CN117143872A - 一种用于抑制根结线虫病的RNAi靶标基因及其应用 - Google Patents
一种用于抑制根结线虫病的RNAi靶标基因及其应用 Download PDFInfo
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明公开了一种用于抑制根结线虫病的RNAi靶标基因及其应用,属于生物技术及农业应用领域,该RNAi的核苷酸序列如SEQ ID No.3所示,其获得步骤如下:(1)提取南方根结线虫的总RNA,通过反转录操作获得cDNA;(2)以步骤(1)中得到的cDNA作为模板,以SEQ ID No.1与SEQ ID No.2所示序列作为引物,通过PCR扩增得到MiMSP30全长序列;(3)以MiMSP30的全长序列作为模板,以SEQ ID No.4与SEQ ID No.5所示序列、或SEQ ID No.6与SEQ ID No.7所示序列分别作为引物,通过PCR反应扩增得到可作为dsRNA正、反链合成模板的DNA序列。对根结线虫游离2龄幼虫饲喂dsRNA‑MSP30的MiMSP30沉默效率达70.02%,根结线虫的侵染虫口数显著下降,根内发育进程出现明显滞后,根结与卵块数均显著减少。
Description
技术领域
本发明属于生物技术及农业应用领域,具体涉及一种用于抑制根结线虫病的RNAi靶标基因及其应用。
背景技术
根结线虫(Meloidogyne spp.)是世界性分布的植物根系专性内寄生物,是威胁农业生产的主要病原物,也是世界各国的对外检疫对象。目前生产上以化学药剂为主防治根结线虫病害,然而化学杀线剂不仅对环境污染严重,对人、畜也不安全。在溴甲烷、涕灭威、除线磷等一系列化学杀线剂被禁止使用后,能够有效防治病害且对环境友好的生物防治技术逐渐成为研究热点。
南方根结线虫食道腺外泌蛋白(M.incognita putative esophageal glandcellsecretory protein 30,MSP30)通过调节植物细胞壁与免疫相关基因的转录水平以减弱植物对根结线虫的防御,促进根结线虫的侵染与发育进程,从而正向调控根结线虫的致病水平,是根结线虫致病的关键蛋白之一,其编码基因可作为根结线虫病防治的新靶标。
RNA干扰(RNA interference,RNAi)是存在于真核生物中的保守性过程,当真核细胞内存在外源或内源的双链RNA(dsRNA)或小干扰RNA(smallinterfering RNA,siRNA)时,其靶标mRNA会在Dicer蛋白与AGO蛋白的作用下被切割,导致靶标mRNA对应编码基因的表达受到干扰。通过在真核细胞内合成dsRNA,或将体外合成的dsRNA或siRNA导入真核细胞内,均能达到沉默特定基因的目的。当前RNAi已成为基因沉默的一种常用手段。
寄主介导的基因沉默(host-induced gene silencing,HIGS)是指植物产生了与病原物或昆虫特定基因序列互补的RNA分子,该RNA分子经植物细胞Dicer蛋白加工后特异性识别病原物基因序列并对其进行沉默,从而干扰病原物的发育或致病进程,抑制病害或虫害的发生与发展。目前,HIGS技术已被运用于多种重要农作物的根结线虫与昆虫防治当中。
发明内容
本发明的目的在于提供一种用于抑制根结线虫病的RNAi靶标基因及其应用,以解决上述背景技术中提出的问题。
为实现上述目的,本发明的第一个方面,提供一种用于抑制根结线虫病的RNAi靶标基因,该RNAi靶标基因的核苷酸序列如SEQ ID No.3所示。
该RNAi靶标基因的的体外合成方法包括以下步骤:
(1)提取南方根结线虫的总RNA,通过反转录操作获得cDNA;
