CN113388593B - Os07g0503900蛋白在调控植物对杂草抗性中的应用 - Google Patents
Os07g0503900蛋白在调控植物对杂草抗性中的应用 Download PDFInfo
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Abstract
本发明公开了Os07g0503900蛋白在调控植物对杂草抗性中的应用。本发明提供了Os07g0503900蛋白或其相关生物材料在如下任一中的应用:调控植物化感作用;调控植物对杂草抗性;调控植物中麦黄酮‑5‑O‑葡萄糖糖苷含量;催化麦黄酮糖基化生成麦黄酮‑5‑O‑葡萄糖糖苷;作为或制备糖基化转移酶;抑制杂草生长。实验证明,Os07g0503900能够催化tricin糖基化生成tricin‑5‑O‑glucopyranoside,tricin‑5‑O‑glucopyranoside能够调控植物化感作用,抑制杂草生长。可见Os07g0503900在调控植物的化感作用方面具有重要应用价值。本发明对于培育具有较高能力化感作用的水稻品种以及对麦黄酮‑5‑O‑葡萄糖糖苷生物合成的调控具有重要指导意义。
Description
技术领域
本发明涉及生物技术领域,具体涉及Os07g0503900蛋白在调控植物对杂草抗性中的应用。
背景技术
黄酮类化合物是一类是广泛分布于植物中的天然代谢产物,含有C6-C3-C6基本骨架。根据中央三碳链的氧化程度、是否构成环状以及B-环连接位置(2-或3-位)等特点,可将主要的天然黄酮类化合物分为黄酮类、黄酮醇类、二氢黄酮类、二氢黄酮醇类、花色素类、异黄酮类等。大部分黄酮类化合物在植物体内与糖结合成苷类,也有一部分以游离态(苷元形式)存在。黄酮类化合物有很强的抗炎和抗癌作用,在心血管疾病、冠心病、肿瘤等疾病治疗中有很大的应用前景。此外,黄酮类化合物在植物生长发育、抗氧化、生物胁迫等方面起着重要作用。如调节激素的运输,作为一种紫外线吸收化合物保护植物免受UV-B辐射,抑制杂草生长等。
目前在水稻中发现的有抑制杂草生长(化感作用)的黄酮类物质主要是麦黄酮,而关于其他黄酮类物质是否具有化感作用,以及水稻化感作用的遗传机制和抑草机制却不是很清楚。能够调控黄酮类物质表达的基因是否能够调控植物的化感作用也不是很清楚。
发明内容
本发明的目的是提供Os07g0503900蛋白在调控植物化感作用中的应用。
第一方面,本发明要求保护Os07g0503900蛋白或其相关生物材料在如下任一中的应用:
(1)调控植物化感作用。
所述调控植物化感作用体现为提高所述植物体内Os07g0503900蛋白的活性和/或含量,则所述植物的化感作用增强。
(2)调控植物对杂草抗性。
所述调控植物对杂草抗性体现为提高所述植物体内Os07g0503900蛋白的活性和/或含量,则所述植物对杂草抗性增强。
(3)调控植物中麦黄酮-5-O-葡萄糖糖苷含量。
所述调控植物中麦黄酮-5-O-葡萄糖糖苷含量体现为提高所述植物体内Os07g0503900蛋白的活性和/或含量,则所述植物中麦黄酮-5-O-葡萄糖糖苷含量升高;和/或降低所述植物体内Os07g0503900蛋白的活性和/或含量,则所述植物中麦黄酮-5-O-葡萄糖糖苷含量降低。
(4)催化麦黄酮(tricin)糖基化生成麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside);
(5)作为或制备糖基化转移酶;
在本发明中,所述糖基化的受体为麦黄酮(tricin)。
在本发明的具体实施方式中,所述糖基化的供体具体为UDPG。
(6)抑制杂草生长。
所述相关生物材料为能够表达所述Os07g0503900蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
所述Os07g0503900蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
上述蛋白质中,所述标签是指利用DNA体外重组技术,与目的蛋白一起融合表达的一种多肽或者蛋白,以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
第二方面,本发明要求保护一种培育化感作用改变的植物品种的方法,可为方法I或方法II:
方法I:一种培育化感作用增强的植物品种的方法,可包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量提高,得到化感作用增强的植物品种。所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
方法II:一种培育化感作用减弱的植物品种的方法,可包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量降低,得到化感作用减弱的植物品种。所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
第三方面,本发明要求保护一种培育对杂草抗性改变的植物品种的方法,可为如下方法III或方法IV:
方法III:一种培育对杂草抗性增强的植物品种的方法,可包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量提高,得到对杂草抗性增强的植物品种。所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
方法IV:一种培育对杂草抗性减弱的植物品种的方法,可包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量降低,得到对杂草抗性减弱的植物品种。所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
第四方面,本发明要求保护一种培育麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量改变的植物品种的方法,可为如下方法V或方法VI:
方法V:一种培育麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量提高的植物品种的方法,可包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量提高,得到麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量提高的植物品种。所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
方法VI:一种培育麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量降低的植物品种的方法,可包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量降低,得到麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量降低的植物品种。所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
第五方面,本发明要求保护一种培育化感作用改变的转基因植物的方法,可为如下方法VII或方法VIII:
方法VII:一种培育化感作用增强的转基因植物的方法,可包括如下步骤:向受体植物中导入能够表达Os07g0503900蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比化感作用增强;所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
方法VIII:一种培育化感作用减弱的转基因植物的方法,可包括如下步骤:对受体植物中能够表达Os07g0503900蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比化感作用减弱;所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
第六方面,本发明要求保护一种培育对杂草抗性改变的转基因植物的方法,可为如下方法IX或方法X:
方法IX:一种培育对杂草抗性增强的转基因植物的方法,可包括如下步骤:向受体植物中导入能够表达Os07g0503900蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比对杂草抗性增强;所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
方法X:一种培育对杂草抗性减弱的转基因植物的方法,可包括如下步骤:对受体植物中能够表达Os07g0503900蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比对杂草抗性减弱;所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
第七方面,本发明要求保护一种培育麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量改变的转基因植物的方法,可为如下方法XI或方法XII:
