CN116622739B - 番茄SlSUVH2或SlSUVH4基因在调控双生病毒中的应用及转基因植物培育方法 - Google Patents
番茄SlSUVH2或SlSUVH4基因在调控双生病毒中的应用及转基因植物培育方法 Download PDFInfo
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Abstract
本发明公开了一种番茄SlSUVH2或SlSUVH4基因在调控双生病毒中的应用及转基因植物培育方法，所述双生病毒为番茄黄曲叶病毒，SlSUVH2和SlSUVH4基因的转录本序列如SEQ ID NO:1‑2所示。本发明通过农杆菌转化的方法，将SlSUVH2或SlSUVH4基因导入目的植物中，获得了番茄SlSUVH2或SlSUVH4基因过表达植物，获得的番茄SlSUVH2或SlSUVH4过表达植物能够显著减轻番茄黄曲叶病毒的侵染。
Description
技术领域
本发明涉及基因工程技术领域，特别是涉及一种番茄SlSUVH2或SlSUVH4基因在调控双生病毒中的应用以及抑制番茄黄曲叶病毒侵染的转基因植物培育方法。
背景技术
番茄黄曲叶病毒(tomato yellow leaf curl virus，TYLCV)属于双生病毒科，菜豆金色花叶病毒属，是目前研究最多的植物病毒之一，同时也是对全球番茄生产危害最大的病毒。流行时可造成农作物的产量和品质下降，对农业经济造成严重损失。
TYLCV在自然界中主要通过烟粉虱进行传播，传播TYLCV的烟粉虱主要有B型和Q型，并且Q型烟粉虱的传毒效率比B型高。除了介体传播外，TYLCV可以通过嫁接传播。TYLCV侵染植物后会使植物生长缓慢，造成植株矮化，还会使叶片卷曲、黄化和皱缩。
农业卫生预防是生产中控制TYLCV的重要措施。然而，由于该病毒具有高度传染性，这就使得农业卫生措施预防病毒成为挑战，尤其是在密集的大田和保护地，该病毒发生严重，造成番茄大量减产或者绝收。虽然目前生产上有一些抗TYLCV的植物遗传材料，但是TYLCV变异速度快、传播介体爆发以及田间经常发生复合侵染原因等，导致这些抗性材料对TYLCV的抗性常常被病毒所突破。因此，目前还没有有效控制TYLCV的方法，急需通过研究获得高效、广谱的抗双生病毒的抗性靶标和抗性资源。
发明内容
本发明的第一目的是提供一种番茄SlSUVH2或SlSUVH4基因在调控双生病毒中的应用。
本发明的第二目的是提供一种番茄SlSUVH2或SlSUVH4转基因植物的培育方法。
本发明发现了番茄中SlSUVH2和SlSUVH4基因是抗病基因，通过农杆菌转化的方法，将SlSUVH2或SlSUVH4基因导入目的植物中，获得的番茄SlSUVH2或SlSUVH4高表达的番茄植物能够显著减轻TYLCV的侵染，有效控制该病毒的危害。
具体而言，本发明采用的技术方案具体如下：
番茄SlSUVH2或SlSUVH4基因在调控双生病毒中的应用，其中，所述双生病毒为番茄黄曲叶病毒。所述SlSUVH2和SlSUVH4基因的转录本序列如SEQ ID NO:1-2所示。
具体的，是通过农杆菌转化的方法，将SlSUVH2或SlSUVH4基因导入目的植物中，获得的番茄SlSUVH2或SlSUVH4高表达植物能够显著减轻番茄黄曲叶病毒的侵染，抑制番茄黄曲叶病毒造成的病害。
一种抗番茄黄曲叶病毒的SlSUVH2或SlSUVH4转基因植物的培育方法：通过农杆菌转化的方法，将SlSUVH2或SlSUVH4基因导入目的植物中，获得番茄SlSUVH2或SlSUVH4基因高表达植物。
具体包括以下步骤：
(1)番茄叶片取样，Trizol法抽提RNA，使用oligo(dT)18(5'-TTTTTTTTTTTTTTTTTT-3')引物反转录获得cDNA；
(2)使用引物对SlSUVH2-F、SlSUVH2-R或SlSUVH4-F、SlSUVH4-R，如SEQ ID NO:4-7所示，以cDNA为模板进行PCR扩增，克隆得到SlSUVH2或SlSUVH4，
SlSUVH2-F：GGGGTACCATGGGTTCTCTAGTCCCATTTCAA；
SlSUVH2-R：CGGGATCCATCACAGATGGCAAGCTTTC；
SlSUVH4-F：GGGGTACCATGGTGGTTCCGTGCGTTG；
SlSUVH4-R：CGGGATCCAAACAAGCGTTTACGACAGTC。
(3)将SlSUVH2或SlSUVH4构建到带有GFP荧光标签的载体上，获得SlSUVH2-GFP或SlSUVH4-GFP重组质粒；
(4)将1μl重组质粒与100μl农杆菌感受态混合转入电击杯中，使用电击装置2500V电击转化，恢复后涂布到抗性培养基上筛选获得带有SlSUVH2-GFP或SlSUVH4-GFP重组质粒的农杆菌；