(2)以步骤(1)中得到的cDNA作为模板,以SEQ ID No.1与SEQ ID No.2所示序列作为引物,通过PCR扩增得到MiMSP30全长序列。
本发明的第二个方面,提供针对所述靶标基因的RNAi在根结线虫病防控中的应用。
对靶标基因MiMSP30具备RNAi效果的dsRNA体外合成方法包括以下步骤:
(1)以MiMSP30的全长序列作为模板,以SEQ ID No.4与SEQ ID No.5所示序列、或SEQ ID No.6与SEQ ID No.7所示序列分别作为引物,通过PCR反应扩增得到可作为dsRNA正、反链合成模板的DNA序列;
(2)以步骤(1)中回收得到的PCR扩增产物作为模板,分别转录合成dsRNA的正链与反链,退火使正、反链杂交形成dsRNA;
(3)酶解去除残余的单链RNA(ssRNA)与DNA,并对酶解产物进行回收与纯化,即获得对靶标基因MiMSP30具备RNAi效果的dsRNA(dsRNA-MSP30)。
针对靶标基因MiMSP30具备RNAi效果的HIGS方法包括如下步骤:
(1)以MiMSP30的全长序列作为模板,以SEQ ID No.14与SEQ ID No.15所示序列作为引物,通过PCR反应扩增得到MiMSP30序列,并将其插入HIGS载体序列中,得到同时携带有MiMSP30序列与MiMSP30反向互补序列的HIGS载体;
(2)将步骤(1)中所得的HIGS载体转化入农杆菌GV3101中;
(3)通过农杆菌介导的植物转基因体系,将步骤(1)中所得的同时携带有MiMSP30序列与MiMSP30反向互补序列的HIGS载体序列转化入植物当中,得到能够在体内合成dsRNA-MSP30的转基因植物。
本发明的第三个方面在于提供一种生物制品,其含有上述RNAi靶标基因。
进一步的,所述生物制品为饲喂式杀虫剂或饵剂。
与现有技术相比,本发明的有益效果是:
以对绿色荧光蛋白编码基因GFP具有RNAi效果的dsRNA(dsRNA-GFP)作为对照,对根结线虫游离2龄幼虫饲喂dsRNA-MSP30,并统计摄入dsRNA后根结线虫的侵染虫口数、根内发育进程、根结数、卵块数以及dsRNA基因沉默效率。实验结果表明,饲喂dsRNA-MSP30的MiMSP30沉默效率达70.02%,根结线虫的侵染虫口数显著下降,根内发育进程出现明显滞后,根结与卵块数均显著减少。
以野生型植株作为对照,对能够在体内合成dsRNA-MSP30的HIGS转基因植物的根结数、卵块数以及dsRNA基因沉默效率进行统计。实验结果表明,野生型根结线虫侵染HIGS-MSP30转基因植株后,MiMSP30沉默效率达46.32%,根结数与卵块数均显著减少。
附图说明
图1为实施例3中用dsRNA-GFP与dsRNA-MSP30分别饲喂南方根结线虫二龄幼虫4h后,MiMSP30的相对表达水平检测结果。
图2为实施例3中经dsRNA-GFP与dsRNA-MSP30分别饲喂后,南方根结线虫接种拟南芥7天后的成功侵染虫口数统计结果。
图3为实施例3中经dsRNA-GFP与dsRNA-MSP30分别饲喂后,南方根结线虫接种拟南芥14天后在根内的虫龄发育进程统计结果。
图4为实施例4中经dsRNA-GFP与dsRNA-MSP30分别饲喂后,南方根结线虫接种番茄45天后的根结数统计结果。
图5为实施例4中经dsRNA-GFP与dsRNA-MSP30分别饲喂后,南方根结线虫接种番茄45天后的卵块数统计结果。
图6为实施例6中用野生型南方根结线虫二龄幼虫侵染HIGS-MSP30转基因植株后,MiMSP30的相对表达水平检测结果。
图7为实施例6中用野生型南方根结线虫二龄幼虫侵染HIGS-MSP30转基因植株45天后的根结数统计结果。
