聚体蛋白，在所有真核生物的进化过程中高度保守。在近年的研究中，水稻NF-Y家族在水稻抽穗期的调控、抗逆性研究和米质改良等方面的研究已经取得一定的进展，例如：在水稻抽穗期的调控方面，长日照下OsNF-YC2和OsNF-YC4作为开花的抑制因子发挥作用，它们可能通过直接与OsNF-YB8、OsNF-YB10和OsNF-YB11蛋白互作，调控水稻光周期开花响应(Kim,S.,Park,H.,Jang,Y.et al.(2016)OsNF-YC2 and OsNF-YC4proteins inhibit flowering under long-day conditions in rice.Planta 243,563–576.)。OsNF-YB9与OsNF-YC12互作形成复合体，并与OsGI一起通过Hd1途径抑制成花素，从而负调控水稻抽穗(Das,S.,Parida,S.K.,Agarwal,P.,&Tyagi,A.K..(2019)Transcription factor osnf-yb9 regulates reproductive growth and developmentin rice.Planta,250(6),1849-1865.)。在抗逆性研究方面，OsNF-YA7在水稻的耐旱性中发挥重要作用，且独立于ABA通路(Lee,D.,Kim,Il.,Geupil,et al.(2015)The NF-YAtranscription factor OsNF-YA7 confers drought stress tolerance of rice in anabscisic acid independent manner.Plant Science,241,199-210.)。OsNF-YA2能赋予水稻耐盐、耐旱和抗病菌的能力，并提高光合能力和分蘖数(Alam,M.,Tanaka,T.,Nakamura,H.,Ichikawa,H.,Kobayashi,K.,Yaeno,T.,et al.(2015).Overexpression of a riceheme activator protein gene(OsHAP2E)confers resistance to pathogens,salinityand drought,and increases photosynthesis and tiller number.PlantBiotechnology Journal,13,85–96.)。在米质改良方面，OsNF-YB1通过与转录因子OsERF115互作，特异调控水稻胚乳发育中下游基因的转录，调控籽粒灌浆和胚乳发育(Xu,J.,Zhang,X.,Xue,H..(2016)Rice aleurone layer specific OsNF-YB1 regulatesgrain filling and endosperm development by interacting with an ERFtranscription factor.Journal of Experimental Botany,67(22):6399-6411.)。OsNF-YB1直接结合并激活Wx转录，从而调控稻谷品质(Bello,B.,Hou,Y.,Zhao,J.,et al.(2019)NF-YB1-YC12-bHLH144 complex directly activates Wx to regulate grain qualityin rice(Oryza sativa L.).Plant Biotechnology Journal,17(7):1222-1235.)。OsNF-YC12协调多种途径来调控水稻胚乳发育和种子贮藏物质的积累，OsNF-YC12与OsNF-YB1互作，在糊粉层中协同调节蔗糖转运基因OsSUT1。发育的胚乳中，OsNF-YC12能直接与FLO6和谷氨酰胺合成酶基因OsGS1；3的启动子结合(Xiong,Y.,Ren,Y.,Li,W.,et al.(2019)NF-YC12 is a key multi-functional regulator of accumulation of seed storagesubstances in rice.Journal of Experimental Botany,70(15):3765-3780.)。
