CN116814647B - 一种提升不结球白菜种子产量的基因cuc2及其载体、宿主细胞和应用 - Google Patents
一种提升不结球白菜种子产量的基因cuc2及其载体、宿主细胞和应用 Download PDFInfo
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Abstract
本发明涉及植物基因工程技术领域，提供了一种提升不结球白菜种子产量的基因CUC2，核苷酸序列如SEQ ID NO.1所示。本发明还提供了一种植物表达重组载体，包括基因CUC2构建的pCAMBIA1301‑35S‑CUC2‑GUS质粒。本发明还提供了一种遗传工程化的宿主细胞，所述宿主细胞包括基因CUC2。本发明还提供了上述基因CUC2在培育高产转基因不结球白菜植株中的应用。本发明基因CUC2可以增加不结球白菜植株的分枝、花序及果荚数，从而提升种子产量和花期观赏价值，为培育高产不结球白菜品种提供理论依据。
Description
技术领域
本发明涉及植物基因工程技术领域，尤其涉及一种提升不结球白菜种子产量的基因CUC2及其载体、宿主细胞和应用。
背景技术
不结球白菜(Brassica rapa ssp.chinensis；Non-heading Chinese Cabbage；NHCC)属十字花科(Cruciferae)芸薹属(Brassica)芸薹种白菜亚种，俗称小白菜、青菜、油菜，具有高维生素、矿物质等营养物质含量，是我国南方地区餐桌上的主要蔬菜。
为了适应当前的蔬菜需求，急需培育高产不结球白菜品种。而对于如何提高当前不结球白菜植株的高产，提升植株种子产量是关键。
因此，若能获得一种有效提高不结球白菜种子产量的方法，将为培育高产不结球白菜品种提供强有力的理论依据。
发明内容
本发明所要解决的技术问题在于提供一种提升不结球白菜种子产量的基因CUC2及其载体、宿主细胞和应用。
本发明采用以下技术方案解决上述技术问题：
一种提升不结球白菜种子产量的基因CUC2，其核苷酸序列如SEQ ID NO.1所示。
作为本发明的优选方式之一，所述基因CUC2的克隆步骤如下：提取不结球白菜总RNA并反转录为cDNA；设计特异性扩增引物CUC2-F和CUC2-R；以cDNA为模板进行PCR扩增，获得目的基因CUC2；其中，特异性扩增引物CUC2-F和CUC2-R核苷酸序列分别如SEQ ID NO.2、SEQ ID NO.3所示。
作为本发明的优选方式之一，所述基因CUC2通过增加不结球白菜植株的分枝、花序及果荚数来提升不结球白菜种子产量。
一种上述提升不结球白菜种子产量的基因CUC2在培育高产转基因不结球白菜植株中的应用。
一种植物表达重组载体，包括上述提升不结球白菜种子产量的基因CUC2构建的pCAMBIA1301-35S-CUC2-GUS质粒。
作为本发明的优选方式之一，所述pCAMBIA1301-35S-CUC2-GUS质粒的构建方法如下：利用同源重组法将基因CUC2序列插入到载体pCAMBIA1301-35S-GUS的酶切位点(XbaI和BamHI)。
一种遗传工程化的宿主细胞，包括上述提升不结球白菜种子产量的基因CUC2的基因序列。
作为本发明的优选方式之一，将所述基因CUC2构建的植物表达重组载体pCAMBIA1301-35S-CUC2-GUS转化至感受态细胞中，即得。
作为本发明的优选方式之一，所述植物表达重组载体pCAMBIA1301-35S-CUC2-GUS利用同源重组法将基因CUC2序列插入到载体pCAMBIA1301-35S-GUS的酶切位点构建而成。
作为本发明的优选方式之一，所述感受态细胞具体采用农杆菌GV3101感受态细胞(常规试剂，直接购买于现有市场)。
本发明相比现有技术的优点在于：分枝是植物提升产量的重要性状之一，花序及果荚数是种子生产的直接影响因素；本发明基因CUC2可以增加不结球白菜植株的分枝、花序及果荚数，从而提升种子产量和花期观赏价值，为培育高产不结球白菜品种提供理论依据。
附图说明
图1是实施例4中野生型植株与转基因植株GUS检测结果图(WT为野生型对照植株叶片，OE5/6/9/11为筛选得到的转基因植株叶片)；
图2是实施例4中野生型与过表达转基因拟南芥中CUC2基因表达量图(WT为野生型对照，OE5/6/9/11为转基因植株)；
图3是实施例4中野生型与过表达转基因拟南芥开花期表型图(WT为野生型对照，OE6/9/11为转基因植株)。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
以下实施例中的所使用的试剂产品、实验方法，未经特别说明的，均为本领域常规试剂、方法，不再赘述。
实施例1
本实施例的一种基因CUC2，核苷酸序列如SEQ ID NO.1所示，克隆步骤为：提取不结球白菜总RNA并反转录为cDNA；设计特异性扩增引物CUC2-F和CUC2-R；以cDNA为模板进行PCR扩增，获得目的基因CUC2；其中，特异性扩增引物CUC2-F和CUC2-R核苷酸序列分别如SEQID NO.2、SEQ ID NO.3所示。
具体克隆过程如下：
(1)将不结球白菜品种“苏州青”播种于32孔穴盘中，于人工气候室中正常生长。生长至6叶期，进行叶片取样，立即放入液氮中，于-80℃保存。
(2)叶片RNA提取及cDNA合成均采用相应试剂盒。
(3)CUC2基因序列如SEQ ID NO.1所示，根据目的基因CDS序列，设计特异性扩增引物CUC2-F和CUC2-R，具体序列如SEQ ID NO.2、SEQ ID NO.3所示；
(4)以cDNA为模板，利用高保酶进行PCR扩增；扩增体系：cDNA 1μL，2X High-Fidelity Master Mix 25μL，特异性引物CUC2-F和CUC2-R各1μL，ddH2O22μL；扩增程序：98℃预变性2min，98℃变性10s，56℃退火15s，72℃延伸20s，循环35次。
(5)对PCR扩增产物进行琼脂糖凝胶电泳，并利用胶回收试剂盒对目的片段进行切胶回收，获得目的基因CUC2片段。
(6)将目的片段构建至pMD19载体，转化大肠杆菌，经公司测序后获得目的基因序列。
实施例2
本实施例的一种植物表达重组载体pCAMBIA1301-35S-CUC2-GUS的构建：