方法XI:一种培育麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量提高的转基因植物的方法,可包括如下步骤:向受体植物中导入能够表达Os07g0503900蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量提高;所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
方法XII:一种培育麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量降低的转基因植物的方法,可包括如下步骤:对受体植物中能够表达Os07g0503900蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量降低;所述Os07g0503900蛋白为前文(A1)-(A4)中任一所示蛋白质。
第八方面,本发明要求保护一种抑制杂草生长的方法。
本发明所要求保护的抑制杂草生长的方法,可包括如下步骤:向所述杂草的种子外施通过使受体植物中Os07g0503900蛋白的活性和/或含量提高培育得到的植物品种的叶片水洗物溶液。
进一步地,所述“使受体植物中Os07g0503900蛋白的活性和/或含量提高”是通过向所述受体植物中导入能够表达所述Os07g0503900蛋白的核酸分子来实现的。
进一步地,所述叶片水洗物可按照包括如下步骤的方法制备得到:将所述叶片按照12g比200mL的比例加入到水中,37℃密封震荡(220rpm)提取4h,室温静置20h,冷冻干燥,得到固态水洗物;每12mL的所述叶片水洗物溶液(工作液)中含有从12g叶片中提取得到的所述固态水洗物。
在上述各方面中,所述“能够表达所述Os07g0503900蛋白的核酸分子”具体可为如下任一所述的DNA分子:
(B1)SEQ ID No.2所示的DNA分子;
(B2)在严格条件下与(B1)限定的DNA分子杂交且编码所述Os07g0503900蛋白的DNA分子;
(B3)与(B1)或(B2)限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述Os07g0503900蛋白的DNA分子。
上述基因中,所述严格条件可为如下:50℃,在7%十二烷基硫酸钠(SDS)、0.5MNa3PO4和1mM EDTA的混合溶液中杂交,在50℃,2×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.5×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在65℃,0.1×SSC,0.1%SDS中漂洗;也可为:在6×SSC,0.5%SDS的溶液中,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
在前文中,所述“对受体植物中能够表达Os07g0503900蛋白的核酸分子进行抑制表达”可通过任何能够实现这一目的的技术手段实现,如通过序列特异核酸酶(如CRISPR/Cas9核酸酶)对所述核酸分子进行特异性剪切,从而降低其在所述受体植株中的表达。
在本发明中,所述“对受体植物中能够表达Os07g0503900蛋白的核酸分子进行抑制表达”具体是通过CRISPER/Cas9技术实现的;以Os07g0503900基因组片段中符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的片段为靶序列;N表示A、G、C和T中的任一种,14≤X≤30,且X为整数,NX表示X个连续的脱氧核糖核苷酸。更加具体的,在本发明的一个具体实施例中,所述X为20。相应的,所述靶序列具体为5'-ATGCTGCGCAGCCGCTGCTG-3'或5'-CAGCAGCGGCTGCGCAGCAT-3'。
在前文中,所述“向受体植物中导入能够表达Os07g0503900蛋白的核酸分子”可通过任何能够实现这一目的的技术手段实现。
在本发明中,所述“向受体植物中导入能够表达Os07g0503900蛋白的核酸分子”具体是通过向所述受体植物中导入含有所述能够表达Os07g0503900蛋白的核酸分子的重组表达载体实现的。
所述重组表达载体可用现有的植物表达载体构建。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等,如pCAMBIA-1300-221、pGreen0029、pCAMBIA3301、pCAMBIA1300、pBI121、pBin19、pCAMBIA2301、pCAMBIA1301-UbiN或其它衍生植物表达载体。所述植物表达载体还可包含外源基因的3’端非翻译区域,即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3’端。使用所述基因构建重组表达载体时,在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子,例如花椰菜花叶病毒(CAMV)35S启动子、泛素基因Ubiquitin启动子(pUbi)、胁迫诱导型启动子rd29A等,它们可单独使用或与其它的植物启动子结合使用;此外,使用本发明的基因构建重组表达载体时,还可使用增强子,包括翻译增强子或转录增强子,这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等,但必需与编码序列的阅读框相同,以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的,可以是天然的,也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所用重组表达载体进行加工,如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因、具有抗性的抗生素标记物或是抗化学试剂标记基因等。也可不加任何选择性标记基因,直接以逆境筛选转化植株。
在本发明的具体实施方式中,所述抑制杂草生长为抑制杂草根的生长。
在上述各方面中,所述植物具体可为水稻;所述杂草具体可为稗草或生菜。
在上述各方面中,所述麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)的结构式如图1所示。
本发明提供了与麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)形成相关的Os07g0503900基因,体外酶活实验表明Os07g0503900能够催化tricin形成糖基化产物tricin-5-O-glucopyranoside。在水稻中改变这个基因的表达量会引起tricin-5-O-glucopyranoside含量的显著改变。另外,化合物外施实验表明麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)具有显著抑制稗草和生菜根生长的作用。可见,Os07g0503900基因在调控植物的化感作用,用于抑制杂草生长方面具有很大的应用价值。本发明对于培育具有较高能力化感作用的水稻品种以及对麦黄酮-5-O-葡萄糖糖苷生物合成的调控具有重要指导意义。
附图说明
图1为麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)结构式。
图2为Os07g0503900蛋白纯化。其中红色箭头为蛋白纯化后重组蛋白的大小。
图3为Os07g0503900体外酶活产物检测的色谱图。Standards为标准品;Emptyvector为空载体对照组。
图4为麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)的二级质谱图。
图5为Os07g0503900突变体核苷酸(A)及氨基酸(B)序列。
图6为Os07g0503900过表达材料鉴定。
图7为突变体及过表达材料中麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)含量。WT为野生型ZH11材料,4-1、4-2、4-3、4-4为Os07g0503900突变体材料,O-1、O-2、O-3、O-4为Os07g0503900过表达材料。
图8为tricin-5-O-glucopyranoside能够抑制稗草或者生菜根的生长。图中,**表示在P<0.01水平上差异极显著。
图9为突变体、过表达及野生型叶片水洗物外施稗草,图中**表示P<0.01水平上差异极显著。
具体实施方式
下述实施例中所使用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材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、化合物纯化
(1)收集播种后约60天的水稻(籼稻)新鲜叶片,用冷冻干燥机干燥后研磨为粉末状。加入8倍体积75%的乙醇,超声提取3次,每次20min。