图8为实施例6中用野生型南方根结线虫二龄幼虫侵染HIGS-MSP30转基因植株45天后的卵块数统计结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
1.供试南方根结线虫饲养
取受南方根结线虫侵染45-60天后的番茄根系,轻柔洗净后挑取成熟卵块,将卵块以滤网隔离浸没入无菌水中,28℃孵育。每3天收取一次无菌水并将卵块浸没入新的无菌水中,以此往复,直至无新线虫孵出。收取得的无菌水中存在孵化出的线虫,即为线虫悬液。
2.南方根结线虫总RNA提取
针对游离南方根结线虫二龄幼虫的总RNA提取步骤如下:
①将线虫悬液(含20,000头线虫)浓缩至100μL体积后,用液氮反复冻融5次。
②加入1mL TRIzol(Invitrogen)后涡旋振荡混匀样品,并用超声波破碎仪(南京以马内利仪器设备有限公司)破碎10min。
③加入200μL氯仿(沪试)涡旋振荡3min后,12,000rpm 4℃离心15min,收集上清并转移至洁净的无RNase 1.5mL离心管中。
④加入上清液2.2倍体积的无水乙醇(沪试),颠倒混匀后置于-20℃过夜孵育。
⑤12,000rpm 4℃离心15min去除上清液,用无RNase的70%乙醇溶液漂洗沉淀2次。
⑥12,000rpm 4℃离心去除乙醇溶液,开盖室温放置5-10min。
⑦加入20-40μL的无RNase去离子水,冰浴30min溶解沉淀后,吸打均匀,得到南方根结线虫的总RNA,保存于-80℃。
3.南方根结线虫cDNA合成
按照III RT SuperMix for qPCR(+gDNA Remover)(擎科生物)说明书进行南方根结线虫cDNA的合成。具体步骤如下:
①将南方根结线虫RNA样品及各反应组分置于冰上溶解后,配置去基因组反应体系(10ng-2μg总RNA,2μL 5×gDNA Romover Mix,无RNase去离子水补足10μL),移液器吸打混匀后42℃孵育2min,60℃孵育1min后置于冰上。
②配置逆转录反应体系(10μL①中反应产物,4μL SynScript III RTSuperMix,无RNase去离子水补足20μL),移液器吸打混匀后50℃孵育15min,85℃孵育5s,得到南方根结线虫cDNA,保存于-20℃。
4.MiMSP30编码基因全序列扩增
按照I-5TM 2×High-Fidelity Master Mix(擎科生物)说明书进行MiMSP30编码基因的全序列扩增,以10倍稀释的南方根结线虫cDNA作为扩增模板,前、后引物序列如表1所示。配置PCR扩增反应体系(12.5μL I-5TM 2×High-Fidelity Master Mix,1μL 10μM前引物,1μL 10μM后引物,1μL模板,去离子水补足25μL)。PCR反应程序:98℃,2min;98℃,10s,55℃,15s;72℃,15s,35个循环;72℃,2min。对PCR产物进行测序分析,序列正确则得到MiMSP30编码基因全序列,即序列SEQ ID NO.3。
表1MiMSP30编码基因全序列扩增引物序列
实施例2
1.dsRNA-MSP30与dsRNA-GFP合成模板扩增
dsRNA合成模板扩增参照T7 RNAi Transcription Kit(诺唯赞)与I-5TM 2×High-Fidelity Master Mix(擎科生物)说明书进行。