CN116143892B - OsGN11基因在改良水稻每穗粒数性状中的应用 - Google Patents
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Abstract
本发明涉及植物基因工程技术领域，具体涉及OsGN11基因在改良水稻每穗粒数性状中的应用。本发明对水稻OsGN11基因采用过表达技术，使该基因表达量显著增加，构建了OsGN11基因的过表达水稻。本发明研究发现，过表达OsGN11基因可显著增加每穗粒数进而显著增加水稻产量。这一结果为培育优良性状水稻提供一种全新的并且简单有效的方法，可投入大规模推广使用。
Description
技术领域
本发明涉及植物基因工程技术领域，具体涉及OsGN11基因在改良水稻每穗粒数性状中的应用。
背景技术
水稻是重要的粮食作物，为全球超过50％的人口提供主食。每穗粒数是产量三要素之一，直接影响水稻的产量。水稻的每穗粒数主要取决于一次枝梗和二次枝梗的数目。调控水稻枝梗数在农业生产和发育生物学上都有重要意义。一次枝梗的数量和位置在水稻植株从营养生长向生殖生长转变的过程中就已经建立，因此挖掘影响水稻一次枝梗和二次枝梗的新基因和优势等位基因不仅有利于拓展调控每穗粒数基因网络，也将丰富和深化水稻NPF家族基因的认知。
植物NPF(NRT1/PTR FAMILY)家族与动物肽转运载体SLC15/PepT/PTR/POT家族同源，在水稻中共有11个成员，其中大部分NPF家族基因的功能和其在育种中的应用方法仍然未知。
发明内容
基于以上技术问题，本发明提供以下技术方案：
本发明提供了OsGN11基因在改良水稻每穗粒数性状中的应用，所述OsGN11基因的核苷酸序列如SEQ ID NO.1所示。
进一步的，所述OsGN11基因正向调控水稻每穗粒数。
更进一步的，通过过表达OsGN11基因，提高水稻每穗粒数。
更进一步的，所述过表达OsGN11基因是通过将OsGN11基因或其相关生物材料转入水稻，或外源施加能够提高OsGN11基因表达的物质实现的。
更进一步的，所述OsGN11基因相关生物材料包括含有所述OsGN11基因的表达盒、重组载体、重组菌或转基因细胞系。
基于同一个发明构思，本发明还提供了所述OsGN11基因或其表达产物在定向选择或鉴定水稻每穗粒数性状中的应用。
进一步的，所述应用包括：鉴定测试水稻中OsGN11基因或其表达产物的水平，若是该测试水稻的OsGN11基因或其表达产物水平显著高于该类水稻的平均值，则为优良每穗粒数性状。
更进一步的，鉴定测试水稻中OsGN11基因水平的方法包括荧光定量PCR，所使用的引物如SEQ ID NO.5-6所示。
更进一步的，所述OsGN11基因表达产物为OsGN11蛋白，氨基酸序列如SEQ ID NO.2所示。
与现有技术相比，本发明具有如下有益效果：
本发明提供了OsGN11基因在改良水稻每穗粒数性状中的应用。采用过表达技术增加OsGN11基因的表达量，构建了OsGN11基因的过表达水稻。研究显示，过表达OsGN11基因可显著增加每穗粒数进而显著增加水稻产量，这一结果为培育优良性状水稻提供一种全新的并且简单有效的方法，可投入大规模推广使用。
附图说明
图1为野生型与OsGN11基因过表达植株的OsGN11基因表达量。
图2为野生型与OsGN11基因过表达植株的穗型(A)和每穗粒数比较(B)。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，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。
本发明采用过表达技术增加OsGN11基因的表达量，获得了一种每穗粒数增加的种质。所述的OsGN11基因的核苷酸序列如序列表SEQ ID NO.1所示，其编码的氨基酸序列如序列表SEQ ID NO.2所示。本发明制备的高产种质，每穗粒数增加，该种质应用于水稻育种，能显著增加水稻产量，因而具有很好的应用前景。
实施例1
过表达OsGN11基因的水稻的构建
1、从水稻基因组注释数据库TIGR检索获得水稻OsGN11基因的完整CDS序列，如SEQID NO.1所示，交由江苏溥博生物科技有限公司进行基因合成；
2、酶切反应：pCAMBIA1300S载体采用双酶切线性化后通过胶回收获得载体片段。酶切反应体系如下：
表1酶切反应体系
3、重组反应：目的DNA片段和载体使用2X HiFi Clone PG-1重组酶37℃反应1个小时后转化Top10感受态细胞并涂板。反应体系如下：
表2重组反应体系
4、基因编辑载体转化农杆菌：将上述构建正确的基因编辑载体转化农杆菌(EHA105)感受态，提取转化后的农杆菌质粒，将其再次转化大肠杆菌(DH 5α)感受态，并提取大肠杆菌质粒进行测序验证，确定构建的基因编辑载体正确转入农杆菌中。