(2)过滤提取液,并用旋转蒸发仪浓缩至无醇味,依次加入等体积的石油醚、二氯甲烷、正丁醇分别萃取3次。
(3)将获得的正丁醇相用旋转蒸发仪浓缩后利用中低压快速制备液相色谱仪(Biotage IsoleraTM Prime)采用湿法上样进行粗分。硅胶柱填料为YMC*GEL C18球形填料,填料粒径为50μm,孔径为12nm。本实施例中所用填料重量为100g。流动相为水(含0.1%的甲酸,%表示体积百分含量)、甲醇。流动相比例为甲醇10%-90%10柱体积,甲醇90%-100%2柱体积。流速:50mL/min,检测波长为210nm和254nm。通过初分,共获得10个组分,编号1-10。
(4)用UPLC-MS(Agilent 1290UPLC-6540Q-TOF)检测各个组分中麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)的含量。将(3)中获得的组分各取1mL于1.5mL的Eppendorf离心管中,用离心浓缩仪器进行浓缩后加入200μL甲醇并涡旋溶解。4℃,12,000rpm离心10min。再吸取上清100μL于含有容量为200μL衬管的Agilent进样瓶中。然后用UPLC-MS进行检测。流动相A相:0.1%甲酸水溶液(%表示体积百分含量);B相:乙腈。洗脱梯度:0-2min:5%B-10%B,2-12min:10%B-25%B,12-18min:25%B-70%B,18-23min:70%B-90%B,23-25min:90%B-100%B,25-30min:100%B,后运行5min,%均表示体积百分含量。流速0.3mL/min,柱温:40℃,进样量:5μL。采用电喷雾离子源(ESI),正离子模式检测,载气为高纯氮气,压力40psi,温度325℃。通过检测发现,组分2-7中含有麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)。
(5)合并含有目的化合物麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)的组分并用旋转蒸发仪浓缩。然后利用HPLC半制备液相色谱(Agilent1260)制备麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)。具体制备过程为:吸取(4)中获得的浓缩组分0.2mL于Agilent进样瓶中,并加入0.8mL的70%甲醇溶液(%均表示体积百分含量)稀释。充分混匀后利用HPLC半制备色谱进行样品制备。色谱柱:AgilentEclipse XDB-C18,规格9.4(内径)×250mm(长度),填料孔径5μm;;柱温:40℃;流速:1.5mL/min;检测波长:210nm,254nm,330nm;流动相:A(H2O+0.1%甲酸,%表示体积百分含量)、B(甲醇);流动相等梯度洗脱程序:A:35%,B:65%,运行60min。将保留时间RT=56.8min的组分接出并保存于-20℃。待所有样品制备完后,将所有的接出组分用离心浓缩仪进行逐步合并,然后使样品完全干燥,获得tricin-5-O-glucopyranoside单品。
(6)将得到的tricin-5-O-glucopyranoside化合物单品利用1mL的CD3OD溶液溶解后,利用核磁共振技术(NMR)(BRUKER 800MHz NMR)进行结构鉴定。tricin-5-O-glucopyranoside的核磁结果见表1和图1。
表1 化合物tricin-5-O-glucopyranoside的核磁结果
实施例2、Os07g0503900基因克隆
(1)提取粳稻中花11(Oryza sativa L.ssp.japonica cv.Zhonghua11,ZH11)幼苗期叶片的基因组DNA。
(2)根据水稻注释网站(Rice Genome Annotation Project)已有的Os07g0503900序列信息,用含有BamHI和HindШ的引物F1/R1以ZH11基因组DNA为模板扩增含有酶切位点的Os07g0503900基因CDS序列。
引物F1:5’-ggatccATGGCTCCAGCGATGGCGAG-3’;
引物R1:5’-aagcttCTATATGGATGACATGTGGGC-3’。
(3)将阳性克隆利用引物F2/R2进行PCR鉴定,扩增片段大小为310bp为阳性克隆。引物如下:
引物F2:5’-AGGACTTCATCTCCCGGTTCATGC-3’;
引物R2:5’-TTCTTCATCACAGGCGTCGGCAACA-3’。
(4)将阳性克隆委托公司进行测序,并将获得的序列与水稻注释网站序列信息一致的阳性克隆提取质粒,重组质粒命名为pEASY-T3-Os07g0503900。
重组载体pEASY-T3-Os07g0503900的结构描述为:将pEASY-T3载体的酶切位点BamHI和HindШ之间的小片段替换为SEQ ID No.2所示DNA片段后的重组质粒。SEQ ID No.2为Os07g0503900基因的CDS序列,编码SEQ ID No.1所示蛋白质。
实施例3、Os07g0503900基因的应用
一、原核表达载体构建
(1)用限制性内切酶BamHI和HindШ将实施例2得到的重组载体pEASY-T3-Os07g0503900完全酶切,同时酶切表达载体pMAL-c2X(华越洋,VECT-570)。酶切体系为:5μg质粒、2.5μL 10×酶切缓冲液、2μL BamHI、2μL HindШ,加ddH2O补充反应体系至50μL。酶切反应条件为:37℃酶切4小时。
(2)用琼脂糖电泳对酶切产物进行分离,回收大小约为1.5Kb包含Os07g0503900的片段,以及6.6Kb左右的pMAL-c2X载体片段,分别溶解于30μL ddH2O中。
(3)将步骤(2)获得的基因片段与载体骨架片段进行连接反应。连接反应体系为:1μL 10×连接酶缓冲液、0.5μL T4 DNA连接酶、1μL pMAL-c2X载体片段、3μL基因片段,加ddH2O补充反应体系至10μL。连接反应条件为:4℃连接12小时。
(4)将连接反应的产物转化大肠杆菌DH5α感受态细胞,用含有Ampicillin的LB平板(Ampicillin浓度为100μg/mL)进行筛选。
(5)将阳性克隆进行PCR鉴定(引物F2/R2)。Os07g0503900阳性克隆的扩增片段为310bp。
引物F2:5’-AGGACTTCATCTCCCGGTTCATGC-3’
引物R2:5’-TTCTTCATCACAGGCGTCGGCAACA-3’。
(6)将获得的阳性克隆委托公司测序,并提取质粒。该质粒为将pMAL-c2X载体BamHI和HindШ两个酶切位点之间的序列替换为Os07g0503900基因(SEQ ID No.2)得到的载体,分别命名为pMAL-Os07g0503900,为重组原核表达载体。
二、Os07g0503900原核表达
(1)将获得的重组原核表达载体pMAL-Os07g0503900转入大肠杆菌NovaBlue(华越洋,WR4478)感受态细胞中。同时转入pMAL-c2X空载体作为对照。用含有Ampicillin的LB平板筛选阳性克隆,然后用PCR进行阳性克隆鉴定引物为前文的F2/R2,扩增片段大小310bp。
(2)将阳性克隆接种于20mL LB液体培养基(含100μg/mL Ampicillin)中,37℃,220rpm振荡培养12-14h。
(3)按1:1000的比例(体积比)将(2)中的菌夜转接至400mL LB液体培养基(含100μg/mL Ampicillin)中,37℃,220rpm,培养至OD600=0.6-0.8。
(4)将菌夜取出置于冰上制冷后加入160μL浓度为500mM的IPTG,使IPTG的终浓度为0.2mM。
(5)16℃,100rpm振荡培养24h,以诱导蛋白表达。
(6)4℃,10,000rpm离心10min收集菌体。然后用柱缓冲液重悬菌体,置于-20℃冷冻过夜。柱缓冲液(1L)配方如下:NaCl 11.7g;DTT 154mg;0.5M EDTA 2mL;1M Tris-HCl(pH=7.4)40mL;余量为水。
(7)次日待样品融化后,用超声破碎仪破碎细胞,然后10,000rpm离心10min。
(8)利用直链淀粉柱纯化目的蛋白。具体纯化过程如下:
a、利用柱缓冲液(配方见上)活化亲和柱填料(流速为1mL/min)。
b、将(7)中获得的粗蛋白上清液缓慢加入已活化的直链淀粉柱中,将流速调至约0.5mL/min,以使目的蛋白与亲和柱填料充分结合。
c、待样品都流过填料后,加入柱缓冲液冲洗多次,以去除未结合的杂蛋白。
d、加入15mL含有麦芽糖的柱缓冲液(1L柱缓冲液中含有3.6g麦芽糖)洗脱目的蛋白,重复该步骤一次。
e、将洗脱液分次加入超滤管中,每次4℃,5000rpm离心15min。
f、加入1mL 100mM Tris-HCl4℃,5000rpm离心15min,倒掉废液。将目的蛋白溶液转入1.5mL Eppendorf离心管中。
g、吸取获得的目的蛋白溶液2μL,加入1mL Bradford(Quick StartTM Bradford 1×Dye Reagent,BioRAD,CAS 67-56-1),利用分光光度计测量待测蛋在595nm(OD595)处的吸收峰并将目的蛋白的吸收值换算为蛋白浓度,得出纯化获得的Os07g0503900重组蛋白的浓度为3.77μg/μL。