尽管NF-YC家族在抽穗期的调控、抗逆性研究和米质改良等方面的研究已经取得一定的进展，同时NF-Y家族基因表达异常或功能缺失的突变体，如OsNF-YB1(Xu,J.,Zhang,X.,Xue,H..(2016)Rice aleurone layer specific OsNF-YB1 regulates grain fillingand endosperm development by interacting with an ERF transcriptionfactor.Journal of Experimental Botany,67(22):6399-6411.)或OsNF-YC12(Xiong,Y.,Ren,Y.,Li,W.,etal.(2019)NF-YC12 is a key multi-functional regulator ofaccumulation of seed storage substances in rice.Journal of ExperimentalBotany,70(15):3765-3780.)的籽粒大小也随之发生改变，但目前关于水稻OsNF-YC1的具体功能尚未见报道。
发明内容
本发明的目的在于提供转录因子OsNF-YC1在调控水稻籽粒大小中的应用及方法。
为了实现上述目的，本发明提供了如下技术方案：
本发明提供了转录因子OsNF-YC1在调控水稻籽粒大小中的应用，所述转录因子OsNF-YC1的CDS序列如SEQ ID No.1所示，编码蛋白的氨基酸序列如SEQ ID No.2所示。
进一步地，所述应用为通过对转录因子OsNF-YC1进行敲除或下调其表达，使水稻籽粒的粒宽和粒厚降低，呈现出轻微的细长粒外形，千粒重降低。
本发明还提供了一种改良水稻籽粒的方法，所述方法为通过对水稻中的转录因子OsNF-YC1进行敲除或下调其表达，使水稻籽粒的粒宽和粒厚降低，呈现出轻微的细长粒外形，千粒重降低。
本发明的有益效果在于：本发明利用CRISPER-Cas9基因靶向编辑技术等基因工程手段对OsNF-YC1基因进行敲除或下调表达，获得该基因功能缺失或下调表达的水稻突变体，与野生型相比，OsNF-YC1基因功能缺失或下调表达的突变体的籽粒粒宽和粒厚降低，呈现出轻微的细长粒外形，千粒重降低，突变体的每穗粒数显著增加，使得其单株产量没有发生明显改变，因此，该基因可用于水稻的籽粒改良。并且，本发明还通过细胞学分析、亚细胞定位、转录激活实验等方式确定了OsNF-YC1调控水稻籽粒的途径，即为OsNF-YC1保守的组蛋白类结构域CBFD-NFYB-HMF可以直接与OsMADS1转录因子保守的结构域MADS-box相互作用，进而增强OsMADS1的转录激活活性，促进共同靶基因OsMADS55的转录激活，从而调节籽粒的大小和形状。可为水稻的籽粒改良提供新的基因和方法。
附图说明
图1为OsNF-YC1的进化树分析和敲除突变体的基因和蛋白序列；
A:OsNF-YC1的进化树分析，OsNF-YC1以红色字体显示；
B:敲除突变体的基因序列，敲除突变体osnf-yc1-g和osnf-yc1-t分别代表鸟嘌呤插入和胸腺嘧啶插入；
C:敲除突变体的蛋白序列，OsNF-YC1的结构域CBFD-NFYB-HMF以蓝色字体显示。
图2为OsNF-YC1蛋白的保守性分析；
A：使用MEME进行OsNF-YC1蛋白的保守基序分析；
B：使用Clustal W进行OsNF-YC1蛋白序列分析。
图3为野生型和敲除突变体osnf-yc1-g/t的表型、农艺性状分析及细胞学观察；
A-E：野生型和敲除突变体osnf-yc1-g/t(A)株高，(B)穗部形态,(C)粒宽,(D)粒厚,(E)粒长的对比，标尺：20cm(A),3cm(B),1cm(C-E)；
F-I：野生型和敲除突变体osnf-yc1-g/t的农艺性状分析，(F-H)粒宽、粒厚、粒长(n＝20)，(I)千粒重(n＝6)；
J：野生型和敲除突变体osnf-yc1-g/t开花前颖壳的对比，标尺：2mm；