分别以MiMSP30编码基因全序列与pSAT5-EGFP-N1作为模板,通过PCR扩增在MiMSP30与GFP目的扩增片段的正义链5’端与反义链5’端添加T7启动子序列得到dsRNA正、反链合成模板,PCR扩增反应的前、后引物序列如表2所示——以SEQ ID No.4与SEQ ID No.5所示序列作为引物扩增得到dsRNA-MSP30正链合成模板,以SEQID No.6与SEQ ID No.7所示序列作为引物扩增得到dsRNA-MSP30反链合成模板,以SEQ ID No.8与SEQ ID No.9所示序列作为引物扩增得到dsRNA-GFP正链合成模板、以SEQID No.10与SEQ ID No.11所示序列作为引物扩增得到dsRNA-GFP反链合成模板。配置PCR扩增反应体系(12.5μL I-5TM 2×High-Fidelity Master Mix,1μL 10μM前引物,1μL 10μM后引物,1μL模板,去离子水补足25μL)。PCR反应程序:98℃,2min;98℃,10s,55℃,15s;72℃,15s,35个循环;72℃,2min。产物回收后得到dsRNA-MSP30与dsRNA-GFP合成模板。
表2dsRNA合成模板扩增引物序列
2.dsRNA-MSP30与dsRNA-GFP合成
按照T7 RNAi Transcription Kit(诺唯赞)说明书分别进行dsRNA-MSP30与dsRNA-GFP的体外合成,配置反应体系(8μL NTP Mix,2μL 10×Transcription Buffer,2μLT7 Enzyme Mix,0.5μg正链模板,0.5μg反链模板,无RNase去离子水补足20μL),移液器轻柔吸打均匀后置于37℃孵育2h,自然冷却退火形成dsRNA。
3.dsRNA双酶消化及纯化
按照T7 RNAi Transcription Kit(诺唯赞)说明书分别进行dsRNA-MSP30与dsRNA-GFP的双酶消化及纯化。具体步骤如下:
①配置双酶消化体系(20μL转录产物,17μL无RNase去离子水,1μLDNase I,2μL10U/μL RNase T1),移液器轻柔吸打均匀后置于37℃孵育30min。
②向消化产物中加入80μL提前室温平衡30min的磁珠溶液,用移液器轻柔吸打混匀,室温孵育8min。
③将样品置于磁力架上约5min待溶液澄清,在不扰动磁珠的情况下去除上清液。
④样品依旧置于磁力架上,在不扰动磁珠的情况下加入200μL新配制的80%乙醇,室温孵育30s后小心去除上清。重复该步骤一次。
⑤室温放置5-10min使样品上残留乙醇挥发。
⑥将样品从磁力架上取下,加入40μL的无RNase去离子水,用移液器将管壁上的磁珠吸打下来,充分混匀去离子水与磁珠,室温孵育3min。
⑦将样品再次置于磁力架上,待溶液澄清后,在不扰动磁珠的情况下将上清转移至新的无RNase EP管中,得到纯化后的dsRNA。
实施例3
以dsRNA-GFP作为对照,用无RNase的1.5mL离心管收集新鲜孵化的二龄幼虫约20000条,用无RNase去离子水清洗虫体2遍后,移液器小心去除上清液。往1.5mL离心管中加入50μL dsRNA-MSP30,使dsRNA终浓度达到2μg/μL,并置于25℃孵育4h,期间不断翻转样品。孵育后将经dsRNA处理的线虫分为两份,其中一份用于dsRNA饲喂的沉默效率检测,另一份分别用于根结线虫的侵染虫口数、根内发育进程统计等。
1.dsRNA饲喂的沉默效率检测
分别提取dsRNA-MSP30与dsRNA-GFP饲喂后线虫的总RNA,并反转录为cDNA,总RNA提取步骤与cDNA合成步骤同实施例1。以MiGAPDH作为内参基因,以稀释10倍的cDNA作为模板,通过荧光定量PCR(qPCR)进行针对dsRNA-MSP30饲喂处理的沉默效率检测,检测引物如表3所示,检测系统为7500Real-Time PCR系统(ABI)。具体步骤如下:按照2×Master qPCR Mix(SYBR Green I)(擎科生物)说明书,配置qPCR反应体系(10μL 2×Master qPCR Mix,0.8μL 10μM前引物,0.8μL10μM后引物,2μL cDNA模板,0.4μL 50×ROX Reference Dye II,去离子水补足20μL)。qPCR反应程序:95℃,1min;95℃,10s;60℃,30s,40个循环(60℃采集荧光信号);95℃,15s;60℃,1min;95℃,15s。数据分析采用ΔΔCT法,进行3次生物学重复,差异性分析采用t检验。