(1)根据已经成功克隆的目的基因CUC2序列设计加接头引物CUC2-S-F和CUC2-S-R(接头为：15bp载体同源臂+酶切位点)，并利用高保酶进行加接头目的片段的扩增，扩增体系及程序同基因克隆。其中，所述CUC2-S-F和CUC2-S-R的核苷酸序列分别如SEQ ID NO.4、SEQ ID NO.5所示。
(2)利用限制性内切酶XbaI和BamHI对载体pCAMBIA1301-35S-GUS进行双酶切，获得线性化载体。
(3)利用胶回收试剂盒分别对目的片段和线性化载体片段进行回收，并通过同源重组试剂盒(购自TAKARA公司)进行目的片段与pCAMBIA1301-35S-GUS载体的连接。其中，连接体系：加接头目的片段2.5μL，线性化载体1.5μL，5X In-Fusion HD Enzyme Premix 2μL，ddH2O补至4μL；反应程序：50℃、20min，4℃保存。
实施例3
本实施例的一种宿主细胞的构建：
(1)将农杆菌GV3101感受态细胞从-80℃冰箱取出，置于冰上融化。
(2)吸取5μL构建好的“植物表达重组载体pCAMBIA1301-35S-CUC2-GUS”至50μL上述感受态细胞中，轻柔吸打混匀。
(3)冰浴30min，然后42℃热激45s，再冰浴3min。
(4)于超净台加入700μL LB液体培养基，37℃培养箱，2000rpm震荡培养1h。
(5)5000rpm离心5min，去除500μL上清。
(6)利用剩余上清液将菌块吸打混匀，涂布于含卡那的固体LB培养基上过夜培养。
实施例4
本实施例的一种过表达CUC2拟南芥株系的构建：
(1)对实施例3获得的宿主细胞进行活化。
(2)利用蘸花法进行拟南芥侵染，收获T0代种子。
(3)利用含潮霉素抗性的MS培养基、GUS染色和荧光定量PCR分别对T1和T2代转基因植株进行筛选鉴定。
图1为野生型植株与转基因植株GUS检测结果(WT为野生型对照植株叶片，OE5/6/9/11为抗性板上筛选得到的转基因植株叶片)。图2为野生型与过表达转基因拟南芥中CUC2基因表达量。由图1和图2可知：CUC2转基因植株具有显著的GUS活性表达；在过表达转基因拟南芥株系中，CUC2基因的转录水平是野生型(WT)的90～150倍，表明转基因拟南芥遗传转化成功，可利用后续试验。
(4)将获得的T3代纯合转基因株系用于表型观察。观察结果：与野生型(WT)植株相比，转基因植株(OE6/9/11)产生更多的分支、花序及果荚，如图3所示。
综合上述各实施例可知，本发明基因CUC2可以增加不结球白菜植株的分枝、花序及果荚数，从而提升种子产量和花期观赏价值，为培育高产不结球白菜品种提供理论依据。
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作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (1)
1. 一种提升不结球白菜种子产量的基因CUC2在培育高产转基因不结球白菜植株中的应用，其特征在于，所述基因CUC2的核苷酸序列如SEQ ID NO.1所示，所述基因CUC2通过增加不结球白菜植株的分枝、花序及果荚数来提升不结球白菜种子产量。
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| CA2804429A1 (en) * | 2010-07-08 | 2012-01-12 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | A plant heat-resistance gene jaz5a and use thereof |
| WO2012005589A1 (en) * | 2010-07-08 | 2012-01-12 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | A plant heat-resistance gene hsf1 and use thereof |
| WO2012005590A1 (en) * | 2010-07-08 | 2012-01-12 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | A plant heat-resistance gene bccdreb2a and use thereof |
| CN113151303A (zh) * | 2021-05-21 | 2021-07-23 | 浙江大学 | 白菜干细胞决定相关基因BrWUS1及其应用 |
| CN114634937A (zh) * | 2022-01-14 | 2022-06-17 | 安徽农业大学 | 一种促进不结球白菜开花的基因及其载体、重组菌和应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2804429A1 (en) * | 2010-07-08 | 2012-01-12 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | A plant heat-resistance gene jaz5a and use thereof |
| WO2012005589A1 (en) * | 2010-07-08 | 2012-01-12 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | A plant heat-resistance gene hsf1 and use thereof |
| WO2012005590A1 (en) * | 2010-07-08 | 2012-01-12 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | A plant heat-resistance gene bccdreb2a and use thereof |
| CN113151303A (zh) * | 2021-05-21 | 2021-07-23 | 浙江大学 | 白菜干细胞决定相关基因BrWUS1及其应用 |
| CN114634937A (zh) * | 2022-01-14 | 2022-06-17 | 安徽农业大学 | 一种促进不结球白菜开花的基因及其载体、重组菌和应用 |
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