K：野生型和敲除突变体osnf-yc1-g/t颖壳的石蜡横切面，标尺：500mm(左)and500um(右)；
L-M:野生型和敲除突变体osnf-yc1-g/t外层薄壁细胞的数量和面积对比；N-S:扫描电子显微镜分析野生型和敲除突变体osnf-yc1-g/t颖片的外表皮细胞和内表皮细胞，标尺：100mm；
T-W:野生型和敲除突变体osnf-yc1-g/t外表皮细胞和内表皮细胞的长度和宽度对比(n＝100)；
其中，数据为平均值±标准差，*代表P<0.05，**代表P<0.01。
图4为野生型和敲除突变体osnf-yc1-g/t的穗部性状及农艺性状考察；
A-C:野生型和敲除突变体osnf-yc1-g/t的一次枝梗对比，图中红框代表图2D的位置，标尺：3cm；
D:野生型和敲除突变体osnf-yc1-g/t的二次枝梗对比，标尺：1cm；
F-K:野生型和敲除突变体osnf-yc1-g/t(F)每穗粒数，(G)一次枝梗数,(H)株高,(I)有效穗数,(J)穗长,(K)单株产量的农艺性状统计；
其中，数据为平均值±标准差，*代表P<0.05，**代表P<0.01。
图5为敲除突变体osnf-yc1-g和OsNF-YC1-COM互补转基因植株的表型和农艺性状分析；
A-H:敲除突变体osnf-yc1-g和OsNF-YC1-COM互补转基因植株(A)株高，(B)穗部形态,(C)粒宽,(D)粒厚,(E-G)一次枝梗，(H)二次枝梗的对比，标尺：20cm(A),3cm(B、E-H),2cm(C-D)；
I：OsNF-YC1-COM互补转基因植株在突变位点的测序峰。
图6为在野生型和敲除突变体osnf-yc1-g/t中相关细胞周期蛋白基因的表达；表达分析使用2cm幼穗进行，以Actin作为对照，野生型的表达水平设为1(n＝3)，数据为平均值±标准差，*代表P<0.05，**代表P<0.01。
图7为OsNF-YC1的RNAi转基因株系；
A-B：野生型和OsNF-YC1-RNAi转基因株系(A)粒宽和(B)粒长的比对，标尺,1cm；
C:通过实时定量荧光PCR检测野生型和OsNF-YC1-RNAi转基因株系中OsNF-YC1的表达(n＝3)；
D-G：野生型和OsNF-YC1-RNAi转基因株系的农艺性状统计，(D-F)粒宽、粒厚、粒长(n＝20)，(G)千粒重(n＝3)；
其中，数据为平均值±标准差，*代表P<0.05，**代表P<0.01。
图8为OsNF-YC1的RNAi转基因株系的株型；
A:野生型的株型，标尺:20cm。
B-D:OsNF-YC1的RNAi株系的株型,标尺:20cm。
图9为OsNF-YC1的亚细胞定位于细胞核与细胞质；
A:在水稻原生质体中OsNF-YC1的亚细胞定位，mCherry代表在细胞核中特异表达的蛋白，标尺：5mm；
B:在本氏烟草中OsNF-YC1的亚细胞定位，标尺：20mm；
C:在OsNF-YC1-GFP转基因植株中OsNF-YC1的亚细胞定位，标尺：20mm。
图10为OsNF-YC1的转录活性分析；
A:在酵母双杂交系统中检测OsNF-YC1的转录活性，将OsNF-YC1的全长的编码区和一系列截断的编码区片段克隆到pGBKT7载体，数字表示OsNF-YC1的氨基酸位置，空载的pGBKT7载体作为阴性对照，pGBKT7-OsTB1作为阳性对照；
B:在水稻原生质体中OsNF-YC1的转录活性分析；
数据为平均值±标准差。经t测验，*代表P<0.05，**代表P<0.01。
图11为OsNF-YC1与OsMADS1相互作用；
A：OsNF-YC1及其截断蛋白与OsMADS1在酵母双杂交试验中的相互作用分析；
B：通过GST下拉试验检测OsNF-YC1与OsMADS1相互作用。黑色箭头表示目标条带的位置；
C：通过双分子萤光互补实验检测OsNF-YC1与OsMADS1在本氏烟草细胞核内相互作用，NYFP和OsMADS1-CYFP共表达的融合蛋白被用作阴性对照。
图12为OsNF-YC1与OsMADS1的MADS1-Box结构域相互作用，M表示MADS区域，I表示中间区域，K表示角蛋白样区域，C表示C端区域。
