CN117126879B - 番茄SlSUVH1基因在调控植物抗病毒中的应用及转基因植物培育方法 - Google Patents
番茄SlSUVH1基因在调控植物抗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SlSUVH1基因在调控植物抗病毒中的应用及转基因植物培育方法,所述病毒为番茄黄化曲叶病毒,SlSUVH1基因的转录本序列如SEQ ID NO:1所示。本发明通过农杆菌转化的方法,将携带目的基因靶点的基因敲除载体导入目的植物中,获得了番茄SlSUVH1基因敲除植物,获得的番茄SlSUVH1敲除植物能够显著减轻番茄黄化曲叶病毒的侵染。
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SlSUVH1基因在调控植物抗病毒中的应用及转基因植物培育方法。
背景技术
番茄黄化曲叶病毒(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。
利用抗病基因防控双生病毒是农业生产中有效、绿色的病毒病害防控方法。然而,由于目前生产上可用的商业化品种具有的抗性基因有限,同时田间病毒的复合侵染会导致抗病基因介导的抗性损失。通过利用隐性抗病基因(也叫绿色抗病突变体):即该基因在病毒侵染中具有重要功能,敲除或者缺失该基因会严重抑制病毒的复制、转录、翻译或移动等病毒的必要生命过程,但是该基因的敲除或者缺失不会影响植物的生长、发育与繁殖。由于隐性抗病基因介导的抗性具有重要的应用价值,因此利用该类基因传递对病毒的抗性具有重要意义。
本发明通过前期研究发现SlSUVH1基因能够促进双生病毒的侵染,因此通过利用这个基因,构建和培育其绿色突变体,可实现对双生病毒的有效防控。
发明内容
本发明的第一目的是提供一种番茄SlSUVH1基因在调控植物抗病毒中的应用。
本发明的第二目的是提供一种番茄SlSUVH1基因敲除的转基因植物的培育方法。
本发明发现了番茄中SlSUVH1基因是一个感病基因。通过农杆菌转化的方法,将携带SlSUVH1靶点的CRISPR载体导入目的植物中,获得的番茄SlSUVH1基因敲除植物能够显著减轻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SlSUVH1基因在调控植物抗病毒中的应用,其中,所述病毒为番茄黄化曲叶病毒TYLCV。所述SlSUVH1基因的转录本序列如SEQ ID NO:1所示,所述植物为番茄。
具体的,通过构建番茄基因SlSUVH1敲除载体,即:针对番茄SlSUVH1基因编码区具备PAM位点设计相应的特异性靶向sgRNA,构建到骨架载体BKG质粒上;以无菌培养的番茄叶片做愈伤组织,使用农杆菌介导的T-DNA方式进行遗传转化,将敲除载体导入目的植物中,获得的番茄SlSUVH1基因突变的纯合植物能够显著减轻番茄黄化曲叶病毒的侵染。
一种抗番茄黄化曲叶病毒的SlSUVH1转基因植物的培育方法:通过叶盘法,将携带SlSUVH1基因靶点的基因敲除载体导入目的植物中,获得番茄SlSUVH1基因突变的纯合植物。
具体包括以下步骤:
(1)分别对SlSUVH1两个拷贝基因公共保守区域进行CRISPR靶点筛选,得到gRNA靶点序列,如SEQ ID NO:2所示;设计引物进行PCR扩增,克隆得到SlSUVH1-gRNA-gRNA片段;引物为SlSUVH1-gRNA-F和SlSUVH1-gRNA-R,序列分别如SEQ ID NO:3-4所示;
(2)将SlSUVH1-gRNA-gRNA构建到带有CRISPR-Cas9载体上,获得重组质粒;
(3)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CRISPR-Cas9重组质粒的农杆菌;
(4)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;
(5)小苗生长稳定后,取叶片样品,使用CTAB法抽提DNA;利用引物对M13-F与BKG-gRNA-R,以提取DNA为模板进行PCR扩增,确定植物中转入了外源CRISPR-Cas9序列;所述M13-F和BKG-gRNA-R的序列如SEQ ID NO:5-6所示;
(6)在阳性转基因材料上,使用引物BKG-SlSUVH1-F与BKG-SlSUVH1-R克隆番茄SlSUVH1基因靶点序列,通过测序,筛选两个拷贝基因均被编辑的株系,留种;所述BKG-SlSUVH1-F、BKG-SlSUVH1-R的序列如SEQ ID NO:7-8所示;