h、经SDS-PAGE电泳后,用考马斯亮蓝染色确认重组蛋白Os07g0503900大小约为110KD(图2)。
三、Os07g0503900的体外酶活
(1)酶活反应体系为:Tris-HCl(pH 7.0,50mM,含有10mM DTT)38μL,10mM糖基受体(Os07g0503900的糖基受体为tricin)1μL,100mM糖基供体(UDPG)1μL,步骤二获得的Os07g0503900重组蛋白10μL(Os07g0503900蛋白浓度为3.77μg/μL)。30℃水浴反应1h,然后用等体积甲醇中止反应。以加入从转入pMAL-c2X空载体的大肠杆菌中按照上述步骤二操作所得蛋白的样品为对照。
(2)酶活产物检测:将(1)中样品4℃,12,000rpm离心10min离心,取30μL用UPLC-MS(Agilent 1290UPLC-6540Q-TOF)进行检测。流动相A相:0.1%甲酸水溶液,%表示体积百分含量;B相:乙腈。洗脱梯度:0-2min:5%B-10%B,2-12min:10%B-25%B,12-18min:25%B-70%B,18-23min:70%B-90%B,23-25min:90%B-100%B,25-30min:100%B,后运行5min,%均表示体积百分含量。流速0.3mL/min,柱温:40℃,进样量:5μL。采用电喷雾离子源(ESI),正离子模式检测,载气为高纯氮气,压力40psi,温度325℃。
检测结果表明,与空载体相比,加入Os07g0503900重组蛋白的反应体系中出现一个新的色谱峰,说明在体外Os07g0503900能催化tricin发生糖基化,糖基化的产物为tricin-5-O-glucopyranoside(如图3、图4所示)。
实施例4、Os07g0503900在水稻中的作用
一、利用CRISPR/Cas技术创制Os07g0503900水稻突变体
利用CRISPR/Cas载体构建试剂盒(BIOGLE,Cat#BGK03)将guide RNA(gRNA)靶点序列,通过一步反应连入CRISPR/Cas质粒中,并将其用于植物转化。具体流程如下:
(1)利用CRISPR-P网站(http://crispr.hzau.edu.cn/CRISPR2/)设计gRNA靶点序列(Oligo),该靶点序列后序列特征序列为NGG序列(即PAM序列),靶点序列长度为20bp。
Os07g0503900的靶序列1:
5'-ATGCTGCGCAGCCGCTGCTG-3'。
对应Os07g0503900靶序列1的Oligo1序列如下:
Oligo1-R:5'-AAACCCGCAGCAGCGGCTGCGCAGCATCA-3'。
Os07g0503900的靶序列2:
5'-CGGTGAGCGACATGGCGGGG-3'。
对应Os07g0503900靶序列2的Oligo2序列如下:
Oligo2-R:5'-AAACCCGCCCCGCCATGTCGCTCACCGCA-3'。
其中,下划线部分为载体酶切后载体特异性识别序列,方框中碱基为PAM序列。
(2)制备Oligo二聚体。将引物溶解为10μM,按照以下体系制备Oligo二聚体:Buffer Aneal 18μL,Oligo-F 1μL,Oligo-R 1μL。混匀后在PCR仪中95℃加热3min,然后降温至20℃。
(3)将Oligo二聚体与CRISPR/Cas载体连接。连接体系为:CRISPR/Cas载体2μL,Oligo二聚体1μL,Enzyme mix 1μL,加ddH2O补充反应体系至10μL。混匀后室温反应1h。
(4)将连接产物转化大肠杆菌DH5α感受态细胞,用含有Kanamycin的LB平板(Kanamycin浓度为50μg/mL)进行筛选。
(5)将阳性克隆进行PCR鉴定并测序。测序引物为:5'-CCCAGTCACGACGTTGTAAA-3'。
(6)将阳性克隆质粒转化农杆菌EHA105感受态细胞,用含有Rifampin和Kanamycin抗性的YEB平板(100mg/L Rif,100mg/L Kan)进行筛选。挑取单菌落,进行菌液PCR鉴定,引物F3/R3,扩增片段大小481bp。。
引物F3:5'-CGAGAGCCTGACCTATTGCAT-3';
引物R3:5'-CTGCTCCATACAAGCCAACCAC-3'。
(7)将阳性克隆菌液按1:100比例(体积比)接种于50mL YEB液体培养基(含100mg/L Rif,100mg/L Kan)中,28℃,220rpm振荡培养至OD600=0.5。
(8)4,000rpm离心10min集菌,并用等体积的AAM-AS培养基重悬菌体。然后侵染水稻ZH11愈伤组织,待长出转基因苗后,用潮霉素(Hyg)抗性基因引物筛选阳性植株。物F3/R3(序列见上),扩增片段大小481bp。
随机选取四个阳性的突变体材料分别记作4-1、4-2、4-3和4-4。其中,4-1、4-2为导入Os07g0503900的Oligo1的阳性突变体,与野生型序列相比,这两个突变体在114bp位置处插入碱基G/T,导致蛋白翻译提前终止;4-3和4-4为导入Os07g0503900的Oligo2的阳性突变体,与野生型序列相比,这两个突变体在第141bp位置插入碱基T/A,导致蛋白翻译提前终止(图5)。
二、创制Os07g0503900基因过表达水稻
(1)以SEQ ID No.4为模板,利用引物F4/R4扩增含有用含有BamHI和KpnI酶切位点的序列,扩增片段大小约为2.3Kb。
引物F4:5'-ggatccGTTCATGTCTGAGGGGTGATTTC-3';
引物R4:5'-ggtaccCTATATGGATGACATGTGGGCCATTTC-3'。
(3)将阳性克隆利用引物F5/R5进行PCR鉴定,扩增片段大小为310bp为阳性克隆。
引物F5:5’-AGGACTTCATGTCCCGGTTCATGC;
引物R5:5’-TTCTTCATCACAGGCGTCGGCAACA-3’。
(4)将阳性克隆委托公司进行测序,并将获得的序列与SEQ ID No.4序列信息一致的阳性克隆提取质粒,重组质粒命名为SEQ4-T3。
重组载体SEQ4-T3的结构描述为:将pEASY-T3载体的酶切位点BamHI和KpnI之间的小片段替换为SEQ ID No.4所示DNA片段后的重组质粒。SEQ ID No.4编码SEQ ID No.3所示蛋白质。
(5)用限制性内切酶BamHI和KpnI将(4)中得到的重组载体SEQ4-T3完全酶切,同时酶切表达载体pCAMBIA1301(华越洋,VECT0080)。酶切体系为:5μg质粒、2.5μL 10×酶切缓冲液、2μL BamHI、2μL KpnI,加ddH2O补充反应体系至50μL。酶切反应条件为:37℃酶切4小时。
(6)用琼脂糖电泳对酶切产物进行分离,回收大小约为2.3Kb包含SEQ ID No.4的片段,以及约12Kb左右的pCAMBIA1301载体片段,分别溶解于30μL ddH2O中。
(7)将步骤(6)获得的基因片段分别与载体骨架片段进行连接反应。连接反应体系为:1μL 10×连接酶缓冲液、0.5μL T4 DNA连接酶、1μL pCAMBIA1301载体片段、3μL基因片段,加ddH2O补充反应体系至10μL。连接反应条件为:4℃连接12小时。
(8)将连接反应的产物转化大肠杆菌DH5α感受态细胞,用含有Kanamycin的LB平板(Kanamycin浓度为50μg/mL)进行筛选。
(9)将阳性克隆进行PCR鉴定(引物F5/R5),扩增片段大小为310bp为阳性克隆。
(10)将获得的阳性克隆委托公司测序,并提取质粒。该质粒为将pCAMBIA1301载体BamHI和KpnI两个酶切位点之间的序列替换为SEQ ID No.4得到的载体,命名为pCAM-SEQ4,为重组原核表达载体。
(11)将阳性克隆菌液按1:100比例(体积比)接种于50mL YEB液体培养基(含100mg/L Rif,100mg/L Kan)中,28℃,220rpm振荡培养至OD600=0.5。
(12)4,000rpm离心10min集菌,并用等体积的AAM-AS培养基重悬菌体。然后侵染水稻ZH11愈伤组织,待长出转基因苗后,用GUS染色方法筛选阳性植株。具体为:用镊子将水稻叶片转移到含GUS染液的2ml Eppendorf管中,抽真空使材料完全下沉,37℃条件下染色过夜。然后将材料转入70%乙醇中脱色,至阴性对照材料为白色时即可。最后在显微镜下观察各个材料的染色情况并拍照记录,如图6中阳性过表达材料O-1、O-2、O-3和O-4。
其中GUS染液(100mL)的配置方法如下:Triton X-100 100μL;0.5M EDTA 2mL;0.5M亚铁氰化钾[K3Fe(CN)]400μL;0.5M铁氰化钾[K4Fe(CN)]400μL;X-Gluc(1mg/mL)1mL,然后用0.1M磷酸缓冲液PBS(pH 7.0)定容至100mL。
三、Os07g0503900相关遗传材料中的化合物检测