图13为OsNF-YC1与OsMADS1相互作用后增强OsMADS1的转录激活活性；
A:在野生型和敲除突变体osnf-yc1-g/t中，通过实时荧光定量PCR检测OsMADS1在转录水平的表达(n＝3)；
B:在野生型和敲除突变体osnf-yc1-g/t中，通过蛋白质免疫印迹法检测OsMADS1在蛋白水平的表达；
C:在野生型和敲除突变体osnf-yc1-g/t中，通过原位杂交实验检测OsMADS1在小穗不同发育阶段的表达部位，标尺：100mm；
D:OsMADS1和OsNF-YC1相互作用对OsMADS1转录激活活性的影响，OsMADS1与GAL4-BD融合，检测GAL4结合元件UAS控制下的萤火虫荧光素酶(LUC)的相对活性，Renilla荧光素酶(REN)的活性被用作内参，空载的GAL4-BD载体被用作阴性对照(n＝3)。
图14野生型和osmads55敲除突变体株系的表型及农艺性状统计；
A-B:野生型和敲除突变体osmads55(A)粒宽，(B)粒厚的对比，标尺：1cm；
C-F:野生型和敲除突变体osmads55的农艺性状分析，(C-E)粒宽、粒厚、粒长(n＝20)，(F)千粒重(n＝6)；
其中，数据为平均值±标准差，经t测验，*代表P<0.05，**代表P<0.01。
具体实施方式
下面将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
实施例1 OsNF-YC1是一个组蛋白类转录因子
OsNF-YC1是NF-Y家族中的转录因子，基因编号为LOC_Os02g07450，位于2号染色体上，CDNA全长1238bp包含2个外显子，CDS全长780bp，核苷酸序列如SEQ ID No.1所示，编码259个氨基酸(NCBI)氨基酸序列如SEQ ID No.2所示，其在104-168aa处包含一个结构域CBFD-NFYB-HMF(https://www.ncbi.nlm.nih.gov/),因此是一种组蛋白类转录因子。
为了确定OsNF-YC1同源基因在不同植物物种中的进化特征，通过使用Gramene网站上的blast分析工具进行蛋白序列比对分析，发现在水稻，玉米，高粱，小米，拟南芥等物种中都含有相似的蛋白保守序列，使用软件MEGA构建系统发育进化树，分析表明在进化过程中，可将OsNF-YC基因家族按亲缘关系分为三支，其中OsNF-YC1与OsNF-YC4、OsNF-YC2亲缘关系较近(图1A)。通过使用MEME进行MOTIFY分析(图2A)，并且在Clustal W上进行同源蛋白氨基酸序列比对表明(图2B)，其结构域CBFD-NFYB-HMF在进化过程中高度保守。但是OsNF-YC1的具体功能还不清晰。
为了研究OsNF-YC1在水稻中的功能，发明人在OsNF-YC1的外显子内设计了sgRNA靶位点(GCAGCAACTCCGTGAGTTCTGG)，利用CRISPR-Cas9技术在中花11的背景下构建了OsNF-YC1的敲除突变体，通过对多株转基因植株的基因组DNA进行PCR扩增、测序，在靶点处鉴定出了两种类型的纯合敲除突变体，分别是在编码区260bp处引起鸟嘌呤插入和胸腺嘧啶插入的osnf-yc1-g和osnf-yc1-t(图1B)。这些突变由于碱基的插入，使其发生移码突变进而导致了编码提前终止并产生了89氨基酸的多态(图1C)。
实施例2 OsNF-YC1通过影响细胞增殖和细胞扩张调控籽粒大小
在田间自然生长条件下，对野生型、敲除突变体osnf-yc1-g和osnf-yc1-t进行全生育期观察。结果发现，osnf-yc1-g/t在苗期和成熟期均表现出相似的表型。但与野生型相比，在营养生长阶段，osnf-yc1-g/t株系与WT植株无明显差异，在成熟期，对其农艺性状统计发现，osnf-yc1-g/t籽粒变小，粒宽减少了8.2％/9.3％，粒厚降低了7.9％/8.3％，粒长无明显差异，千粒重降低了8.5％/7.8％(图3A-I)。同时，每穗粒数增加13.0％/12.2％，一次枝梗数不变，二次枝梗数增加，株高、单株产量等其他性状与野生型相比无明显差异(图4)。为了进一步确定osnf-yc1-g/t的表型是由OsNF-YC1的突变引起的，将OsNF-YC1的完整基因及3.1Kb的启动子区域的互补结构引入到敲除突变体osnf-yc1-g中，在成熟期观察发现，转基因阳性植株都基本恢复了osnf-yc1-g的表型(图5)。这些结果表明了在水稻中OsNF-YC1调控籽粒大小。