(7)在得到的T1代种子中筛选纯合编辑株系,并在纯合株系中选择剔除CRISPR-Cas9骨架的植物进行收种,获得SlSUVH1基因敲除番茄。
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SlSUVH1基因是感病基因,并克隆了该基因,并且通过农杆菌转化的方法,将携带SlSUVH1靶点的CRISPR载体导入目的植物中,获得了转基因剔除的SlSUVH1基因敲除番茄。获得的SlSUVH1基因敲除植物能够显著减轻番茄黄化曲叶病毒的侵染,抑制番茄黄化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SlSUVH1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SlSUVH1沉默效率的鉴定。
其中,(A)TRV-VIGS沉默番茄中SlSUVH1基因的表型图,TRV-GUS作为对照;(B)RT-qPCR检测各株系中SlSUVH1的沉默效率;
图2为通过TRV沉默SlSUVH1基因对番茄黄化曲叶病毒(TYLCV)的抗性分析。
其中,(A)TRV-VIGS沉默番茄中SlSUVH1基因后,TYLCV侵染20天后症状图;(B)qPCR分析TYLCV侵染沉默SlSUVH1基因的番茄20天后,TYLCV基因组DNA的积累水平。
图3为SlSUVH1基因敲除载体示意图。
图4为SlSUVH1基因敲除番茄的基因突变类型(A)及突变植物的表型(B)。
图5为SlSUVH1基因敲除植物对番茄黄化曲叶病毒(TYLCV)的抗性分析。
其中,(A)SlSUVH1基因敲除番茄植物在TYLCV侵染20天后症状图;(B)qPCR分析TYLCV侵染SlSUVH1基因敲除番茄植物番茄20天后,TYLCV基因组DNA的积累水平。
具体实施方式
一种抗番茄黄化曲叶病毒的SlSUVH1转基因植物的培育方法,具体包括以下步骤:
(1)首先通过烟草脆裂病毒诱导的基因沉默系统(TRV-VIGS)对番茄SlSUVH1进行了沉默,如图1所示,以TRV-GUS作为对照,TRV-SlSUVH1接种番茄植株中的SlUVH1被有效沉默,并对番茄生长无明显影响;
(2)在SlSUVH1沉默植株上接种TYLCV侵染性克隆,在接种20天后发现,SlSUVH1被沉默后对TYLCV具备较强的抗性(图2A)。取发病叶提取DNA,并通过qPCR检测病毒积累量,结果显示通过TRV沉默SlSUVH1的植株上病毒积累量显著低于对照(图2B)。以上结果说明SlSUVH1调控植物抗病毒,可作为基因编辑的重要靶点,培育抗病品种。
(3)针对番茄SlSUVH1基因编码区具备PAM位点(NGG特征,N=A,T,C,或G),利用在线软件http://cbi.hzau.edu.cn/crispr/,设计能够特异性靶向番茄内源基因SlSUVH1的gRNA,得到gRNA靶点序列,SlSUVH1:TTTGTGTGTGTTCCTCCAAGTGG,如SEQ ID NO:2所示;
设计引物进行PCR扩增,克隆得到SlSUVH1-gRNA-gRNA片段;其中,引物为
SlSUVH1-gRNA-F和SlSUVH1-gRNA-R序列分别如SEQ ID NO:3-4所示。
SlSUVH1-gRNA-F:tgattgtttg tgtgtgttcc tccaag;
SlSUVH1-gRNA-R:aaaccttgga ggaacacaca caaaca。
(4)然后将得到的引物经过退火得到双链DNA,使用碧云天Annealing Buffer forDNA Oligos(D0251)的方法。将步骤(3)中所述的SlSUVH1-gRNA-gRNA退火合成双链,具体步骤如下:
①将SlSUVH1-gRNA-gRNA干粉用ddH2O配置为50μM;
②在PCR管中配置反应体系:
ddH2O | 4μL |
Annealing Buffer for DNA Oligo | 2μL |
Oligo A | 2μL |
Oligo B | 2μL |
总共 | 10μL |
③用PCR仪器控温:95℃2分钟;每8秒下降0.1℃,降到25℃;4℃5分钟暂时存放;
④退火产物用于后续的连接。
(5)将BKG载体进行酶切与以上产物通过T4连接酶连接,转入DH5α大肠杆菌,菌液涂板于卡那霉素筛选的培养平板,鉴定阳性后进行质粒提取,获得重组质粒。SlSUVH1基因敲除载体示意图见图3。
(6)将1μl重组质粒与50μl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已经转入重组质粒的农杆菌。
