CN111606977B - 一种利用GF14f蛋白靶向多肽配体调控水稻弱势籽粒灌浆的方法 - Google Patents
一种利用GF14f蛋白靶向多肽配体调控水稻弱势籽粒灌浆的方法 Download PDFInfo
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Abstract
本发明公开了一种利用GF14f蛋白靶向多肽配体溶液调控水稻弱势籽粒灌浆的方法。所述多肽配体溶液由60mg/L的14‑3‑3蛋白成员GF14f靶向多肽配体R18和6ml/L的表面活性剂吐温‑20组成。在水稻灌浆期外源喷施GF14f蛋白靶向多肽配体溶液，多肽配体R18能与弱势籽粒中的GF14f蛋白竞争性结合，从而对灌浆过程中淀粉合成代谢起到调控作用，能够显著提高弱势籽粒千粒重及结实率。
Description
技术领域
本发明属于作物生长调控技术领域，具体涉及一种利用GF14f蛋白靶向多肽配体调控水稻弱势籽粒灌浆的方法。
背景技术
水稻籽粒的充实及粒重大小与颖花在穗上着生的部位有密切关系。生长发育早、灌浆充实快且稳定，粒重高，称为强势籽粒；生长发育迟、灌浆充实差且易受环境所调控、粒重低，称为弱势籽粒。水稻强弱势粒的结实率和充实度差异很大，有的达 25% 左右。前人大量的研究结果和生产实践均证明，弱势籽粒充实差和粒重低不仅限制了水稻产量潜力的发挥，而且严重影响籽粒品质，弱势粒充实度差已成为现代水稻品种产量潜力进一步提高的关键限制因素。同时，这种灌浆差异也在其他禾谷类作物中也普遍存在，例如小麦，玉米。一直以来，籽粒灌浆差异现象都是禾谷类作物科学和生产上长期未解决的难题。
水稻籽粒灌浆是个复杂的生理生态过程，涉及大量基因（蛋白）的表达调控。申请人前期的研究已表明14-3-3蛋白家族成员GF14f蛋白在弱势籽粒中的表达量显著高于强势籽粒是导致弱势籽粒灌浆差的一个重要原因，该蛋白会通过蛋白互作机制，进而对弱势籽粒淀粉合成过程中关键酶起到调控作用，采用反向遗传学的方法特异性的降低籽粒灌浆期GF14f的表达量，能够显著提升籽粒灌浆过程中蔗糖合成酶（SuS），可溶性淀粉合成酶（SSS），ADPG焦磷酸化酶（AGPase）三个蔗糖转化及淀粉合成关键酶的活性，进而有利于弱势籽粒的灌浆。田间产量数据表明，GF14f-RNAi 植株与野生型相比，有效穗数及每穗粒数未有显著的差异，但籽粒的粒长增加了12.9%，千粒重提高约10%，弱势籽粒的结实率提高10.85%，产量提高 15.2%，证实 GF14f 在籽粒灌浆过程中发挥着重要的功能（zhang etal. The 14-3-3 protein GF14f negatively affects grain filling of inferiorspikelets of rice (Oryza sativa L.). The Plant Journal, 2019, 99, 344–358）。
近年来，随着生物技术的不断发展，多肽配体（peptide aptamers，PA)等人造小分子已成为直接影响蛋白质功能的有效技术。肽配体是在惰性支架蛋白中展示肽环的短（8-20个氨基酸）肽，对给定靶标具有高度特异性结合亲和力。肽配体可特异性结合并失活目标蛋白质，因此作为强竞争性抑制剂。它们可以特异性地阻断蛋白质-蛋白质相互作用的形成，并且在不改变蛋白质水平或结构的情况下干扰基因功能。多肽配体技术的本质就是在蛋白水平上特异抑制靶蛋白，从而使其在体内失去其生物学功能。利用多肽配体特异结合靶蛋白的方法，可以在蛋白水平上对所感兴趣的植物基因进行特异干扰，有助于精确地理解靶基因在复杂性状起源和形态建成中的生物学功能。通过筛选到的靶蛋白的多肽配体，并通过体外喷施调控，能够起到调节作物生长及抗逆的作用，这种策略已在作物抗病性上以及生长发育上已有广泛的运用，例如外源喷施番茄斑萎病毒TSWV的靶向多肽配体能显著增强番茄抗斑萎病的能力。
