CN117126880B - 本氏烟NbKTI1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
本氏烟NbKTI1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
本发明公开了一种本氏烟NbKTI1基因在调控植物抗病毒中的应用及转基因植物培育方法,所述病毒为芜菁花叶病毒,所述NbKTI1基因序列如SEQ ID NO:1所示。本发明使用Gateway体系构建了NbKTI1‑YFP的重组克隆载体,从烟草的cDNA扩增基因NbKTI1,通过BP反应连入pDONR221载体,然后通过LR反应连入含有YFP的重组载体,从而获得含有NbKTI1的过表达载体,通过农杆菌转化的方法将基因导入到烟草中并获得稳定遗传的过表达株系,实验结果表明,NbKTI1的过表达植物能够显著抑制芜菁花叶病毒的侵染。
Description
技术领域
本发明涉及基因工程领域,特别是涉及本氏烟NbKTI1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
芜菁花叶病毒(Turnip mosaic virus,TuMV),属于马铃薯Y病毒科、马铃薯Y病毒属的典型性病毒,也是农业生产中最具有危害性的植物病毒之一。TuMV的寄主范围十分广泛,能够侵染至少43个双子叶植物科植物,包括油菜、甘蓝、白菜、青花菜等十字花科作物和蔬菜。TuMV侵染植株后易造成植物矮化、黄萎、花叶等,严重影响经济作物产量和质量,危害我国农业生产,对我国农业造成非常严重的经济损失。
TuMV在自然界主要依靠昆虫介体传播,如蚜虫传播;此外也可通过汁液摩擦传播,多样的传播途径和极高的传播效率使得该病毒极难防治。我国每年花费大量人力物力用于TuMV的防控和防治但都收效甚微,至今还没有找到一种较为显著的抵御TuMV侵染、降低经济损失的高效途径。
植物只具有天然免疫系统,一旦感病,无药可治,所以植物病毒也被称为农作物的“绝症”,随着基因工程领域的全面快速发展,新型抗病毒手段层出不穷,而植物内源抗性基因的寻找与利用就是其中十分重要的技术手段,为病毒防控提供了很多新思路。
本发明发现了一种在植物抵御TuMV侵染中扮演重要角色的基因,并通过获取其稳定过表达的遗传株系实现了对病毒侵染的高效抑制,对TuMV的防控具有重要意义,也为病毒防控事业提供了借鉴意义。
发明内容
本发明的第一目的是提供一种本氏烟NbKTI1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NbKTI1转基因植物的培育方法。
本发明发现了一个抗病基因——本氏烟NbKTI1基因,构建了该基因融合YFP荧光标签的过表达载体NbKTI1-YFP,通过农杆菌转化的方法将其导入本氏烟中,获得了稳定遗传的高表达材料,可以实现对TuMV侵染的有效抑制,在一定程度上降低了TuMV的危害。
具体而言,本发明采用的技术方案具体如下:
本氏烟NbKTI1基因在调控植物抗病毒中的应用,其中,所述病毒为芜菁花叶病毒;所述NbKTI1基因序列如SEQ ID NO:1所示。
具体为,通过农杆菌转化将NbKTI1基因导入本氏烟中,获得的本氏烟NbKTI1高表达植株能够显著抑制TuMV的侵染。
一种NbKTI1转基因植物的培育方法,包括以下步骤:
(1)取健康的本氏烟叶片,用Trizol法提取RNA,使用oligo(dT)18
(5'-TTTTTTTTTTTTTTTTTT-3')引物反转录获得cDNA;
(2)使用引物对221-NbKTI1-F和221-NbKTI1-R,如SEQ ID NO:2-3所示,以上述步骤获得的cDNA为模板进行PCR扩增,克隆得到NbKTI1;
(3)通过BP反应将所获片段连接到中间载体pDONR221上;再通过LR反应将NbKTI1连接到携带有YFP荧光标签的双源表达载体上,获得NbKTI1-YFP的重组质粒;
(4)将500ng上述质粒加入100μl农杆菌感受态细胞中,通过化学转化法转化后摇菌培养,涂布到抗性培养基上,培养两天后,筛选出长出的阳性克隆,获得转入了NbKTI1-YFP的农杆菌;