表3qPCR检测特异性引物序列
实验结果表明,经dsRNA-MSP30饲喂处理后,根结线虫体内MiMSP30的沉默效率为70.02%。
2.侵染7天后根结线虫侵染虫口数统计
以dsRNA-GFP饲喂后的线虫作为对照,将dsRNA-MSP30饲喂后的线虫悬液沉降去除上清,与提前4℃预冷的23%Pluronic F-127(Sigma)混合,备用。用镊子小心夹取在Murashige and Skoog(MS)培养基上培养至苗龄2星期的拟南芥Col-0幼苗,将幼苗根部浸没入线虫混合胶中,每棵幼苗接种线虫200头。将盛有拟南芥幼苗的线虫混合胶置于25℃植物培养箱(10h光照、14h黑暗)培养7天。用蒸馏水冲洗病根组织数次以去除残留于组织上的胶体,对病根组织进行次氯酸钠-酸性品红染色并统计根内线虫虫口数。该实验进行3次生物学重复,差异性分析采用t检验。
实验结果表明,dsRNA-MSP30饲喂后根结线虫侵染虫口数(7天)平均为19.86头/株;对照组的根结线虫侵染虫口数(7天)平均为37.64头/株。dsRNA-MSP30饲喂后根结线虫侵染虫口数显著低于对照组,虫口数降低了47.24%。
3.侵染14天后根结线虫根内发育进程统计
以dsRNA-GFP饲喂后的线虫作为对照,将dsRNA-MSP30饲喂后的线虫悬液线虫悬液与1%羧甲基纤维素钠溶液等体积混匀,于苗龄6星期、沙土培养的拟南芥Col-0茎基部进行灌根处理,每棵苗接种线虫500头,并置于25℃植物培养箱(10h光照、14h黑暗)培养。用蒸馏水冲洗病根组织数次以去除残留于组织上的沙土,对病根组织进行次氯酸钠-酸性品红染色并统计根内处于不同虫龄阶段的线虫虫口数,并分析其在总虫口数中的占比,从而评估线虫的根内发育进程。该实验进行3次生物学重复,差异性分析采用t检验。
实验结果表明,dsRNA-MSP30饲喂后根结线虫不同龄期占比(14天)为:二龄幼虫74.02%、三龄幼虫15.01%、四龄幼虫10.79%;对照组根结线虫不同龄期占比(14天)为:二龄幼虫63.67%、三龄幼虫17.67%、四龄幼虫18.18%。与对照组相比,dsRNA-MSP30饲喂后根结线虫虫龄发育速度显著减慢。
实施例4
以dsRNA-GFP作为对照。选取苗龄4星期的番茄幼苗进行经dsRNA饲喂后根结线虫接种,dsRNA-MSP30与dsRNA-GFP的线虫饲喂方法与线虫接种方式同实施例3。接种后的番茄幼苗置于25℃温室(14h光照、10h黑暗)培养45天,用蒸馏水冲洗病根组织数次以去除残留于组织上的土壤,并对每棵幼苗上的根结数与卵块数进行统计。
实验结果表明,dsRNA-MSP30饲喂后受侵染番茄株系的根结数为246.67个/株,卵块数为23.91个/株;而对照组的根结数为330.50个/株,卵块数为50.82个/株。dsRNA-MSP30饲喂后根结线虫侵染番茄导致的根结与卵块数量均显著低于对照组,根结数减少了25.36%,卵块数减少了52.95%。
实施例5
1.针对MiMSP30与GFP的HIGS载体构建