(5)将带有重组质粒的农杆菌侵染番茄叶片，经分化培养获得愈伤组织，再经生根培养获得小苗，转移继续培养；
(6)继续培养的小苗生长稳定后，取叶片样品，使用Trizol法抽提RNA，反转录获得cDNA。利用引物对q-SlSUVH2–F、q-SlSUVH2-R或q-SlSUVH4–F、q-SlSUVH4-R，如SEQ ID NO:8-11所示，以cDNA为模板进行RT-qPCR扩增，检测植物中SlSUVH2或SlSUVH4的表达水平；
q-SlSUVH2-F：CGGGATCCTGCTGATAATTGTTTTTGTGC；
q-SlSUVH2-R：CCGCTCGAGTCACCATTCATTGTAAAAATTTGGG；
q-SlSUVH4-F：CGGGATCCTAAGGGTTACACGTGGACAT；
q-SlSUVH4-R：CCGCTCGAGCCGGTGGATCATCAACTAAA。
另外，取叶片抽提总蛋白，进行SDS-PAGE凝胶电泳，再使用GFP抗体进行Westernblot确定植物表达了带GFP荧光标签的SlSUVH2-GFP或SlSUVH4-GFP；对以上阳性植株留种。
同现有技术相比，本发明的突出效果在于：
本发明发现了番茄中SlSUVH2和SlSUVH4基因是抗病基因，并克隆了该基因，通过农杆菌转化的方法，将SlSUVH2或SlSUVH4基因导入目的植物中，获得了番茄SlSUVH2或SlSUVH4高表达转基因植物，获得的SlSUVH2或SlSUVH4转基因植物能够显著减轻番茄黄曲叶病毒的侵染，抑制番茄黄曲叶病毒造成的病害。
下面结合附图说明和具体实施例对本发明所述的番茄SlSUVH2或SlSUVH4基因在调控植物抗病毒中的应用及转基因植物培育方法作进一步说明。
附图说明
图1为载体构建示意图。
图2为SlSUVH2-GFP转基因植物的鉴定。
其中，(A)T1代SlSUVH2-GFP转基因植物与野生型番茄表型对比，WT为野生型番茄，SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3为SlSUVH2-GFP转基因番茄两个不同高表达株系；
(B)RT-qPCR检测各株系中SlSUVH2的表达水平；
(C)Western blot分别分析来自于野生型番茄、SlSUVH2-GFP转基因高表达株系SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3植物叶片中的蛋白表达量，丽春红染色的RuBisCO大亚基用于表示上样量的水平。
图3为SlSUVH4-GFP转基因植物的鉴定。
其中，(A)T1代SlSUVH4-GFP转基因植物与野生型番茄表型对比，WT为野生型番茄，SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2为SlSUVH4-GFP转基因番茄两个不同高表达株系；
(B)RT-qPCR检测各株系中SlSUVH4的表达水平；
(C)Western blot分别分析来自于野生型番茄、SlSUVH4-GFP转基因高表达株系SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2植物叶片中的蛋白表达量，丽春红染色的RuBisCO大亚基用于表示上样量的水平。
图4为SlSUVH2-GFP转基因植物对番茄黄曲叶病毒(TYLCV)的抗性分析。
其中，(A)TYLCV接种野生型番茄及SlSUVH2-GFP转基因高表达株系SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3后14天的症状图；
(B)RT-qPCR分析TYLCV接种野生型番茄及SlSUVH2-GFP转基因高表达株系SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3 14天系统叶片病毒DNA积累量；
(C)Western blot检测TYLCV接种野生型番茄及SlSUVH2-GFP转基因高表达株系SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3 14天系统叶病毒外壳蛋白(CP)的积累量，丽春红染色的RuBisCO大亚基用于表示上样量水平。
图5为SlSUVH4-GFP转基因植物对番茄黄曲叶病毒(TYLCV)的抗性分析。
其中，(A)TYLCV接种野生型番茄及SlSUVH4-GFP转基因高表达株系SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2后14天的症状图；