(7)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。
(8)继续培养的小苗生长稳定后,取叶片样品,使用CTAB法抽提DNA;利用引物对M13-F与BKG-gRNA-R,以提取DNA为模板进行PCR扩增,确定植物中转入了外源
CRISPR-Cas9序列;引物M13-F和BKG-gRNA-R的序列分别如SEQ ID NO:5-6所示;
M13-F:GTTGTAAAACGACGGCCAG;
BKG-gRNA-R:GGCCATTTGTCTGCAGAATTGG。
(9)在阳性转基因材料上,采用BKG-SlSUVH1-F与BKG-SlSUVH1-R引物(如SEQ IDNO:7-8所示),克隆番茄SlSUVH1的基因靶点序列,
BKG-SlSUVH1-F:ttcagaactc ctgctgcgaa;
BKG-SlSUVH1-R:tccactgtag ccactaccga;
通过测序,筛选基因被编辑的株系,留种;如图4A所示,获得了一个碱基缺失的材料,命名为:Cas9-SlUVH1#1;获得了一个3碱基缺失的材料,命名为Cas9-SlUVH1#3;如图4B所示,两种基因编辑植物对于番茄生长发育无影响。
(10)将获得的SlUVH1基因敲除植物接种TYLCV侵染性克隆,在接种第20天观察发现,基因敲除植物具备较强的抗TYLCV的表型(图5A)。同时对发病毒叶进行取样,提取DNA,通过qPCR检测病毒积累量,结果显示两种SlUVH1基因敲除番茄植物株系的病毒积累量显著低于野生型番茄(图5B)。同时使用TYLCV外壳蛋白CP的特异性抗体,通过west blot检测了发病叶中的病毒积累量,结果显示,SlSUVH1敲除的两种不同番茄植物株系中的病毒外壳蛋白积累量要低于野生型番茄(图5C)。
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SlSUVH1基因在调控植物抗病毒中的应用,其特征在于:所述病毒为番茄黄化曲叶病毒;所述SlSUVH1基因的转录本序列如SEQ ID NO:1所示;所述植物为番茄;
采用农杆菌介导的T-DNA方式进行遗传转化,将基因敲除载体导入目的植物中,获得的番茄SlSUVH1敲除植株能够显著减轻番茄黄化曲叶病毒的侵染。
2.一种抗番茄黄化曲叶病毒的SlSUVH1转基因植物的培育方法,其特征在于:使用农杆菌介导的T-DNA方式进行遗传转化,将基因敲除载体导入目的植物中,获得的番茄SlSUVH1敲除植株,所述SlSUVH1基因的转录本序列如SEQ ID NO:1所示。
3.根据权利要求2所述的SlSUVH1转基因植物的培育方法,其特征在于,包括以下步骤:
(1)分别对SlSUVH1两个拷贝基因公共保守区域进行CRISPR靶点筛选,得到gRNA靶点序列,如SEQ ID NO:2所示;设计引物进行PCR扩增,克隆得到SlSUVH1-gRNA-gRNA片段;
(2)将SlSUVH1-gRNA-gRNA构建到带有CRISPR-Cas9载体上,获得重组质粒;
(3)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CRISPR-Cas9重组质粒的农杆菌;
(4)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;
(5)小苗生长稳定后,取叶片样品,使用CTAB法抽提DNA;利用引物对M13-F与BKG-gRNA-R,以提取DNA为模板进行PCR扩增,确定植物中转入了外源CRISPR-Cas9序列;
(6)在阳性转基因材料上,使用引物BKG-SlSUVH1-F与BKG-SlSUVH1-R克隆番茄SlSUVH1基因靶点序列,通过测序,筛选两个拷贝基因均被编辑的株系,留种;
(7)在得到的T1代种子中筛选纯合编辑株系,并在纯合株系中选择剔除CRISPR-Cas9骨架的植物进行收种,获得SlSUVH1基因敲除番茄。
4.根据权利要求3所述的SlSUVH1转基因植物的培育方法,其特征在于:所述步骤(1)中的引物为SlSUVH1-gRNA-F和SlSUVH1-gRNA-R,序列分别如SEQ ID NO:3-4所示。
5.根据权利要求4所述的SlSUVH1转基因植物的培育方法,其特征在于:所述M13-F和BKG-gRNA-R的序列如SEQ ID NO:5-6所示。
6.根据权利要求5所述的SlSUVH1转基因植物的培育方法,其特征在于:所述BKG-SlSUVH1-F、BKG-SlSUVH1-R的序列如SEQ ID NO:7-8所示。
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