(1)取材:分别取转基因水稻过表达材料(4-1、4-2、4-3和4-4,O-1、O-2、O-3、O-4)和野生型水稻四叶期叶片,每个遗传材料各含四个生物学重复。取样后立即置于液氮中冷冻,然后用低温真空冷冻机干燥。待样品完全干燥后准确称取20mg于2.0mL Eppendorf管中。加入一粒不锈钢钢珠,用MM400球磨仪(Roach,Germany)振动频率为20Hz,打磨10min。
(2)代谢物提取。将样品用球磨仪打碎后,加入1mL预存于-20℃的提取液(甲醇)及5μL伞形酮内酯(2mg/mL)(内标),37℃220rpm振荡提取2h。然后将提取液12,000rpm高速离心10min。吸取上清500μL于Agilent进样瓶中,然后用UPLC-MS(Agilent 1290UPLC-6540Q-TOF)进行检测。流动相A相:0.1%甲酸水溶液,%表示体积百分含量;B相:乙腈。洗脱梯度:0-2min:5%B-10%B,2-12min:10%B-25%B,12-18min:25%B-70%B,18-23min:70%B-90%B,23-25min:90%B-100%B,25-30min:100%B,后运行5min,%均表示体积百分含量。流速0.3mL/min,柱温:40℃,进样量:5μL。采用电喷雾离子源(ESI),正离子模式检测,载气为高纯氮气,压力40psi,温度325℃。
(3)Os07g0503900相关遗传材料中化合物含量分析
与野生型相比,在Os07g0503900突变体材料中麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)的含量显著降低,而在该基因过表达材料中麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)的含量显著升高(图7),表明Os07g0503900可以催化麦黄酮(tricin)生成麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside),该结果与体外生化结果一致。在突变体中,Os07g0503900基因序列中有碱基插入,导致蛋白翻译提前终止(图5),Os07g0503900酶功能丧失,最终使得Os07g0503900不能催化麦黄酮(tricin)生成麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside),因此在突变体中麦黄酮-5-O-葡萄糖糖苷(tricin-5-O-glucopyranoside)的含量很低
实施例5、化合物tricin-5-O-glucopyranoside外施生菜和稗草
一、化合物tricin-5-O-glucopyranoside外施生菜和稗草实验流程
外施实验选用生菜和稗草的原因是:生菜是文献中报道的做化感实验外施的常用指示作物,而稗草是水稻田中主要的杂草。
(1)将化合物tricin-5-O-glucopyranoside用离心浓缩仪干燥至粉末状并称重,加入适量无菌水,使其终浓度为10mg/mL。
(2)配制0.8%琼脂500mL,并将其置于高压灭菌锅中灭菌。
(3)将灭好菌的琼脂置于超净台冷却至室温(注意:不要使培养基凝固),然后准确吸取4.75mL培养基于已灭菌的10mL离心管中。加入250μL 10mg/mL化合物溶液,使化合物的终浓度为500mg/L。将含有化合物的培养基快速倒于10cm×10cm的方形皿中,迅速摇晃方形皿,使培养基均匀的平铺于方形皿中。以在4.75mL培养基中加入250μL无菌水的培养基作为对照。
(4)将稗草和生菜种子分别用蒸馏水洗数次,然后置于无菌滤纸上干燥。
(5)待培养基凝固后,用无菌镊子放入生菜和稗草种子,每个培养皿中放入26粒种子(分为两行,每行13粒种子)。每个处理包含3个生物学重复。
(6)将放有稗草和生菜的方形皿置于28℃培养箱中黑暗培养3天。
(7)表型统计:3天后统计每个处理材料的根长。
实验同时设置了以tricin替代tricin-5-O-glucopyranoside的对照。
二化合物外施稗草和生菜表型
tricin为文献中报道的水稻中具有化感作用的主要黄酮类物质,但在本实施案例中发现,在外施浓度为500mg/L时,与对照相比,tricin对于稗草和生菜的生长的抑制作用并不明显。但是与对照和tricin相比,tricin-5-O-glucopyranoside具有极显著的抑制生菜和稗草生长的能力(图8)。
实施例6、Os07g0503900基因敲除水稻突变体叶片水洗物外施稗草
一、突变体及野生型材料叶片收集
将Os07g0503900基因敲除水稻突变体、过表达及野生型ZH11材料种植于水稻生长池,待种植约60天后,收取突变体及野生型材料的叶片。
二、突变体及野生型材料叶片水洗物提取
(1)分别取突变体4-1、4-2、4-3,过表达材料O-1、O-2、O-3(见实施例4)及野生型ZH11叶片12g。
(2)将称好的叶片置于250mL锥形瓶中,加入200mL蒸馏水,用封口膜封好瓶口。将材料置于37℃摇床220rpm震摇提取4h后,将材料取出并置于室温静置约20h(注:叶片水洗物提取总时间约为24h)。
(3)将每个材料的叶片水洗物分装于4个50.0mL无菌离心管带盖中,然后置于-20℃冷冻保存。
(4)取出于-20℃冷冻保存的水洗物,并置于冷冻干燥机干燥,得到固态水洗物。
(5)待水洗物将冻干后,将每个材料对应的4个离心管中的水洗物用适量蒸馏水润洗,然后合并各个管中的水洗物,并将每个材料的水洗物定容至12.0mL,使每12mL的所述叶片水洗物溶液中含有从12g叶片中提取得到的固态水洗物。然后12,000rpm离心10min,转移上清至新的10mL离心管中。然后置于-20℃冰箱备用。
三、叶片水洗物外施稗草流程
将上述获得的水洗物外施稗草,具体如下:
(1)配制0.8%琼脂500mL,并将其置于灭菌锅中高压灭菌。
(2)将灭好菌的琼脂置于超净台冷却至室温后准确吸取5.00mL培养基于已灭菌的10mL离心管中。加入1mL已制备的叶片水洗物溶液,充分颠倒混匀并快速倒于10cm×10cm的方形皿中,迅速摇晃方形皿,使培养基均匀的平铺于方形皿中。以在5mL培养基中加入1mL无菌水的培养基作为对照。
(3)待培养基凝固后,用无菌镊子放入稗草种子,每个培养皿中放入26粒种子(分为两行,每行13粒种子)。每个处理包含3个生物学重复。
(4)将放有稗草的方形皿置于28℃培养箱中黑暗培养3天。
(5)表型统计:3天后统计每个处理材料的根长。
四叶片水洗物外施稗草表型
如图9所示,与野生型相比,用突变体的叶片水洗物外施时稗草的根显著增长,而在过表达材料的叶片水洗物外施时稗草根显著变短。这是由于突变体中tricin-5-O-glucopyranoside的含量降低,而过表达材料中tricin-5-O-glucopyranoside的含量高(图7),所以突变体叶片水洗物外施稗草时会引起稗草的根变长,而在过表达材料叶片水洗物外施稗草时会引起稗草的根变短。说明tricin-5-O-glucopyranoside在抑制稗草生长,尤其是抑制稗草根的生长方面发挥着重要作用。
<110> 中国科学院植物研究所
<120> Os07g0503900蛋白在调控植物对杂草抗性中的应用
<130> GNCLN200664
<160> 4
<170> PatentIn version 3.5
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Met Ala Pro Ala Met Ala Ser Ser Ala Ala Thr Val Val Leu Ile Pro
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Phe Cys Val Ser Gly His Leu Thr Pro Met Leu Glu Val Gly Lys Arg
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Met Leu Arg Ser Arg Cys Cys Gly Asp Asp Asp Asp Gly Arg Pro Ala
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Met Ser Leu Thr Val Leu Leu Ala Gln Leu Pro Glu Ser His Arg Ala
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Pro Glu Ile Asp Glu Ile Ile Arg Arg Glu Ala Ala Gly Ala Ser Glu
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His Ser Gly Phe Asp Val Arg Phe His Cys Leu Pro Ala Glu Glu Leu
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Pro Asp Phe Arg Gly Gly Glu Asp Phe Ile Ser Arg Phe Met Gln Gln
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His Ala Ser His Ala Arg Glu Ala Ile Ala Gly Leu Glu Ser Arg Val