5、组织培养
1)愈伤组织诱导：选取饱满、无霉斑的成熟Sasanishiki水稻种子去壳，先用无菌水冲洗3遍，然后用体积分数70％的乙醇处理1min，用无菌水冲洗3遍，每次30s，用0.1％升汞溶液消毒12min，用无菌水冲洗7遍，每次30s，将种子放在灭菌滤纸上吸干水分，然后用无菌镊子夹到诱导培养基上，28℃，暗培养4周以上。
2)愈伤组织继代培养：挑选颜色鲜亮、紧实且干燥的胚性愈伤，放于继代培养基上暗培养10天，温度28℃。
3)农杆菌侵染：将构建好的农杆菌，划线培养活化一次，再预培养2天。然后转移至装有悬浮培养基的离心管里，涡旋振荡重悬农杆菌，调节农杆菌的悬浮液至OD600为0.9；挑选颜色鲜亮、紧实且干燥的胚性愈伤与农杆菌悬浮液混合，浸泡30分钟；转移愈伤至灭菌好的滤纸上，超净工作台上吹干；将愈伤放置在已铺有一层滤纸的共培养基上28℃培养3天。
4)选择培养：将共培养的愈伤转移至灭菌好的三角瓶内，用灭菌的蒸馏水充分洗涤愈伤；将愈伤浸泡在含400mg/L羧苄青霉素的灭菌水中30min，不时摇动；转移愈伤至灭菌好的滤纸上吸干水分，并在超净工作台中晾干；转移愈伤至含有50mg/L潮霉素的选择培养基上选择培养2次，每次2周(第一次羧苄青霉素筛选浓度为400mg/L，第二次为250mg/L)。
5)分化培养：将抗性愈伤转移至分化培养基上，光照下培养，温度28℃。
6)生根培养：剪掉分化时产生的根，然后将其转移至生根培养基中，光培养2周以上，温度28℃。
7)待苗高10cm左右，根系发生较好，打开瓶盖，向瓶内注入无菌水，炼苗2天后，洗掉根上的残留培养基，移栽。在最初的几天，蒙上保鲜膜，避免强光照射，保持水分湿润。
实施例2：OsGN11过表达植株的鉴定
1、阳性转基因株系鉴定
将实施例1共获得的水稻再生苗，提取叶片DNA进行潮霉素标记PCR鉴定，获得转基因阳性株系，PCR扩增引物如下所示：
OsGN11-F(SEQ ID NO.3)：5′-TTCGAGCTCGGTACCCGGGGATCCATGGATGTTGAGTCAAGGCAA-3′；
OsGN11-R(SEQ ID NO.4)：5′-CTTGCATGCCTGCAGGTCGACTCAGTGACCCATGTTGACCT-3′。
PCR反应扩增体系为：
模板DNA1μl；2×taq buffer 10μl；dNTP 0.5μl；正向引物0.5μl；反向引物0.5μl；taq酶0.5μl；ddH2O 7μl。
PCR反应扩增程序为：
94℃预变性5min；94℃变性30s，56℃退火30s，72℃延伸1min，31次循环；72℃延伸5min。
2、OsGN11过表达株系鉴定
以上述转基因阳性株系为材料，取其抽穗期剑叶提取RNA，反转录成cDNA后进行表达量检测，RT-PCR检测引物序列如下：
正向引物(SEQ ID NO.5)：5′-CGGTAACCAAGAGGAAACAAGTG-3′；
反向引物(SEQ ID NO.6)：5′-CACCACGCACAGTAGCACCTT-3′。
结果：OsGN11过表达株系的OsGN11表达量显著高于野生型，其中两个代表性过表达株系OX-1和OX-2的OsGN11表达量如图1所示。
野生型与过表达株系在成熟期进行每穗粒数和穗数的调查，如图2所示，过表达株系的每穗粒数显著增加，这一结果为培育高产水稻提供一种全新的并且简单有效的方法。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (5)
1.OsGN11基因在改良水稻每穗粒数性状中的应用，其特征在于，所述OsGN11基因的核苷酸序列如SEQ ID NO .1所示，所述OsGN11基因正向调控水稻每穗粒数，通过过表达OsGN11基因，提高水稻每穗粒数，所述过表达OsGN11基因是通过将OsGN11基因或其相关生物材料转入水稻实现的，所述相关生物材料为含有所述OsGN11基因的表达盒、重组载体、重组菌或转基因细胞系。
2.根据权利要求1中所述的应用，其特征在于，将所述OsGN11基因或其表达产物用于定向选择或鉴定水稻每穗粒数性状。
3.根据权利要求2所述的应用，其特征在于，所述应用包括：鉴定测试水稻中OsGN11基因或其表达产物的水平，若是该测试水稻的OsGN11基因或其表达产物水平显著高于该类水稻的平均值，则为优良每穗粒数性状。
4.根据权利要求3所述的应用，其特征在于，鉴定测试水稻中OsGN11基因水平的方法包括荧光定量PCR，所使用的引物如SEQ ID NO .5-6所示。
5.根据权利要求4所述的应用，其特征在于，所述OsGN11基因表达产物为OsGN11蛋白，氨基酸序列如SEQ ID NO .2所示。
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