(5)将带有NbKTI1-YFP的农杆菌侵染烟草叶片,经分化培养获得愈伤组织,再经过生根培养获得转入NbKTI1-YFP的本氏烟小苗,转移到土里继续培养;
(6)培养至4-5叶期时取样,CTAB法抽提DNA作为模板,利用引物221-NbKTI1-R与35S-F,如SEQ ID NO:3-4所示,选取扩得条带的株系,这些株系转入了NbKTI1-YFP,对这些株系再次取样,用尿素法提取蛋白质,进行SDS-PAGE凝胶电泳,Western blot检测荧光标签是否表达,使用GFP抗体检测确定植物表达了携带YFP荧光标签的NbKTI1-YFP;对以上阳性植株留种,获取T1代阳性种子进行实验。
同现有技术相比,本发明的突出效果在于:
本发明发现了抗病基因NbKTI1,将其克隆出并构建双源表达载体NbKTI1-YFP,将其转入植物后,获得了抵抗TuMV侵染的本氏烟株系,这种高表达抗病基因的株系可以实现对TuMV的有效抑制,从而减轻TuMV造成的危害,而且不需要施加任何药物,是一种高效、绿色的抗病技术。
下面结合附图说明和具体实施例对本发明所述的本氏烟NbKTI1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NbKTI1-YFP载体构建及表达鉴定。其中,
(A)NbKTI1-YFP载体示意图,35S promoter为花椰菜花叶病毒35S启动子,能够启动下游融合目标基因NbKTI1-YFP的表达,NOS为转录终止位点;
(B)将获得的NbKTI1-YFP的农杆菌注射到RFP-H2B(细胞核定位的转基因植物,细胞核发出红色荧光)转基因本氏烟草中观察亚细胞定位,在接种后的48小时在激光共聚焦显微镜下观察,分别拍摄NbKTI1-YFP,Bright,和RFP,以及Merge后的叠加图;
(C)将观察过亚细胞定位的注射过NbKTI1-YFP的RFP-H2B材料重新取样,提取蛋白质,进行SDS-PAGE凝胶电泳,Western blot检测荧光标签的表达,GFP抗体检测到50KDa蛋白大小,NbKTI1-YFP也是50KDa,Ponceau S为上样量分析,WT为野生型本氏烟对照。
图2为NbKTI1-YFP转基因植物的鉴定。其中,
(A)野生型本氏烟和NbKTI1-YFP转基因植物在4-5叶期的生长状况,比例尺=3cm;
(B)Western blot检测转基因植物中NbKTI1-YFP的正常表达,WT为野生型本氏烟对照,Ponceau S为上样量水平分析;
(C)NbKTI1-YFP转基因植物的亚细胞定位,共聚焦观察转基因本氏烟叶片确定NbKTI1-YFP能够正常表达;
(D)RT-qPCR检测NbKTI1-YFP表达水平,WT为野生型本氏烟对照,**代表显著性水平P<0.01。
图3为瞬时过表达NbKTI1抑制TuMV侵染。将携带Myc标签的Myc-NbKTI1和Myc-GUS接种到长势一致的本氏烟24小时后,取接种过TuMV-GFP的发病植物研磨出汁液摩擦接种,摩擦后60小时取被摩擦的叶片。其中,
(A)Western blot分析TuMV的蛋白积累量,WT为野生型本氏烟对照,Ponceau S为上样量水平分析;
(B)病毒积累量的RNA水平分析,提取60小时样品的RNA,反转录为cDNA,RT-qPCR检测病毒的表达水平,**代表显著性水平P<0.01;
(C)摩擦后6天的发病植物表型图,分别在紫外灯和白光下拍摄;
(D)摩擦后6天取系统叶,Western blot分析TuMV的蛋白积累量,WT为野生型本氏烟对照,Ponceau S为上样量水平分析;
(E)系统叶病毒积累量的RNA水平分析,提取6天样品的RNA,反转录为cDNA,RT-qPCR检测病毒的表达水平,**代表显著性水平P<0.01。
图4为NbKTI1-YFP转基因植物对TuMV的抗性分析。在野生型本氏烟和NbKTI1-YFP的Line 2、Line 12上接种TuMV-GFP。其中,
(A)为接种后6天的发病植物表型图,分别在紫外灯和白光下拍摄,比例尺=3cm;
(B)为图(4A)中的系统叶病毒积累量的RNA水平分析,提取6天系统叶样品的RNA,反转录为cDNA,RT-qPCR检测病毒的表达水平,**代表显著性水平P<0.01;
