CN116837004A - 嵌合结构imc及其在黄瓜绿斑驳花叶病毒抗性烟草培育中的应用 - Google Patents
嵌合结构imc及其在黄瓜绿斑驳花叶病毒抗性烟草培育中的应用 Download PDFInfo
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Abstract
本发明公开嵌合结构IMC及其在黄瓜绿斑驳花叶病毒基因抗性烟草培育中的应用。针对现有技术未公开对CGMMV基因间隔区域的注释,也没有构思过从间隔区域筛选目的片段用于抗性烟草培育,本发明提供CGMMV基因间隔区域在介导抗性烟草培育中的应用。本发明构建嵌合结构IMC,由CGMMV基因间隔区域碱基片段IR4、MP基因片段MP1、CP基因片段CP1连接而成。成功用于植物干涉载体实现抗性烟草培育。本发明还提供利用这些目的片段介导转基因抗性烟草的完整技术方案。
Description
本申请是中国发明专利申请CN2021114824401(黄瓜绿斑驳花叶病毒基因间隔区域在抗性烟草培育中的应用,申请日20211206)的分案申请。
技术领域
本发明涉及一种植物抗性育种,特别是涉及一种由黄瓜绿斑驳花叶病毒CGMMV基因介导的抗性烟草培育技术,属于基因工程、遗传育种领域。
背景技术
黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)属于烟草花叶病毒属(Tobamovirus spp.)。CGMMV能够以西瓜、甜瓜、南瓜、瓠瓜等较多种葫芦科(Cucurbitaceae)植物作为其自然寄主,且是葫芦科植物最主要病毒之一。由于葫芦科是世界范围内仅次于禾本科、豆科与茄科的重要食用植物科,因而黄瓜绿斑驳花叶病毒容易引发田间连续大面积感染与农业生产的规模性损失,已被世界许多国家与地区都列入检疫对象。CGMMV也可侵染苋色藜(Chenopodium Amaranticolor)、曼陀罗(Datura stramonium)、菟丝子(Cuscuta chinensis)、本氏烟(Nicotiana benthamiana)、珊西烟(Nicotiana tabacum var Xanthi nc)等。本氏烟是黄瓜绿斑驳花叶病毒的试验寄主,以本氏烟作为研究对象,通过转基因的手段可以更快的解析该病毒的侵染机制。构建本氏烟草抗性材料在CGMMV研究中意义突出。
CGMMV是正义单链RNA病毒,基因组全长6423nt,5′端有60nt的非编码区,3′端有175nt的非编码区,中间部分有三个编码区。第一个编码区是与病毒复制相关的129KDa和186KDa蛋白,统称为复制相关蛋白。这两个蛋白具有共同的N端。129KDa复制相关蛋白包含甲基转移酶(Vmethyltransforase)与RNA解旋酶(RNA Helicase)结构域以及两者之间的间隔区域(Internal Region,IR)。186KDa复制相关蛋白通读区域对应依赖RNA的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)结构域。第二编码区是运动蛋白MP(MovementProtein)主要介导病毒细胞间运动。第三编码区是外壳蛋白CP(Coat Protein)主要介导病毒长距离运动。
利用病毒基因介导获得转基因植株抗性材料是植物抗性育种重要手段。在CGMMV的该类现有技术中,用以介导植物抗性的病毒基因目标片段均来自于运动蛋白MP与外壳蛋白CP。