MiMSP30与GFP目的扩增片段的扩增参照Nimble Cloning试剂盒(壹田生物)与I-5TM2×High-Fidelity Master Mix(擎科生物)说明书进行。分别以MiMSP30编码基因全序列与pSAT5-EGFP-N1作为模板,通过PCR扩增在MiMSP30与GFP目的扩增片段的5’端与3’端添加20bp的通用接头序列,PCR扩增反应的前、后引物序列如表4所示——以SEQ ID No.14与SEQID No.15所示序列作为引物扩增出5’与3’端添加了通用接头序列的MSP30目的扩增片段,以SEQ ID No.16与SEQ ID No.17所示序列作为引物扩增出5’与3’端添加了通用接头序列的GFP目的扩增片段。目的片段扩增的反应体系与扩增程序同实施例2,扩增产物回收后分别得到5’与3’端带有通用接头序列的MiMSP30与GFP目的扩增片段。
针对MiMSP30与GFP的HIGS载体构建均参照Nimble Cloning试剂盒(壹田生物)说明书进行,所选择的HIGS载体为pNC-Cam1304-RNAi(壹田生物)。配置NC克隆反应体系(10-80ng目的扩增片段、20-120ng HIGS表达载体、5μL Nimble Mix,无菌去离子水补足10μL),用移液枪吸打混匀,50℃孵育30-60min后,取2-5μL反应产物转化入100μL转化效率大于108的大肠杆菌DH5α感受态细胞,并涂于含有50μg/mL卡那霉素与5μg/mL氯霉素的LuriaBertani(LB)平板上37℃过夜培养。
挑选平板上生长出的单克隆进行PCR鉴定,鉴定引物如表4所示——分别用SEQ IDNo.18与SEQ ID No.20以及SEQ ID No.19与SEQ ID No.21两对引物对单克隆进行鉴定。挑取单菌落于20-30μL无菌去离子水中重悬,作为PCR鉴定模板。按照2×T5 Super PCR Mix(擎科生物)说明书配置PCR鉴定反应体系(12.5μL 2×T5 Super PCR Mix、1μL 10μM前引物、1μL 10μM后引物、3μL模板、无菌去离子水补足25μL);反应程序:98℃,5min;98℃,10s,55℃,15s;72℃,15s,35个循环;72℃,2min。将PCR产物电泳后,选取阳性克隆进行测序验证,2段插入序列均测序正确则将阳性克隆与等体积的80%甘油混匀,并冻存于-80℃。
表4单克隆PCR检测引物序列
2.针对MiMSP30与GFP的HIGS农杆菌构建
分别从阳性克隆中提取出针对MiMSP30与GFP的HIGS载体。将农杆菌LBA4404化转感受态细胞(擎科生物)置于冰上,取2-5μL HIGS载体滴加于未融化的感受态细胞上,置于37℃孵育5min后,轻柔转移至冰上孵育2-5min。往冰浴后的感受态细胞与载体混合物中加入1mL LB液体培养基,并将菌悬液置于28℃200rpm振荡培养2-4h。分别取100μL振荡培养后的菌悬液原液与100μL经室温5000rpm离心1min并去除上清的浓缩液涂于含有50μg/mL利福平、50μg/mL卡那霉素与5μg/mL氯霉素的LB平板上28℃培养2d。挑选平板上生长出的单克隆进行PCR鉴定,鉴定引物、反应体系与反应程序同实施例5第1点。
3.针对MiMSP30与GFP的HIGS转基因植株构建
HIGS转基因植株构建采用农杆菌介导的叶盘转化法,所使用植物材料为番茄品种Money Maker,具体步骤修改自曹慧颖等(2008)与梁超等(2009)。详细步骤如下:
①将番茄种子先后用75%乙醇溶液消毒1min、2%次氯酸钠溶液浸泡15min,使用无菌水洗涤种子6-10次。将种子以适当间距播种于MS固体培养基,置于24-26℃光照培养箱中暗培养3-4d后转移到16h光照/8h黑暗下培养至子叶完全展开(约为苗龄2d)。