(C)为图(4A)中的系统叶的病毒蛋白积累水平分析,Western blot分析TuMV的蛋白积累量,WT为野生型本氏烟对照,Ponceau S为上样量水平分析。
具体实施方式
一种抗芜菁花叶病毒的转基因植物培育方法,具体步骤为:
(1)取健康的本氏烟叶片,用Trizol法提取RNA,使用oligo(dT)18
(5'-TTTTTTTTTTTTTTTTTT-3')引物反转录获得cDNA;
(2)使用引物对221-NbKTI1-F和221-NbKTI1-R,如SEQ ID NO:2-3所示,
221-NbKTI1-F:
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAAGACCAAACAACTTTTT;
221-NbKTI1-R:
GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCCTTCTTAAACATAACCTT。
以上述步骤获得的cDNA为模板进行PCR扩增,克隆得到NbKTI1;
(3)通过BP反应将所获片段连接到中间载体pDONR221上;
回收产物 | 4μL |
pDONR 221 | 0.5μL |
BP酶mix | 0.5μL |
总共 | 5μL |
①混合上述体系,轻轻吸打混匀后放入PCR仪中,25℃恒温孵育4小时;
②将反应产物取出加入到100μL大肠杆菌感受态细胞中进行转化,摇菌培养后涂布到抗性培养基上过夜生长;
③挑选单菌落进行PCR验证,引物为221-NbKTI1-R和M13F,如SEQ ID NO:3和SEQIDNO:5所示;
M13F:TGTAAAACGACGGCCAGT。
2×taq mix | 10μL |
M13F,10μM | 0.5μL |
221-NbKTI1-R,10μM | 0.5μL |
ddH2O | 9μL |
④将鉴定成功的菌落摇菌培养,提取质粒,获得pDONR221-NbKTI1。
(4)再通过LR反应将NbKTI1连接到携带有YFP荧光标签的双源表达载体上,获得NbKTI1-YFP的重组质粒,如图1A所示;
①将pDONR221-NbKTI1质粒用MluI酶切;
②将纯化回收的产物用LR酶(Invitrogen)连接到pEarleygate 101载体,该载体携带YFP标签;
回收产物 | 4μL |
pEarleygate 101 | 0.5μL |
LR酶mix | 0.5μL |
总共 | 5μL |
③混合上述体系,轻轻吸打混匀后入PCR仪中,25℃恒温孵育4小时;
④将反应产物取出加入到100μL大肠杆菌感受态细胞中进行转化,摇菌培
养后涂布到抗性培养基上过夜生长;
⑤挑选单菌落进行PCR验证,引物为221-NbKTI1-R和35s-F,如SEQ ID NO:3-4所示;
35S-F:CGCAAGACCCTTCCTCTATATAAGGAA。
2×taq mix | 10μL |
35s-F,10μM | 0.5μL |
221-NbKTI1-R,10μM | 0.5μL |
ddH2O | 9μL |
⑥将鉴定成功的菌落摇菌培养,提取质粒,获得NbKTI1-YFP重组质粒。
(5)将500ng质粒加入100μl农杆菌感受态细胞中,使用化学转化法转化后摇菌培养,涂布到抗性培养基上,28度培养两天后,筛选出长出的阳性克隆,获得转入了NbKTI1-YFP的农杆菌。
(6)将转入NbKTI1-YFP的农杆菌注射到细胞核定位的转基因植物RFP-H2B中,表达48小时后观察亚细胞定位(图1B),然后取注射叶片Western blot检测荧光标签是否表达,使用GFP抗体检测确定植物表达了携带YFP荧光标签的NbKTI1-YFP(图1C)。