为了阐明osnf-yc1-g/t籽粒变小的细胞学机制，对野生型和突变体开花前的颖壳进行石蜡切片观察发现，osnf-yc1-g/t外层薄壁细胞数量显著降低，细胞面积显著减小。同时，取植株抽穗前的小穗颖壳通过扫描电镜观察其内表层细胞和外表层细胞(图3)发现，osnf-yc1-g/t内表层和外表层的细胞长度没有显著变化，但细胞宽度都显著减小。这表明osnf-yc1-g/t形成小籽粒可能是由细胞数量降低和细胞面积减小造成的。在osnf-yc1-g/t中检测参与细胞周期蛋白相关基因的表达，发现cdc2Os-1、CDKB1、CycA1；1、CycA2；1、CycD3；1这些基因在osnf-yc1-g/t中表达下调(图6)，这表明osnf-yc1-g/t细胞数量的减小可能与促进细胞增殖的基因表达下调有关。这些数据进一步表明了OsNF-YC1可能是通过影响细胞增殖和细胞扩张来调节籽粒大小。
实施例3 OsNF-YC1表达降低导致籽粒变小
为了进一步证实OsNF-YC1在水稻籽粒发育中的调控作用，构建OsNF-YC1-RNAi载体，并将其转化到野生型植株中，通过对多株转基因植株的基因组DNA进行PCR扩增、测序，获得4株独立的阳性转基因株系，并且通过qRT-PCR鉴定选择其中OsNF-YC1表达量显著低于WT的3个株系(OsNF-YC1-RNAi-1、OsNF-YC1-RNAi-2、OsNF-YC1-RNAi-3)，其表达量分别降低了22.1％、25.2％、32.9％(图7C)。在正常田间条件下，在营养生长阶段，OsNF-YC1-RNAi株系与野生型植株无明显差异(图8)，在成熟期，对其进行农艺性状统计，结果表明，较野生型相比，OsNF-YC1-RNAi转基因株系产生的籽粒变小(图7A-B)。其中，OsNF-YC1-RNAi-1籽粒变小，粒宽减少了4.8％，粒厚降低了5.1％，粒长无明显差异，千粒重降低了7.1％(图7D-G)。这与敲除突变体osnf-yc1-g/t株系相似。
实施例4 OsNF-YC1蛋白定位于细胞核和细胞质
为了确定OsNF-YC1蛋白在细胞中定位情况，将OsNF-YC1的编码区与含有GFP的PAN580载体融合，将构建好的p35S::OsNF-YC1::GFP转化到水稻原生质体中瞬时表达，通过观察融合蛋白的绿色荧光信号表明，OsNF-YC1定位在细胞的细胞核和细胞质(图9A)。为了进一步验证其定位情况，将OsNF-YC1的编码区与含有eGFP的pCAMBIA 1300载体融合，利用农杆菌介导法将融合载体转化到烟草细胞，在细胞核与细胞质中可以观察到荧光信号(图9B)。同时，在OsNF-YC1-GFP的转基因植株中，发现在其细胞的细胞质和细胞核中都可检测到绿色荧光信号(图9C)。因此，这些结果也都表明OsNF-YC1蛋白定位在细胞核与细胞质。将编码osnf-yc1-g和osnf-yc1-t突变后形成的89个氨基酸的序列与含有GFP的PAN580载体融合，并且转化到水稻原生质体中瞬时表达，其结果表明仍然定位在细胞核和细胞质(图9A)。
实施例5 OsNF-YC1是一个具有转录激活活性的转录因子
OsNF-YC1作为组蛋白类转录因子，为探究其转录活性，首先利用酵母双杂交系统进行OsNF-YC1的转录活性验证，通过将OsNF-YC1的编码区构建到酵母表达载体pGBKT7中，使其在酵母感受态细胞中进行表达并观察到酵母菌斑在缺陷培养基上生长(图10A)。同时，将OsNF-YC1的编码区构建到载体GAL4BD中，构建35S::OsNF-YC1::GAL4 BD载体，以VP16作为阳性对照，空载GAL4BD为阴性对照，转化在水稻原生质体中瞬时表达检测其LUC活性，发现与阴性对照相比OsNF-YC1的LUC活性明显增加(图10B)。这些结果也证实了OsNF-YC1是一个具有转录激活活性的转录因子。