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Ala Ala Val Val Leu Asp Trp Phe Cys Thr Thr Leu Leu Asp Val Thr
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Arg Asp Leu Gly Leu Pro Gly Tyr Val Phe Phe Thr Ser Ala Ala Ser
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Val Asp Phe Glu Glu Met Gly Gly Ala Val Asp Leu Pro Gly Leu Pro
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Pro Val Pro Ala Ala Leu Leu Pro Thr Pro Val Met Lys Lys Gly Cys
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Asn Tyr Glu Trp Leu Val Tyr His Gly Ser Arg Phe Met Glu Ala Ala
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Gly Ile Ile Val Asn Thr Val Ala Glu Leu Glu Pro Ala Val Leu Glu
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Ala Ile Ala Asp Gly Arg Cys Val Pro Gly Arg Arg Val Pro Ala Ile
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Tyr Thr Val Gly Pro Val Leu Ser Phe Lys Thr Pro Pro Glu Lys Pro
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His Glu Cys Val Arg Trp Leu Asp Ala Gln Pro Arg Ala Ser Val Val
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Phe Leu Cys Phe Gly Ser Met Gly Ser Phe Ala Pro Pro Gln Val Leu
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atggctccag cgatggcgag ctcagcagcg acggtggtgc tgatcccgtt ctgcgtctcc 60
ggccacctca cgcccatgct ggaagtcggc aagcggatgc tgcgcagccg ctgctgcggc 120
gacgacgacg acggccgccc cgccatgtcg ctcaccgtgc tcctcgcgca gctgccggag 180
tcccaccgcg cgcccgagat cgacgagatc atccgccgtg aagcggccgg cgcgtcggag 240
cactccggct tcgacgtccg gttccactgc ctccccgccg aggagctccc ggacttccgc 300
ggcggcgagg acttcatctc ccggttcatg cagcagcacg cgtcgcacgc cagggaggcc 360
atcgccggcc tcgagtcccg cgtcgccgcc gtggtcttgg actggttctg caccacgctc 420
ctcgacgtca cccgcgacct cggcctcccc gggtacgtgt tcttcacgtc cgccgcctcc 480
atgctctcgc tcctgctgcg gttgccggcg ctggacaagg aggtggccgt ggatttcgag 540
gagatgggag gcgccgtcga cttaccgggg ttgccgcctg tgccggcggc tctgttgccg 600
acgcctgtga tgaagaaggg ttgcaactac gagtggctcg tgtaccacgg gagccgcttc 660
atggaggctg cggggatcat cgtcaacacg gtggccgagc tcgagccggc cgtcctcgag 720
gccatcgccg acggccggtg cgtgccggga cgccgcgtcc cggccatcta cacggtcggc 780
cccgtgctgt cgttcaagac gccgcccgag aagccgcacg agtgcgtgcg gtggctcgac 840
gcgcagccgc gagcgtcggt cgtgttcctc tgcttcggga gcatgggcag cttcgcgccg 900
ccgcaggtgc tcgagatagc cgccggcctc gagcgcagcg ggcaccgctt cctgtgggtg 960
ctgcgcggcc gtccacccgc cggctcgccg tacccgacgg acgccgacgc cgacgagctc 1020
ctcccggagg ggttcctgga gaggaccaag gggaggggca tggtgtggcc gacgtgggcg 1080
ccgcagaagg acatcctcgc ccacgccgcc gtgggaggct tcgtgacgca cggcgggtgg 1140
aactcgacgc tggagagcct gtggcacggc gtgccgatgg cgccgtggcc gctgtacgcg 1200
gagcagcacc tgaacgcgtt cgagctcgtg cgcgacatgg gcgtcgccgt ggagatggag 1260
gtggacagga agcggggcaa cttggtggag gcggcggagc tggagcgcgc ggtgcggtgc 1320
ctgatggacg agggatcgga ggaggggagg atggcgaggg agaaggcggc ggcggcgaag 1380
gcggcgtgcc ggaacgccgt ggacggaggc gggtcgtcga tagcggcgtt gcggaagctc 1440
acgcaagaaa tggcccacat gtcatccata tag 1473
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Met Ala Ser Ala Met Ala Ser Ser Ala Ala Thr Val Val Leu Ile Pro
1 5 10 15
Phe Cys Val Ser Gly His Leu Thr Pro Met Leu Glu Val Gly Lys Arg
20 25 30
Met Leu Arg Ser Arg Cys Cys Gly Asp Asp Asp Asp Gly Arg Pro Ala
35 40 45
Met Ser Leu Thr Val Leu Leu Ala Gln Leu Pro Glu Ser His Arg Ala
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Pro Glu Ile Asp Glu Ile Ile Arg Arg Glu Ala Ala Gly Ala Ser Glu
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His Ser Gly Phe Asp Val Arg Phe His Cys Leu Pro Ala Glu Glu Leu
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Pro Asp Phe Arg Gly Gly Glu Asp Phe Met Ser Arg Phe Met Gln Gln
100 105 110
His Ala Ser His Ala Arg Glu Ala Ile Ala Gly Leu Glu Ser Arg Val
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Ala Ala Val Val Leu Asp Trp Phe Cys Thr Thr Leu Leu Asp Val Thr
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Arg Asp Leu Gly Leu Pro Gly Tyr Val Phe Phe Thr Ser Ala Ala Ser