(7)将携带Myc标签的Myc-NbKTI1和Myc-GUS接种到长势一致的本氏烟24小时后,取接种过TuMV-GFP的发病植物研磨出汁液摩擦接种,摩擦后60小时取被摩擦的叶片,Western blot分析TuMV的蛋白积累量,发现Myc-NbKTI1可以显著抑制病毒积累,如图3A;WT为野生型本氏烟对照,Ponceau S为上样量水平分析。同时分析病毒的RNA水平积累,提取60小时样品的RNA,反转录为cDNA,RT-qPCR检测病毒的表达水平,RNA同蛋白水平一样显著下调,如图3B,**代表显著性水平P<0.01。以上摩擦接种的植物在第6天进行紫外灯和白光下拍照(图3C),通过图3C可以看出,过表达Myc-NbKTI1相对于Myc-GUS,能够显著抑制病毒症状的发展与抑制病毒携带荧光GFP的亮度;Western blot和RT-qPCR进一步分析TuMV侵染6天后系统叶片的蛋白(图3D)与RNA积累量(图3E),发现Myc-NbKTI1可以显著抑制病毒蛋白(图3D)与RNA积累(图3E)。
(8)确定瞬时过表达NbKTI对TuMV有抑制效果后,将带有NbKTI1-YFP的农杆菌侵染烟草叶片,经分化培养获得愈伤组织,再经过生根培养获得转入NbKTI1-YFP的本氏烟小苗,转移到单独的盆子中培养,如图2A;培养至4-5叶期时取样,CTAB法抽提DNA作为模板,利用引物35S-F与221-NbKTI1-R,如SEQ ID NO:4和SEQ ID NO:3所示,选取扩得条带的株系,这些株系转入了NbKTI1-YFP,对这些株系再次取样,用尿素法提取蛋白质,进行SDS-PAGE凝胶电泳,Western blot检测荧光标签是否表达,使用GFP抗体检测确定植物表达了带YFP荧光标签的NbKTI1-YFP,如图2B、2C、2D,获取了Line 2、Line 12两个株系,对以上阳性植株留种。
(9)播种本氏烟野生型及NbKTI1-YFP转基因植物株系Line 2、Line 12。生长4周后,如图4A所示,接种TuMV-GFP,接种后60小时取接种叶分别提取蛋白质和RNA,Westernblot检测蛋白水平的病毒积累,RT-qPCR检测病毒RNA水平积累(如图4B),接种后6天取发病系统叶,分别提取蛋白质和RNA,Western blot检测蛋白水平的病毒积累,RT-qPCR检测病毒RNA水平积累,实验表明转基因两个株系的病毒蛋白积累量和RNA表达水平显著低于野生型本氏烟,如图4C。
实验结果表明:通过农杆菌遗传转化的方法获得的本氏烟NbKTI1过表达植物能够显著减轻TuMV的侵染,抑制TuMV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 (3)
1.本氏烟NbKTI1基因在调控植物抗病毒中的应用,其特征在于:所述病毒为芜菁花叶病毒;所述NbKTI1基因序列如SEQ ID NO:1所示;所述植物为本氏烟。
2.根据权利要求1所述本氏烟NbKTI1基因在调控植物抗病毒中的应用,其特征在于:通过农杆菌转化将NbKTI1基因导入本氏烟中,获得的本氏烟NbKTI1高表达植株能够显著抑制芜菁花叶病毒的侵染。
3.一种NbKTI1转基因植物的培育方法,其特征在于,包括以下步骤:
(1)取野生型的本氏烟材料,使用Trizol法抽提取RNA,使用序列为5'-TTTTTTTTTTTTTTTTTT-3'的oligo(dT)18引物,反转录获得cDNA;
(2)使用引物对221-NbKTI1-F和221-NbKTI1-R,如SEQ ID NO:2-3所示,以步骤(1)获得的cDNA为模板进行PCR扩增,克隆得到NbKTI1;
(3)通过BP反应将所获片段连接到中间载体pDONR221上,再通过LR反应将NbKTI1连接到携带有YFP荧光标签的双源表达载体上,获得NbKTI1-YFP的重组质粒;
(4)将上述质粒加入到100微升农杆菌感受态细胞中,通过化学转化法转入农杆菌中,培养后涂布到抗性培养基上筛选获得带有NbKTI1-YFP重组质粒的农杆菌;
(5)将上述农杆菌侵染烟草叶片,经分化培养获得愈伤组织,生根培养获得烟草幼苗,转移到土里继续培养;
(6)幼苗生长稳定后,取叶片样品,使用CTAB法抽提DNA,利用引物对221-NbKTI1-R与35S-F,如SEQ ID NO:3-4所示,以提取的DNA为模板进行PCR扩增,确定植物中转入了外源NbKTI1-YFP的转录本;同时提取总蛋白,进行SDS-PAGE凝胶电泳,使用GFP抗体进行Westernblot检测确定植物正常表达了携带YFP荧光标签的NbKTI1-YFP;将以上阳性植株留种。
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