分别是:将CGMMV的外壳蛋白CP基因构建到植物表达载体,通过农杆菌转化西瓜砧木“gongdae”,获得具有抗性的转基因砧木材料(Transgenic watermelon rootstockresistant to CGMMV (cucumber green mottle mosaic virus) infection. Plant Cell Rep (2005) 24: 350-356);采用限制性酶切连接的方法将CGMMV的外壳蛋白CP基因构建到载体中,RNA沉默转基因烟草具有抗性(Transgenic Nicotiana benthamiana plantsresistant to cucumber green mottle mosaic virus based on RNA silencing. Plant Cell Rep (2007) 26:1283-1288);将CGMMV的运动蛋白MP基因正向重复构建到植物表达载体,通过农杆菌转化甜瓜,获得了具有抗性的转基因甜瓜(Molecular analysis oftransgenic melon plants showing virus resistance conferred by direct repeatof movement gene of Cucumber green mottle mosaic virus. Plant Cell Rep (2012)31:1371-1377)。
从已知的CGMMV基因组看,第一编码区相较于第二编码区运动蛋白MP与第三编码区外壳蛋白CP序列更长,能够为筛选具备介导植株抗性功能的目的基因片段提供更丰富的资源库。同时,在第一编码区中,位于含甲基转移酶与RNA解旋酶结构域两者之间的间隔区域(IR)尚未被现有研究注释功能,若能从中有效地筛选出能够介导植株抗性的目的基因片段,则能扩展现有研究对间隔区域功能的认识,表明其并非单纯间隔甲基转移酶与RNA解旋酶,可能有比较重要的功能。
发明内容
本发明的目的就是针对现有技术的不足,提供CGMMV基因间隔区域基因在抗性烟草培育中的应用。
本发明发现利用独立的CGMMV基因间隔区域能够构建植物干涉表达载体介导抗性烟草,并且利用CGMMV基因间隔区域与CGMMV非间隔区域片段组成嵌合结构所构建的植物干涉表达载体也能介导抗性烟草。
本发明首先提供如下技术方案如下:
嵌合结构IMC,其特征在于:由CGMMV基因间隔区域碱基片段IR4(序列如SEQ IDNO.1所示)、MP基因片段MP1(序列如SEQ ID NO.2所示)、CP基因片段CP1(序列如SEQ IDNO.3所示)构成。
上述嵌合结构IMC是由CGMMV基因间隔区域碱基片段IR4(间隔区域193bp,如SEQID NO.1所示)、MP基因片段MP1(MP 142bp,如SEQ ID NO.2所示)、CP基因片段CP1(CP128bp,如SEQ ID NO.3所示)连接组成,序列如SEQ ID NO.4所示。
本发明试验证明,嵌合结构IMC能够介导转基因烟草抗性。因而,本发明同时提供如下技术方案:
包含上述嵌合结构IMC的载体。
上述载体包括重组质粒、植物干涉表达载体等。
上述嵌合结构IMC在CGMMV基因介导烟草抗性中的应用。
本发明基于试验数据,提供利用黄瓜绿斑驳花叶病毒基因组间隔区域片段构建重组质粒培育抗性烟草的技术方案,具体如下:
利用黄瓜绿斑驳花叶病毒基因组间隔区域片段培育抗性烟草的方法,其特征在于:依如下步骤实施:
首先,根据CGMMV目的片段设计PCR扩增引物对,经PCR扩增后回收纯化得到各PCR产物,所述CGMMV目的片段序列如SEQ ID NO.4所示;
其次,将各PCR产物与干涉载体pHellsgate2进行重组反应,重组产物转化大肠杆菌DH5α,提取质粒,筛选序列正确的阳性重组质粒;
再次,各阳性重组质粒转化农杆菌筛选含阳性ihpRNAi的农杆菌;
最后,用含阳性ihpRNAi的农杆菌转化烟草。
综合上述各方案,本发明提供如下技术方案:
黄瓜绿斑驳花叶病毒间隔区域在抗性烟草培育中的应用。
与现有技术相比,本发明的有益效果是:(1)现有技术未公开过CGMMV基因组第一编码区的间隔区域(IR)的功能注释,更未构思过从该区域筛选目的片段建立病毒内源基因介导转基因烟草抗性技术体系,本发明部分地注释了CGMMV基因间隔区域的功能,该功能在于能够应用于CGMMV转基因抗性烟草培育,且其技术效果不低于由MP基因、CP基因参与介导的抗性烟草培育。(2)构建了利用CGMMV基因间隔区域、MP基因、CP基因组成的碱基嵌合结构IMC,能够用于构建植物干涉载体,实现转基因烟草培育。(3)建立了多个利用CGMMV病毒基因间隔区域及其与MP基因、CP基因组合介导转基因抗性烟草的完整技术方案。