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Met Leu Ser Leu Leu Leu Arg Leu Pro Ala Leu Asp Lys Glu Val Ala
165 170 175
Val Asp Phe Glu Glu Met Gly Gly Ala Val Asp Leu Pro Gly Leu Pro
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Pro Val Pro Ala Ala Leu Leu Pro Thr Pro Val Met Lys Lys Gly Cys
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Asn Tyr Glu Trp Leu Val Tyr His Gly Ser Arg Phe Met Glu Ala Ala
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Gly Ile Ile Val Asn Thr Val Ala Glu Leu Glu Pro Ala Val Leu Glu
225 230 235 240
Ala Ile Ala Asp Gly Arg Cys Val Pro Gly Arg Arg Val Pro Ala Ile
245 250 255
Tyr Thr Val Gly Pro Val Leu Ser Phe Lys Thr Pro Pro Glu Lys Pro
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His Glu Cys Val Arg Trp Leu Asp Ala Gln Pro Arg Ala Ser Val Val
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Phe Leu Cys Phe Gly Ser Met Gly Ser Phe Ala Pro Pro Gln Val Leu
290 295 300
Glu Ile Ala Ala Gly Leu Glu Arg Ser Gly His Arg Phe Leu Trp Val
305 310 315 320
Leu Arg Gly Gln Pro Ala Ala Gly Met Pro Tyr Pro Thr Asp Ala Val
325 330 335
Val Asp Glu Leu Leu Pro Glu Gly Phe Leu Glu Arg Thr Lys Glu Lys
340 345 350
Gly Leu Val Trp Ser Lys Trp Ala Pro Gln Lys Glu Ile Leu Ala His
355 360 365
Pro Ala Val Gly Gly Phe Ala Thr His Cys Gly Trp Asn Ser Thr Leu
370 375 380
Glu Ser Leu Trp Asn Gly Val Pro Leu Leu Pro Trp Pro Leu Tyr Ala
385 390 395 400
Glu Gln His Leu Asn Ala Phe Glu Leu Val Arg Asp Met Gly Val Ala
405 410 415
Val Glu Met Glu Val Asp Arg Lys Arg Gly Asn Leu Val Glu Ala Ala
420 425 430
Glu Leu Glu Arg Ala Val Arg Cys Leu Met Asp Glu Gly Ser Glu Gly
435 440 445
Arg Met Ala Arg Glu Lys Ala Ala Ala Ala Lys Ala Ala Cys Arg Asn
450 455 460
Ala Val Asp Gly Gly Gly Ser Ser Ile Ala Ala Leu Arg Lys Leu Thr
465 470 475 480
Gln Glu Met Ala His Met Ser Ser Ile
485
<210> 4
<211> 2237
<212> DNA
<213> Artificial sequence
<400> 4
gttcatgtct gaggggtgat ttctgtagat ttagaccatt aaatttaata gaatagcttt 60
ggcgtacatt atggttattt tttatcgacg agtaggccta cttgttctga ataaacatga 120
cgccatgtgc ggcgtatttc tttttttttt ttcaaagcaa cccgggaaat ctggacagtc 180
agcgggaaaa acaagggtag tcagcatcca ttgtgacgtg gtccattgac tgcattcatg 240
ttacccctcc cctctggcac ccgatgtgca cgccaaaatg acaagcctcc gcagggtctg 300
tttagtttcc aaataaaaat tttccacaat gtcacactag atgtttggtt atatatatat 360
atatatatat atatatatat atatatatat atatatatat gtgtgtgtgt gtgtaaaata 420
ttaaatatat aaaaaaacta attatatata ttacatgtaa attacgagat aaatctttta 480
agctcaatta ctccataatt tgataattta ttaatgacgg attaattaga cttaataaat 540
tcttctcgca gttcttagac gtaatatgta atttatttta ttattaattt atatttaata 600
tatatttaat actttaaata tgtatctata tccgatgtta catttcaaaa attttcgttt 660
gcgaactaaa aaggccctca gtcctcagct tcagtgcctc acgcttgcaa caatatcgtc 720
ctccctgtcc gtgtactgtg aatctgccat ctgtgacttc gtgagacatg gcttcagcga 780
tggcgagctc agcagcgacg gtcgtgctga tcccgttctg cgtctccggc cacctcacgc 840
ccatgctgga agtcggcaag cggatgctgc gcagccgctg ctgcggcgac gacgacgacg 900
gccgccccgc catgtcgctc accgtgctcc tcgcgcagct gccggagtcc caccgcgcgc 960
ccgagatcga cgagatcatc cgccgtgaag cggccggcgc gtcggagcac tccggcttcg 1020
acgtccggtt ccactgcctc cccgccgagg agctcccgga cttccgcggc ggcgaggact 1080
tcatgtcccg gttcatgcag cagcacgcgt cgcacgccag ggaggccatc gccggcctcg 1140
agtcccgcgt cgccgccgtg gtcttggact ggttctgcac cacgctcctc gacgtcaccc 1200
gcgacctcgg cctccccggg tacgtgttct tcacgtccgc cgcctccatg ctctcgctcc 1260
tgctgcggtt gccggcgctg gacaaggagg tggccgtgga tttcgaggag atgggaggcg 1320
ccgtcgactt accggggttg ccgccggtgc cggcggctct gttgccgacg cctgtgatga 1380
agaagggttg caactacgag tggctcgtgt accacgggag ccgcttcatg gaggctgcgg 1440
ggatcatcgt caacacggtg gccgagctcg agccggccgt cctcgaggcc atcgccgacg 1500
gccggtgcgt gccgggacgc cgcgtcccgg ccatctacac ggtcggcccc gtgctgtcgt 1560
tcaagacgcc gcccgagaag ccgcacgagt gcgtgcggtg gctcgacgcg cagccgcgag 1620
cgtcggtcgt gttcctctgc ttcgggagca tgggcagctt cgcgccgccg caggtgctcg 1680
agatagccgc cggcctcgag cgcagcggcc accgcttcct gtgggtactg cgcggccagc 1740