②在番茄苗龄2d时,通过平板划线法于LB平板上活化农杆菌,置于28℃培养箱中倒置培养24h后在,将农杆菌接种于LB液体培养基中28℃200rpm振荡培养近24h。
③取完全伸展的番茄子叶,切除其前后两段,留取中间约0.5cm叶片,近轴面朝上置于预培养基(在MS固体培养基的基础上,添加终浓度为0.2mg/L的生长素和终浓度为2mg/L的玉米素)上,24-26℃暗培养2d。
④5000rpm离心富集农杆菌,用MS培养液重悬农杆菌并将菌浓度调整为OD600=0.4-0.6,置于5mL离心管中,加入终浓度为100μM/L的乙酰丁香酮避光静置30min。
取番茄外植体浸泡于菌悬液中25-30min后,用无菌滤纸吸干组织块上残余的菌液,并将其近轴面朝上置于新的预培养基上,24-26℃黑暗条件共培养2d。
⑤夹取暗培养后的外植体,采用无菌水漂洗外植体6-10次,每次30min,最后用终浓度为250mg/L的羧苄青霉素溶液漂洗外植体30min。用无菌滤纸吸干外植体上残余的水分后,将其近轴面朝上置于新的预培养基上,并置于24-26℃光照培养箱(16h光照/8h黑暗)中培养3d。
⑥将外植体近轴面朝上转移到分化培养基(在MS固体培养基的基础上,添加终浓度为0.2mg/L的生长素、终浓度为2mg/L的玉米素、终浓度为400mg/L的羧苄青霉素以及不同浓度的潮霉素)上,每2星期继代一次,直至抗性芽分化。
⑦当抗性芽长到约1cm长时,切下抗性芽并将茎基部插入生根培养基(在MS固体培养基的基础上,添加终浓度为0.1mg/L的生长素与终浓度为400mg/L的羧苄青霉素)中,培养至有大量根系长出时,洗净根部培养基并移栽到土壤中,24-26℃16h光照/8h黑暗条件培养。
⑧参照快速DNA提取检测试剂盒(TIANGEN)说明书提取抗性植株的DNA并以此为模板进行PCR扩增,扩增所用引物同实施例5第1点。PCR扩增反应体系(10μL 2×Det PCRMaster Mix、0.5μL 10μM前引物、0.5μL 10μM后引物、1μL模板DNA、去离子水补足20μL),反应程序:94℃,3min;94℃,30s,55℃,30s;72℃,1min,35个循环;72℃,5min。电泳检测PCR扩增结果。
实施例6
以HIGS-GFP转基因番茄幼苗作为对照。选取苗龄4星期的HIGS转基因番茄幼苗进行野生型根结线虫接种,接种方式同实施例3。接种线虫7天后进行HIGS-MSP30基因沉默效率检测,接种线虫45天后进行根结数与卵块数统计。
1.HIGS的沉默效率检测
取已接种根结线虫7天的HIGS转基因番茄幼苗根部,进行番茄组织与侵染后根结线虫的混合总RNA提取,提取步骤参照RNAsimple总RNA提取试剂盒(TIANGEN)说明书进行。详细步骤如下:
①液氮研磨受根结线虫侵染的番茄根系组织,每100mg组织加入1mL裂解液RZ,涡旋振荡1min后,将样品置于室温静置5min。
②向样品中加入体积为裂解液RZ 0.2倍的氯仿,涡旋振荡1min后,室温静置3min。
③将样品置于4℃12000rpm离心10min,将上层水相转移至新的无RNase离心管中。
④向水相中加入体积为水相0.5倍的无水乙醇,吸打混匀后转移入吸附柱CR3中,4℃12000rpm离心30s,去除滤液。
⑤向吸附柱中加入500μL去蛋白液RD,4℃12000rpm离心30s,去除滤液。
⑥向吸附柱中加入700μL去蛋白液RW,4℃12000rpm离心30s,去除滤液。重复该步骤一次。
⑦将吸附柱转移至新的无RNase离心管中,将40μL无RNase的去离子水滴加到吸附膜上,室温放置2min后,4℃12000rpm离心2min,收集洗脱液即得到总RNA,保存于-80℃。
cDNA合成步骤同实施例1。通过荧光定量PCR(qPCR)进行HIGS转基因番茄对根结线虫的沉默效率检测,反应体系、反应程序与所使用引物同实施例3。