附图说明
图1是IR4、MP1、CP1目的片段PCR扩增结果。
图2是IR-F/CP-R扩增全长(a)的酶切验证结果(b)。
图3是转化农杆菌的PCR验证结果(IMC)。
图4是实施例五nptII引物检测结果。
图5是35S、CP-R引物对检测结果。
图6是病毒接种攻毒试验结果(示接种后1月叶片症状)
具体实施方式
下面结合附图,对本发明的优选实施例作进一步的描述。
实施例一
构建包含IR间隔区域片段的嵌合结构的植物干涉表达载体。
选取CGMMV病毒中三个区段作为目的片段,包括:IR间隔区域193bp(IR4,如SEQ IDNO.1所示),MP基因142bp(MP1,如SEQ ID NO.2所示),CP基因128bp(CP1,如SEQ ID NO.3所示)。根据三条目的片段序列特征分别设计扩增引物对(见表1),
用IR-F与IR+MP-R,IR+MP-F与MP+CP-R,MP+CP-F与CP-R三对引物PCR分别扩增对应目的片段,最后用IR-F与CP-R扩增重组片段,得到嵌合结构IMC序列(如SEQ ID NO.4所示,463bp)。
PCR反应体系、PCR条件及程序、试剂/材料/操作记载于本申请母案CN2021114824401实施例一、实施例五。
图1是IR4、MP1、CP1目的片段PCR扩增结果。
表1 IR4、MP1、CP1目的片段PCR引物对
目的片段 | PCR扩增引物对 |
IR4(IR 193bp) | IR-F(CN 2021114824401,SEQ ID NO.15)IR+MP-R(CN 2021114824401,SEQ ID NO.16) |
MP1(MP 142bp) | IR+MP-F(CN 2021114824401,SEQ ID NO.17)MP+CP-R(CN 2021114824401,SEQ ID NO.18) |
CP1(CP 128bp) | MP+CP-F(CN 2021114824401,SEQ ID NO.19)CP-R(CN 2021114824401,SEQ ID NO.20) |
PCR产物回收纯化后构建包含IMC序列的阳性质粒pHe-IMC、干涉植物表达载体ihpRNAi载体。构建试剂/材料/方法记载于本申请母案CN 2021114824401实施例一、实施例五。
图2是IR-F/CP-R扩增全长的酶切验证结果;图3是转化农杆菌的PCR验证结果(IMC)。
实施例二
烟草遗传转化。
利用实施例一所得ihpRNAi载体转化烟草(本氏烟(Nicotiana benthamiana)),得转基因烟草植株,并采集T0代种子备用。转化试剂/材料/操作记载于本申请母案CN2021114824401实施例一、实施例五。
实施例三
转基因烟草植株PCR阳性检测。
检测实验例二所得转基因烟草植株。检测试剂/材料/操作记载于本申请母案CN2021114824401实施例一、实施例五。
图4是nptII引物检测结果;图5是CP-R引物检测结果(35S)。
实施例四
病毒接种攻毒试验
接种攻毒实施例二所得转基因烟草植株,并观察记录病毒病发生情况。试剂/材料/操作记载于本申请母案CN 2021114824401实施例一、实施例五。
WT材料:在接种10d时全部发病,表现为所有的材料心叶和第二片叶均呈花叶、皱缩。
转基因材料:接种10d时,10个株系发病植株少,IMC-11,IMC-13,IMC-5,IMC-17,IMC-18没有发病植株,IMC-5发病1株,IMC-3,IMC-11,IMC-16发病2株,IMC-2发病3株;接种20d时,转基因材料只有IMC-2、IMC-5株系发病增加,达到5株;接种30d时,各株系发病情况与接种后20d时维持一样,没有增加趋势,表明30d统计发病率能够表现T1代材料的分离比例。10个株系发病率不同,说明分离比不同,在T1代转基因植株健康生长,均表现抗性。
图6是病毒接种攻毒试验结果(示接种后1月叶片)。图6显示野生型和转基因株系第5片叶子的表型,WT显示花叶皱缩,转基因株系IMC-13,IMC-17,IMC-18叶片表现为健康状态。