cagccgccgg catgccatac ccgacggacg ccgtcgtcga cgagctcctc cccgaggggt 1800
tcttggagag gaccaaggag aagggcctcg tgtggtccaa gtgggcgccg cagaaggaga 1860
tcctcgccca ccctgccgtc ggcggcttcg cgacgcactg cgggtggaac tcgacgctgg 1920
agagcctgtg gaacggcgtg ccgctactgc cgtggccgct gtacgcggag cagcacctga 1980
acgcgttcga gctcgtgcgc gacatgggcg tcgccgtgga gatggaggtg gacaggaagc 2040
ggggcaactt ggtggaggcg gcggagctgg agcgcgcggt gcggtgcctg atggacgagg 2100
gatcggaggg gaggatggcg agggagaagg cggcggcggc gaaggcggcg tgccggaacg 2160
ccgtggacgg aggcgggtcg tcgatagcgg cgttgcggaa gctcacgcaa gaaatggccc 2220
acatgtcatc catatag 2237
Claims (8)
1.Os07g0503900蛋白或其相关生物材料在如下任一中的应用:
(1)调控植物化感作用;
(2)调控植物对杂草抗性;
(3)抑制杂草生长;
所述植物为水稻;所述杂草为稗草或生菜;
所述相关生物材料为能够表达所述Os07g0503900蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系;
所述Os07g0503900蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
2.根据权利要求1所述的应用,其特征在于:能够表达所述Os07g0503900蛋白的核酸分子为SEQ ID No.2所示的DNA分子。
3.一种培育化感作用改变的植物品种的方法,为如下方法I或方法II:
方法I:一种培育化感作用增强的植物品种的方法,包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量提高,得到化感作用增强的植物品种;
方法II:一种培育化感作用减弱的植物品种的方法,包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量降低,得到化感作用减弱的植物品种;
所述Os07g0503900蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述植物为水稻。
4.一种培育对杂草抗性改变的植物品种的方法,为如下方法III或方法IV:
方法III:一种培育对杂草抗性增强的植物品种的方法,包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量提高,得到对杂草抗性增强的植物品种;
方法IV:一种培育对杂草抗性减弱的植物品种的方法,包括如下步骤:使受体植物中Os07g0503900蛋白的活性和/或含量降低,得到对杂草抗性减弱的植物品种;
所述Os07g0503900蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述植物为水稻;所述杂草为稗草或生菜。
5.一种培育化感作用改变的转基因植物的方法,为如下方法VII或方法VIII:
方法VII:一种培育化感作用增强的转基因植物的方法,包括如下步骤:向受体植物中导入能够表达Os07g0503900蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比化感作用增强;
方法VIII:一种培育化感作用减弱的转基因植物的方法,包括如下步骤:对受体植物中能够表达Os07g0503900蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比化感作用减弱;
所述Os07g0503900蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述植物为水稻。
6.一种培育对杂草抗性改变的转基因植物的方法,为如下方法IX或方法X:
方法IX:一种培育对杂草抗性增强的转基因植物的方法,包括如下步骤:向受体植物中导入能够表达Os07g0503900蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比对杂草抗性增强;
方法X:一种培育对杂草抗性减弱的转基因植物的方法,包括如下步骤:对受体植物中能够表达Os07g0503900蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比对杂草抗性减弱;
所述Os07g0503900蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述植物为水稻;所述杂草为稗草或生菜。
7.根据权利要求5或6所述的方法,其特征在于:能够表达所述Os07g0503900蛋白的核酸分子为SEQ ID No.2所示的DNA分子。
8.根据权利要求5或6所述的方法,其特征在于:对所述受体植物中能够表达所述Os07g0503900蛋白的核酸分子进行抑制表达是通过序列特异核酸酶对所述核酸分子进行特异性剪切,从而降低所述核酸分子在所述受体植物中的表达;
向所述受体植物中导入能够表达所述Os07g0503900蛋白的核酸分子是通过向所述受体植物中导入含有能够表达所述Os07g0503900蛋白的核酸分子的重组表达载体实现的。
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EP20924536.4A EP4119507A4 (en) | 2020-03-12 | 2020-08-10 | USE OF FLAVONOID GLYCOSIDE SUBSTANCE AND GLYCOSYL TRANSFERASE GENE FOR REGULATING THE RESISTANCE OF PLANTS TO WEEDS |
PCT/CN2020/108151 WO2021179530A1 (zh) | 2020-03-12 | 2020-08-10 | 一种黄酮苷类物质及其糖基转移酶基因在调控植物对杂草抗性中的应用 |
AU2020434699A AU2020434699A1 (en) | 2020-03-12 | 2020-08-10 | Use of flavonoid glycoside substance and glycosyltransferase gene therefor in regulating resistance of plant to weeds |
BR112022018201A BR112022018201A2 (pt) | 2020-03-12 | 2020-08-10 | Métodos para cultivar uma variedade de planta, uso, variedade de planta cultivada e produto |
CA3175043A CA3175043A1 (en) | 2020-03-12 | 2020-08-10 | Use of flavonoid glycoside substance and glycosyltransferase gene therefor in regulating resistance of plant to weeds |
JP2022554422A JP2023516479A (ja) | 2020-03-12 | 2020-08-10 | フラボノイド配糖体類物質とその糖転移酵素遺伝子の、植物の雑草に対する抵抗力を制御するための使用 |
US17/905,980 US20230203524A1 (en) | 2020-03-12 | 2020-08-10 | Use of flavonoid glycoside substance and glycosyltransferase gene for regulating resistance of plants to weeds |
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PREDICTED: Oryza sativa Japonica Group anthocyanidin 3-O-glucosyltransferase 2,(LOC4343325), mRNA,NCBI Reference Sequence: XM_015789990.2,1944bp mRNA linear;NCBI genbank;《NCBI genbank》;20180807;1-2 * |
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