(B)RT-qPCR分析TYLCV接种野生型番茄和SlSUVH4-GFP转基因高表达株系SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2 14天系统叶片病毒DNA积累量；
(C)Western blot检测TYLCV接种野生型番茄及SlSUVH4-GFP转基因高表达株系SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2 14天系统叶病毒外壳蛋白(CP)的积累量，丽春红染色的RuBisCO大亚基用于表示上样量水平。
具体实施方式
一种抗番茄黄曲叶病毒的SlSUVH2、SlSUVH4转基因植物的培育方法，具体包括以下步骤：
(1)番茄叶片取样，使用Trizol法抽提RNA，去除基因组DNA后，使用oligo(dT)18(5'-TTTTTTTTTTTTTTTTTT-3'，如SEQ ID NO:3所示)引物反转录获得cDNA。
(2)使用引物对SlSUVH2-F、SlSUVH2-R以及SlSUVH4-F、SlSUVH4-R，如SEQ ID NO:4-7所示，以cDNA为模板进行PCR扩增，克隆得到SlSUVH2和SlSUVH4，
SlSUVH2-F：GGGGTACCATGGGTTCTCTAGTCCCATTTCAA；
SlSUVH2-R：CGGGATCCATCACAGATGGCAAGCTTTC；
SlSUVH4-F：GGGGTACCATGGTGGTTCCGTGCGTTG；
SlSUVH4-R：CGGGATCCAAACAAGCGTTTACGACAGTC。
(3)将SlSUVH2和SlSUVH4构建到带有GFP荧光标签的载体上，获得SlSUVH2-GFP和SlSUVH4-GFP重组质粒；如图1所示；
(4)将1μl重组质粒与100μl农杆菌感受态混合转入电击杯中，使用电击装置2500V电击转化，恢复后涂布到含有卡那(50μg/ml)和利福平(100μg/ml)抗性的培养基上筛选获得带有SlSUVH2-GFP和SlSUVH4-GFP重组质粒的农杆菌；
(5)挑取阳性单菌落转入液体抗性培养基中，28℃，200rpm过夜培养，调节农杆菌OD600至1.0。然后将带有重组质粒的农杆菌侵染番茄叶片，经分化培养获得愈伤组织，再经生根培养获得小苗，转移继续培养；
(6)继续培养的小苗生长稳定后，取叶片样品，使用Trizol法抽提RNA，反转录获得cDNA。利用引物对q-SlSUVH2-F、q-SlSUVH2-R以及q-SlSUVH4-F、q-SlSUVH4-R，如SEQ IDNO:8-11所示，以cDNA为模板进行RT-qPCR扩增，检测植物中SlSUVH2和SlSUVH4的表达水平；如图2B、3B所示；
q-SlSUVH2-F：CGGGATCCTGCTGATAATTGTTTTTGTGC；
q-SlSUVH2-R：CCGCTCGAGTCACCATTCATTGTAAAAATTTGGG；
q-SlSUVH4-F：CGGGATCCTAAGGGTTACACGTGGACAT；
q-SlSUVH4-R：CCGCTCGAGCCGGTGGATCATCAACTAAA。
(7)以上样品抽提总蛋白，进行SDS-PAGE凝胶电泳，再使用GFP抗体进行Westernblot检测，确定植物表达了带GFP荧光标签的SlSUVH2-GFP和SlSUVH4-GFP，如图2C、3C所示，SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3以及SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2均能检测到SlSUVH2-GFP和SlSUVH4-GFP蛋白，对以上阳性植株留种；
(8)播种野生型番茄及SlSUVH2-GFP和SlSUVH4-GFP高表达株系SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3以及SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2。生长4周后，如图2A、3A所示，三者植物表型没有显著差异。然后分别接种TYLCV。接种14天后观察病毒症状，如图4A、5A所示，相对于野生型番茄，SlSUVH2-GFP-OE1与SlSUVH2-GFP-OE3以及SlSUVH4-GFP-OE1与SlSUVH4-GFP-OE2两个高表达株系表现出的病毒症状更轻。RT-qPCR检测系统叶病毒DNA积累量，转基因两个株系的病毒DNA积累量显著低于野生型番茄，如图4B、5B所示，结果显示SlSUVH2-GFP和SlSUVH4-GFP转基因植物能够明显降低TYLCV的DNA水平。Western blot检测病毒外壳蛋白(CP)，如图4C、5C所示，转基因两个株系的病毒蛋白积累量显著低于野生型番茄，结果显示SlSUVH2-GFP和SlSUVH4-GFP转基因植物能够明显降低TYLCV的蛋白积累量。