表2 IMC转基因植株对CGMMV病毒抗性鉴定
接种一个月后,用DAS-ELISA检测转基因IMC-13、IMC-15、IMC-18三个株系与WT材料接种CGMMV的情况,WT的ELISA检测指数高于3,平均值高达11.9。三个转基因株系的ELISA检测指数均小于3,说明在转基因株系当中并未检测到CGMMV病毒。
表3 DAS-ELISA分析结果(30dpi)
株系 | I/H | 株系 | I/H | 株系 | I/H | 株系 | I/H |
WT-1 | 10.9 | IMC-13-1 | 1.7 | IMC-15-1 | 2.4 | IMC-18-1 | 1.8 |
WT-2 | 11.7 | IMC-13-2 | 1.6 | IMC-15-2 | 1.6 | IMC-18-2 | 1.3 |
WT-3 | 13.0 | IMC-13-3 | 1.5 | IMC-15-3 | 1.8 | IMC-18-3 | 1.6 |
注:I/H比值≥3.0 为阳性材料. 阳性对照I/H比值为11.9。
CGMMV侵染烟草后,感染病毒的WT不仅叶片表现花叶、皱缩,而且整个植株比三个转基因株系矮,影响植株生长,造成不可逆的伤害。
表4 接种30d后统计株高结果(30dpi)
株系 | 株高(cm) | 株系 | 株高(cm) | 株系 | 株高(cm) | 株系 | 株高(cm) |
WT-1 | 26.7 | IMC-13-1 | 32.5 | IMC-15-1 | 32 | IMC-18-1 | 32.5 |
WT-2 | 26.2 | IMC-13-2 | 29.2 | IMC-15-2 | 33.5 | IMC-18-2 | 32.6 |
WT-3 | 25.5 | IMC-13-3 | 28.5 | IMC-15-3 | 31.6 | IMC-18-3 | 33 |
以上实施例一~四结果表明:利用CGMMV病毒IR间隔区域碱基片段能够与病毒CP、MP结构域的片段组成嵌合结构,也能够应用于病毒基因介导抗性烟草培育。
技术方案所得抗性植株在整个生育期调查均没发病。这表明,CGMMV病毒IR间隔区域能够作为内源基因介导CGMMV抗性烟草,且技术效果不低于传统由病毒MP片段、CP片段为内源基因的介导效果。
Claims (9)
1.嵌合结构IMC,其特征在于:由CGMMV间隔区域碱基片段IR4(SEQ ID NO.1)、MP基因片段MP1(SEQ ID NO.2)、CP基因片段CP1(SEQ ID NO.3)构成,序列如SEQ ID NO.4所示。
2.权利要求1所述的嵌合结构IMC在CGMMV基因介导烟草抗性中的应用。
3.根据权利要求2所述的应用,其特征在于:所述烟草是本氏烟(Nicotiana benthamiana)。
4.烟草干涉表达载体,其特征在于:包含权利要求1所述嵌合结构IMC。
5.根据权利要求4所述的烟草干涉表达载体,其特征在于:是具有发夹结构的反向重复序列载体ihpRNAi。
6.根据权利要求5所述的烟草干涉表达载体,其特征在于:回收纯化的所述黄瓜绿斑驳花叶病毒间隔区域基因片段PCR产物与干涉载体pHellsgate2进行BP重组反应构建。
7.重组质粒,其特征在于:包含权利要求1所述嵌合结构IMC。
8.利用嵌合结构IMC培育抗性烟草的方法,其特征在于:依如下步骤实施:
首先,根据CGMMV目的片段设计PCR扩增引物对,经PCR扩增后回收纯化得到各PCR产物,所述目的片段是序列SEQ ID NO.4所示嵌合结构IMC;
其次,将各PCR产物与干涉载体pHellsgate2进行重组反应,重组产物转化大肠杆菌DH5α,提取质粒,筛选序列正确的阳性重组质粒;
再次,各阳性重组质粒转化农杆菌筛选含阳性ihpRNAi的农杆菌;
最后,用含阳性ihpRNAi的农杆菌转化烟草。
9.根据权利要求8所述的培育抗性烟草的方法,其特征在于:所述烟草是本氏烟(Nicotiana benthamiana)。
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