实验结果表明,野生型根结线虫二龄幼虫侵染HIGS-MSP30转基因植株后,根结线虫体内MiMSP30的沉默效率为46.32%。
2.根结数与卵块数统计
HIGS-GFP与HIGS-MSP30转基因番茄幼苗接种线虫45天后的根结数与卵块数统计方法同实施例4。实验结果表明:野生型线虫接种HIGS-MSP30后根结数为299.92个/株,卵块数为60.17个/株;而对照组的根结数为389.50个/株,卵块数为83.17个/株。野生型线虫侵染HIGS-MSP30转基因番茄幼苗导致的根结与卵块数量均显著低于对照组,根结数减少了23.00%,卵块数减少了27.65%。
由实施例3、实施例4与实施例6可知,两种实施方式均可达成对MiMSP30基因的沉默效果,对根结线虫的侵染能力与根内发育进程具有明显抑制效果,并有效降低了根结线虫病的发病情况,应用前景极高。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (6)
1.一种用于抑制根结线虫病的dsRNA,其特征在于,所述dsRNA的制备方法包括以下步骤:
以SEQ ID No.3所示的核苷酸为模板,以SEQ ID No.4与SEQ ID No.5所示序列、或SEQID No.6与SEQ ID No.7所示序列分别作为引物,分别进行PCR扩增获得正、反向产物;以前述回收得到的PCR扩增产物作为模板,转录合成dsRNA的正链与反链,而后退火使正、反链杂交形成dsRNA。
2.根据权利要求1所述的一种用于抑制根结线虫病的dsRNA,其特征在于,所述dsRNA的制备方法包括以下步骤:
(1)提取南方根结线虫的总RNA,通过反转录操作获得cDNA;
(2)以步骤(1)中得到的cDNA作为模板,以SEQ ID No.1与SEQ ID No.2所示序列作为引物,通过PCR扩增得到MiMSP30全长序列;
(3)以MiMSP30的全长序列作为模板,以SEQ ID No.4与SEQ ID No.5所示序列、或SEQID No.6与SEQ ID No.7所示序列分别作为引物,通过PCR反应扩增得到可作为dsRNA正、反链合成模板的DNA序列;
(4)以步骤(3)中回收得到的PCR扩增产物作为模板,分别转录合成dsRNA的正链与反链,退火使正、反链杂交形成dsRNA;
(5)酶解去除残余的单链RNA与DNA,并对酶解产物进行回收与纯化,即获得对靶标基因MiMSP30具备RNAi效果的dsRNA。
3.根据权利要求2所述的一种用于抑制根结线虫病的dsRNA,其特征在于,所述针对靶标基因MiMSP30具备RNAi效果的HIGS方法,包括如下步骤:
(1)以MiMSP30的全长序列作为模板,以Seq ID No.14与Seq ID No.15所示序列作为引物,通过PCR反应扩增得到MiMSP30序列,并将其插入HIGS载体序列中,得到同时携带有MiMSP30序列与MiMSP30反向互补序列的HIGS载体。
(2)将步骤(1)中所得的HIGS载体转化入农杆菌GV3101中。
(3)通过农杆菌介导的植物转基因体系,将步骤(1)中所得的同时携带有MiMSP30序列与MiMSP30反向互补序列的HIGS载体序列转化入植物当中,得到能够在体内合成dsRNA-MSP30的转基因植物。
4.权利要求1-3任一项所述的dsRNA在用于抑制根结线虫病的应用。
5.一种生物制品,含有权利要求1-3任一项所述的dsRNA。
6.根据权利要求5所述的生物制品,其特征在于,所述生物制品为饲喂式杀虫剂或饵剂。
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