以上实验结果表明：通过农杆菌遗传转化的方法获得的番茄SlSUVH2或SlSUVH4过表达植物能够显著减轻番茄黄曲叶病毒的侵染，抑制番茄黄曲叶病毒造成的病害。
以上所述的实施例仅仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案作出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (6)
1.番茄SlSUVH2或SlSUVH4基因在调控双生病毒中的应用，其特征在于：所述SlSUVH2和SlSUVH4基因的转录本序列如SEQ ID NO:1-2所示；所述双生病毒为番茄黄曲叶病毒；
通过农杆菌转化的方法，将SlSUVH2或SlSUVH4基因导入番茄中，获得的番茄SlSUVH2或SlSUVH4高表达植株能够显著减轻番茄黄曲叶病毒的侵染。
2.一种抗番茄黄曲叶病毒的SlSUVH2或SlSUVH4转基因植物的培育方法，其特征在于：通过农杆菌转化的方法，将SlSUVH2或SlSUVH4基因导入目的植物中，获得番茄SlSUVH2或SlSUVH4基因高表达植物，所述SlSUVH2和SlSUVH4基因的转录本序列如SEQ ID NO:1和SEQID NO:2所示，所述目的植物为番茄。
3.根据权利要求2所述的SlSUVH2或SlSUVH4转基因植物培育方法，其特征在于，包括以下步骤：
(1)番茄叶片取样，使用Trizol法抽提RNA，使用oligo(dT)18引物，反转录获得cDNA；
(2)使用引物对SlSUVH2-F、SlSUVH2-R或SlSUVH4-F、SlSUVH4-R，以cDNA为模板进行PCR扩增，克隆得到SlSUVH2或SlSUVH4；
(3)将SlSUVH2或SlSUVH4构建到带有GFP荧光标签的载体上，获得SlSUVH2-GFP或SlSUVH4-GFP重组质粒；
(4)将1μl重组质粒与100μl农杆菌感受态混合转入电击杯中，使用电击装置2500V电击转化，恢复后涂布到抗性培养基上筛选获得带有SlSUVH2-GFP或SlSUVH4-GFP重组质粒的农杆菌；
(5)将带有重组质粒的农杆菌侵染番茄叶片，经分化培养获得愈伤组织，再经生根培养获得小苗，转移继续培养；
(6)继续培养的小苗生长稳定后，取叶片样品，使用Trizol法抽提RNA，反转录获得cDNA；利用引物对q-SlSUVH2–F、q-SlSUVH2-R或q-SlSUVH4–F、q-SlSUVH4-R，以cDNA为模板进行RT-qPCR扩增，检测植物中SlSUVH2或SlSUVH4的表达水平；
取少许叶片组织，抽提总蛋白，进行SDS-PAGE凝胶电泳，再使用GFP抗体进行Westernblot确定植物表达了带GFP荧光标签的SlSUVH2-GFP或SlSUVH4-GFP，对以上阳性植株留种。
4.根据权利要求3所述的SlSUVH2或SlSUVH4转基因植物培育方法，其特征在于：所述oligo(dT)18引物序列为5'-TTTTTTTTTTTTTTTTTT-3'，如SEQ ID NO:3所示。
5.根据权利要求4所述的SlSUVH2或SlSUVH4转基因植物培育方法，其特征在于：所述SlSUVH2-F、SlSUVH2-R以及SlSUVH4-F、SlSUVH4-R的序列如SEQ ID NO:4-7所示；
SlSUVH2-F：GGGGTACCATGGGTTCTCTAGTCCCATTTCAA；
SlSUVH2-R：CGGGATCCATCACAGATGGCAAGCTTTC；
SlSUVH4-F：GGGGTACCATGGTGGTTCCGTGCGTTG；
SlSUVH4-R：CGGGATCCAAACAAGCGTTTACGACAGTC。
6.根据权利要求5所述的SlSUVH2或SlSUVH4转基因植物培育方法，其特征在于：所述q-SlSUVH2–F、q-SlSUVH2-R以及q-SlSUVH4–F、q-SlSUVH4-R的序列如SEQ ID NO:8-11所示；
q-SlSUVH2-F：CGGGATCCTGCTGATAATTGTTTTTGTGC；
q-SlSUVH2-R：CCGCTCGAGTCACCATTCATTGTAAAAATTTGGG；
q-SlSUVH4-F：CGGGATCCTAAGGGTTACACGTGGACAT；
q-SlSUVH4-R：CCGCTCGAGCCGGTGGATCATCAACTAAA。
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