为了进一步确定OsNF-YC1的转录激活活性区域，构建基于CBFD-NFYB-HFM结构域缺失的不同载体，通过酵母双杂交系统进行检测，结果表明OsNF-YC1的N端有较强的转录激活活性，C端有较弱的转录激活活性，而CBFD-NFYB-HFM结构域没有转录激活活性(图10A)。OsNF-YC1的N端包含103个氨基酸(aa)，osnf-yc1-g/t植株的突变位于该N端末端，导致编码提前终止产生了89氨基酸的多肽(图1C)。为了确定这些截断多肽的转录激活活性，将编码OsNF-YC1-G和OsNF-YC1-T多肽的序列与载体pGBKT7和GAL4BD融合，然后分别转化到的酵母感受态细胞和水稻原生质体中。实验结果表明，OsNF-YC1-G/T保留了其转录激活活性(图10A-B)。
实施例6 OsNF-YC1与OsMADS1转录因子相互作用
为了探究OsNF-YC1调控籽粒发育的分子机理，将OsNF-YC1的结构域CBFD-NFYB-HMF的编码区与载体pGBKT7融合，通过酵母双杂交筛选与其相互作用的蛋白，实验发现CBFD-NFYB-HMF能够和参与花发育调控的E类MADS-box基因OsMADS1相互作用，接下来，将OsMADS1的编码区与载体pGBKT7融合，将OsNF-YC1的编码区与载体pGADT7融合，使其在酵母双杂交菌株中共同表达，结果表明OsNF-YC1与OsMADS1相互作用(图11A)。同时，在大肠杆菌中表达了GST标记的OsMADS1(GST-OSMADS1)和His标记的OsNF-YC1(His-OsNF-YC1)，发现GST-OsMADS1能拉下His-OsNF-YC1，但GST不能。体外GST下拉实验进一步验证了OsNF-YC1与OsMADS1之间的相互作用(图11B)。然后，通过双分子荧光互补实验检测OsNF-YC1与OsMADS1在体内的相互作用。在OsMADS1-CYFP和OsNF-YC1-NYFP共表达的烟草叶片表皮细胞的细胞核中检测到黄色荧光蛋白信号。而在与OsMADS1-CYFP和OsNF-YC1-G-NYFP、OsNF-YC1-T-NYFP共表达的融合蛋白在烟草叶片表皮细胞的细胞核中没有检测到黄色荧光蛋白信号(图11C)。因此，这些结果进一步证实了OsNF-YC1与OsMADS1在体外和体内都相互作用。
为了验证OsNF-YC1与OsMADS1相互作用的具体结构域，基于OsNF-YC1结构域CBFD-NFYB-HMF的缺失构建不同的载体，通过酵母双杂交实验进一步证明OsMADS1与OsNF-YC1相互作用的区域为OsNF-YC1的结构域CBFD-NFYB-HMF，而突变后的OsNF-YC1-G和OsNF-YC1-T则不能与OsMADS1相互作用(图11A)。然后，构建各种基于OsMADS1结构域缺失的不同载体，发现OsMADS1结构域MADS-box对于OsMADS1与OsNF-YC1的相互作用都是必要的(图12)。
实施例7 OsNF-YC1与OsMADS1相互作用后增强OsMADS1的转录激活活性
为了揭示OsNF-YC1和OsMADS1相互作用后对籽粒发育的影响，通过RT-qPCR检测在osnf-yc1-g/t中OsMADS1转录水平的表达，结果表明，OsMADS1在转录水平的表达没有发生明显改变(图13A)；接下来，通过蛋白质印迹法检测在osnf-yc1-g/t中OsMADS1蛋白水平的表达，结果表明，OsMADS1蛋白水平的表达也没有发生明显改变(图13B)；然后通过原位杂交技术检测在osnf-yc1-g/t中OsMADS1的表达位置，结果表明OsMADS1也没有发生明显异位表达的情况(图13C)。
近来研究发现，GS3和DEP1与MADS转录因子相互作用，通过增强OsMADS1的转录活性，继而调控籽粒大小(Liu,Q.,Han,R.,Wu,W.,et al.(2018)G-proteinβγsubunitsdetermine grain size through interaction with MADS-domain transcriptionfactors in rice.Nature Communications,9:852.Mantovani,R..(1999)Review:Themolecular biology of the CCAAT binding factor NF-Y.Gene,239(1):15.)