本发明针对14-3-3蛋白家族成员GF14f在水稻籽粒灌浆期的表达模式及其功能，筛选了一种能靶向与GF14f蛋白结合的多肽配体片段R18，通过灌浆期外源喷施于籽粒上，靶向作用于GF14f上，从而显著提高弱势籽粒千粒重及结实率，实现对弱势籽粒灌浆的靶向调控，国内外尚未见相关的研究报道。
发明内容
本发明提供了一种利用GF14f蛋白靶向多肽配体调控水稻弱势籽粒灌浆的方法，该多肽配体能够与籽粒中14-3-3蛋白成员GF14f蛋白靶向竞争性结合，使其与互作的蛋白不结合，从而起到调控水稻籽粒灌浆过程中淀粉合成代谢，达到提高弱势籽粒千粒重及结实率的方法。
一种调控水稻弱势籽粒灌浆的多肽配体溶液，其组分包括：60 mg/L的GF14f蛋白靶向多肽配体R18和6ml/L的表面活性剂。
上述GF14f蛋白靶向多肽配体R18，其氨基酸序列为PHCVPRDLSWLDLEANMCLP。
上述表面活性剂为吐温-20。
一种调控水稻弱势籽粒灌浆的方法：在水稻灌浆期外源喷施上述一种调控水稻弱势籽粒灌浆的多肽配体溶液。
优选的，在水稻灌浆期，即花后5~7天，外源喷施1~1.5L/亩上述一种调控水稻弱势籽粒灌浆的多肽配体溶液。
上述一种调控水稻弱势籽粒灌浆的多肽配体溶液在改良水稻粒重及结实率中的应用。
本发明具有以下有益的效果：
1、 本发明的14-3-3蛋白家族成员GF14f蛋白多肽配体R18是喷施剂的有效成分，成分简单，无污染。
2、 本发明的多肽配体能够靶向与籽粒中GF14f蛋白结合，通过竞争性互作与靶向蛋白GF14f结合，使其与互作的蛋白不结合，在不影响蛋白表达的情况下，对蛋白的功能产生调控作用。
附图说明：
图1 GF14f蛋白靶向多肽配体R18与GF14f的分子对接示意图。A：多肽配体R18与GF14f蛋白对接示意图；B：GF14f蛋白与多肽配体R18结合氨基酸位点示意图。
图2 GF14f蛋白靶向多肽配体R18与GF14f蛋白互作激光共聚焦荧光成像图。
图3 GF14f蛋白靶向多肽配体R18与GF14f蛋白及其淀粉合成相关互作蛋白的竞争性pull-down实验结果。（a）：GF14f蛋白靶向多肽配体R18与GF14f蛋白及蔗糖合成酶（SuS）的竞争性pull-down实验结果；（b）：GF14f蛋白靶向多肽配体R18与GF14f蛋白及可溶性淀粉合成酶（SSS）的竞争性pull-down实验结果；（c）GF14f蛋白靶向多肽配体R18与GF14f蛋白及ADPG焦磷酸化酶（AGPase）的竞争性pull-down实验结果。
图4 外源GF14f蛋白靶向多肽配体R18对水稻籽粒蔗糖合成酶（SuS）酶活的影响。
图5外源喷施GF14f蛋白靶向多肽配体R18对籽粒蔗糖合成酶（SuS）、可溶性淀粉合成酶（SSS）及ADPG焦磷酸化酶（AGPase）酶活性的影响。
图6 外源喷施GF14f蛋白靶向多肽配体R18对成熟期弱势籽粒结实率及千粒重的影响。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是下述的实例仅仅是本发明其中的例子而已，并不代表本发明所限定的权利保护范围，本发明的权利保护范围以权利要求书为准。
实施例1
已有的报道发现14-3-3蛋白成员GF14f能够与淀粉合成相关蛋白蔗糖合成酶（SuS），可溶性淀粉合成酶（SSS），ADPG焦磷酸化酶（AGPase）互作，进而抑制相关酶的活性。为了评价多肽配体R18，首先在ICM-DOCK环境下，以水稻GF14f蛋白为靶标，对已有报道的14-3-3蛋白多肽配体进行分子对接，如图1所示发现多肽配体R18与GF14f蛋白具有潜在的结合位点，分别位于GF14f蛋白序列中的R59 、R63、 N53、 E15 、N45、 D218 、和K217。之后，采用人工合成的方式获得纯度95%以上的R18肽段。在此基础上，采用BiFC技术，在体内证实多肽配体R18能够与GF14f蛋白结合（图2）。