。因此，发明人推测osnf-yc1-g/t籽粒的改变是否是因为OsMADS1转录活性的改变进而影响其下游靶基因的表达而造成的。为证明这一推测，通过瞬时转录活性测定发现，当OsNF-YC1和OsMADS1共表达时，OsMADS1的转录激活活性显著增强，而当OsNF-YC1-G/T和OsMADS1共表达时，OsMADS1的转录激活活性并没有发生明显改变(图13D)。这一结果表明，OsNF-YC1与转录因子OsMADS1的相互作用增强了OsMADS1的转录激活活性。
OsMADS1作为含有MADS结构域的转录因子，已经被报道能够直接调控OsMADS17,OsMADS55,OsKANADI4,OsPIN1,OsHB4,OsETTIN2 and OsARF9等下游靶基因的表达(Arora,R.,Agarwal,P.,Ray,S.,et al.(2007)MADS-box gene family in rice:genome-wideidentification,organization and expression profiling during reproductivedevelopment and stress.BMC Genomics,8:242.；Khanday,I.,Yadav,S.,Vijayraghavan,U..(2013)Rice LHS1/OsMADS1 controls floret meristem specification bycoordinated regulation of transcription factors and hormone signalingpathways.Plant Physiology,161(4):1970-1983.；Liu,Q.,Han,R.,Wu,W.,et al.(2018)G-proteinβγsubunits determine grain size through interaction with MADS-domain transcription factors in rice.Nature Communications,9:852.)。因此，发明人在osnf-yc1-g/t中对这些基因进行了RT-qPCR分析，结果发现仅OsMADS55在osnf-yc1-g/t中表达极显著降低，其表达量仅为WT的34.8％/39.5％(图13E)，为进一步证明这个结论，通过改进的瞬时转录活性测定表明发现，OsMADS55启动子驱动的LUC活性受到OsMADS1的诱导，但当OsNF-YC1和OsMADS1共表达时，OsMADS55启动子驱动的LUC活性显著增强，当OsNF-YC1-G/T和OsMADS1共表达时，OsMADS55启动子驱动的LUC活性并没有发生明显改变(图13F)。同时，使用CRISPR-Cas9技术创建OsMADS55敲除突变体(osmads55)，通过农艺性状考察发现它们的籽粒变小，千粒重降低，这与osnf-yc1-g/t的表型相似(图14)。因此,这些证据表明OsNF-YC1与OsMADS1相互作用，通过增强OsMADS1的转录激活活性，进而调控共同靶基因OsMADS55的表达进而调控水稻籽粒的发育。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所有的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (2)
1.转录因子OsNF-YC1在调控水稻籽粒大小中的应用，其特征在于，所述转录因子OsNF-YC1的CDS序列如SEQ ID No.1所示，所述转录因子OsNF-YC1的氨基酸序列如SEQ IDNo.2所示。
2.根据权利要求1所述的转录因子OsNF-YC1在调控水稻籽粒大小中的应用，其特征在于，所述应用为通过对转录因子OsNF-YC1进行敲除或下调其表达，使水稻籽粒的粒宽和粒厚降低，呈现出轻微的细长粒外形，千粒重降低。
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