其次，采用Pull-down实验，开展了体外竞争性实验，评价多肽配体R18对GF14f蛋白及其互作靶蛋白中与淀粉合成相关蛋白蔗糖合成酶（SuS），可溶性淀粉合成酶（SSS），ADPG焦磷酸化酶（AGPase）的竞争性调控机制。分别纯化表达GST-GF14f融合表达蛋白以及His-SuS、His-SSS、His-AGPase上述三个酶的亚基蛋白。之后，将纯化后His-SuS与结合在纯化介质上的GST-GF14f混合在一起，之后按照0 mg/L，1 mg/L, 6 mg/L,12 mg/L,60 mg/L的量添加多肽配体R18，结果表明随着多肽配体R18浓度的增加，GF14f与SuS蛋白间的结合逐渐减弱，在多肽配体R18浓度达到60 mg/L的时候能够使GF14f与SuS间不互作，进一步选用纯化后的His-SSS、His-AGPase蛋白，外源添加60 mg/L 多肽配体R18，均发现GF14f与SSS及AGPase这两个蛋白不互作（图3），可见多肽配体R18能够竞争性的与GF14f互作，进而影响其与SuS、SSS及AGPase间的结合。此外，通过体外酶活实验发现，随着外源添加到籽粒酶活提取液中多肽配体R18浓度的升高，SuS酶活性逐渐升高，在浓度达到60mg/L时，活性最高（图4），证实多肽配体R18能够竞争性的与GF14f蛋白结合，从而释放SuS酶的活性。
实施例2
通过籽粒灌浆期外源喷施多肽配体R18的混合溶液，验证其对籽粒灌浆过程中酶活、千粒重及结实率的影响。
具体方法为：
第一步: 选用大穗型水稻“金恢809”，在其花后7天，外源喷施60 mg/L多肽配体R18及6ml/L的表面活性剂吐温-20的混合溶液。
第二步：在花后10天（前期）、20天（中期）、30天（后期）测定籽粒中蔗糖合成酶（SuS），可溶性淀粉合成酶（SSS），ADPG焦磷酸化酶（AGPase）的活性，结果表明外源喷施多肽配体R18的混合溶液后，三个淀粉合成相关酶的酶活性显著提高（图5）。
第三步：在成熟期考察弱势籽粒的千粒重及结实率，发现外源喷施多肽配体R18的混合溶液能显著提升弱势籽粒的千粒重及结实率（图6）。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建农林大学
<120> 一种利用GF14f蛋白靶向多肽配体调控水稻弱势籽粒灌浆的方法
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> PRT
<213> GF14f蛋白靶向多肽配体R18
<400> 1
Pro His Cys Val Pro Arg Asp Leu Ser Trp Leu Asp Leu Glu Ala Asn
1 5 10 15
Met Cys Leu Pro
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Claims (4)
1.一种调控水稻弱势籽粒灌浆的多肽配体溶液，其特征在于：所述多肽配体溶液其组分包括60mg/L的GF14f蛋白靶向多肽配体R18和6ml/L的表面活性剂；
所述GF14f蛋白靶向多肽配体R18，其氨基酸序列为：PHCVPRDLSWLDLEANMCLP；所述的表面活性剂为吐温-20。
2.一种调控水稻弱势籽粒灌浆的方法，其特征在于：在水稻灌浆期外源喷施权利要求1所述的一种调控水稻弱势籽粒灌浆的多肽配体溶液。
3.根据权利要求2所述的一种调控水稻弱势籽粒灌浆的方法，其特征在于：在水稻灌浆期，即花后5~7天，外源喷施1~1.5L/亩权利要求1所述的一种调控水稻弱势籽粒灌浆的多肽配体溶液。
4.如权利要求1 所述的一种调控水稻弱势籽粒灌浆的多肽配体溶液在改良水稻粒重及结实率中的应用。
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