CN112195178B - 番茄抗晚疫病长链非编码RNA-lncRNA40787及其克隆方法与应用方法 - Google Patents
番茄抗晚疫病长链非编码RNA-lncRNA40787及其克隆方法与应用方法 Download PDFInfo
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Abstract
本发明提供了一种通过沉默miR394来提高番茄晚疫病抗性的长链非编码RNA‑lncRNA40787,所述DNA分子的核苷酸序列如SEQ ID NO.1所示;提供了该长链非编码RNA的克隆方法:以野生型感晚疫病番茄早粉2号的cDNA为模板进行PCR扩增,将得到的PCR产物与pMD‑18T克隆载体连接,转化大肠杆菌DH5α,挑取单菌落进行测序:还提供了该长链非编码RNA的应用方法,用于番茄抵御病原菌侵染,即在番茄中瞬时过表达所述番茄抗晚疫病的长链非编码RNA‑lncRNA40787。本发明得到的长链非编码RNA在番茄瞬时过表达后,番茄miR394的表达量显著降低,其抗晚疫病效果更优:其用于沉默miR394使番茄对晚疫病的抗性增强,对培育抗晚疫病番茄品种,从而提高番茄产量具有重要意义。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及番茄抗晚疫病长链非编码RNA-lncRNA40787及其克隆方法与应用方法。
背景技术
番茄是番茄属的草本植物,是世界范围内广泛种植的重要经济园艺作物。含丰富的番茄红素、维生素等,具有极高的营养价值。但在其生长过程中经常遭受着多种病原物的侵害,给番茄生产造成巨大的经济损失。其中,由致病疫霉导致的晚疫病,是一种毁灭性的病害,在番茄的各个阶段均产生了恶劣影响,发病严重时导致番茄茎部腐烂,植株萎蔫,最终导致番茄严重减产。目前防治方法存在环境污染、农药残留、效率低、病菌遗传结构改变的问题。通过分子生物学手段发现抗病基因来抵御病原菌侵染,是增强番茄对晚疫病抗性的一种经济有效的方法。
非编码RNA是一类在转录组中不编码蛋白的RNA,包括管家型RNA及调控型RNA两类,其中调控型RNA被认为是关键调控RNA分子。长链非编码RNA(LncRNAs)作为一种长度大于200nt的调控型RNA存在,通过染色质重塑,转录及转录后调控参与植物生命进程。LncRNAs在小麦白粉病、拟南芥尖刀镰胞杆菌,番茄黄化曲叶病毒等多种抗病过程中均有参与。近年来的研究发现lncRNAs在调控植物抗病过程中,与microRNA(miRNA)间存在着相互作用的关系。LncRNAs可作为miRNA的竞争性内源RNA(competing endogenous,ceRNA)也可以被miRNA靶向并剪切,lncRNAs也可以作为miRNA的前体产生miRNA。在玉米中通过共表达网络发现了86个可以与miRNA共同作用的lncRNA。拟南芥中的LncRNA-IPS1可作为ceRNA沉默miR399,进而调控生物过程。
MiRNA作为一类长约20-24nt内源单链小分子RNA,是另一种发挥重要生物学功能的调控型RNA。MiR394作为一种高度保守的miRNA,广泛存在于水稻、番茄、玉米等多种植物中,在植株对生物胁迫的响应中扮演关键角色。在番茄感染灰霉病后,植株体内miR394呈现上调趋势,其靶基因LEAF CURLING RESPONSIVENESS(LCR)表达量下降;在尖孢镰刀菌侵染的大蒜中也得到了相似的结果,miR394表达量上调,靶向LCR,参与了茉莉酸信号通路;miR394在棉花抗曲叶病中也可影响相关致病基因的存在。miR394与三个包含其前体序列的lncRNAs呈现差异表达,响应了油菜核盘菌的感染。说明miR394在介导真菌病原体的应答过程中,lncRNA也参与其中,但二者间具体关系及调控方式还未见报道。
发明内容
本发明的目的在于开发一种番茄抗晚疫病长链非编码RNA-lncRNA40787及其克隆的方法;并提供该基因在沉默miR394及增强番茄对晚疫病抗性中的应用方法。
为了达到上述目的,本发明提供了一种番茄抗晚疫病长链非编码RNA-lncRNA40787,其序列如SEQ ID NO.1所示。
本发本发明还提供了上述番茄抗晚疫病长链非编码RNA-lncRNA40787的克隆方法,以野生型感晚疫病番茄早粉2号的cDNA为模板,以lncRNA40787-FP、lncRNA40787-RP为特异性引物,进行PCR扩增;所述的特异性引物lncRNA40787-FP、lncRNA40787-RP序列分别如SEQ ID NO.2、SEQ ID NO.3所示;
克隆所用特异性引物具体如下:
lncRNA40787-FP:CGCGGATCCGCGATCACAAACAAGAAAATGCTTC;
lncRNA40787-RP:CGAGCTCGCGAAGGGATCTGCTGGTTCTGTG;
将得到的PCR产物与PMD-18T克隆载体连接,得到的克隆载体连接产物转化大肠杆菌DH5α并涂布在含有氨苄青霉素的LB固体培养基上,挑取单菌落,进行测序,测序结果如SEQ ID NO.1所示。优选挑取单菌落于LB液体培养基后,提取含有番茄抗晚疫病长链非编码RNA-lncRNA40787菌体中的质粒进行测序。
本发明还提供了番茄抗晚疫病长链非编码RNA-lncRNA40787的应用方法,用于沉默番茄miR394。
优选方式下,上述番茄抗晚疫病长链非编码RNA-lncRNA40787提高番茄对晚疫病的抗性。
具体通过以下技术方案实现:
将含有番茄抗晚疫病长链非编码RNA-lncRNA40787的重组表达载体转化农杆菌GV3101并涂布在含有卡那霉素、链霉素及利福平的YEB固体培养基上,挑取单菌落进行菌液PCR验证;选取阳性工程菌瞬时侵染番茄叶片,并检测叶片中miR394的表达量,更进一步地,检测叶片对晚疫病的抗性。
本发明的目的植物为双子叶植物番茄。
优选方式下,所述重组表达载体由番茄抗晚疫病长链非编码RNA-lncRNA40787插入表达载体得到。
进一步优化,所述重组表达载体的具体制备方法为:
①克隆番茄抗晚疫病长链非编码RNA-lncRNA40787:以野生型感晚疫病番茄早粉2号的cDNA为模板、以lncRNA40787-FP和lncRNA40787-RP为特异性引物进行PCR扩增;所述特异性引物的序列分别如SEQ ID NO.2和SEQ ID NO.3所示;将得到的PCR产物与pMD-18T克隆载体连接,克隆载体连接产物转化大肠杆菌DH5α并涂布在含有氨苄青霉素的LB固体培养基上,挑取单菌落于LB液体培养基后,提取含有番茄抗晚疫病长链非编码RNA-lncRNA40787菌体中的质粒进行测序;
②构建含有番茄抗晚疫病长链非编码RNA-lncRNA40787的重组表达载体:用限制性内切酶BamHI及SacI对上述测序正确的质粒进行双酶切,回收得到目的片段,将其与同样经BamHI及SacI双酶切的pBI121表达载体连接,表达载体连接产物即为重组表达载体。
将上述表达载体连接产物转化大肠杆菌DH5α,涂布在含有卡那霉素的LB固体培养基上,挑取单菌落进行PCR及双酶切验证,结果显示表达载体构建成功。
本发明的技术创新在于:
1、本发明克隆得到了一个番茄抗晚疫病长链非编码RNA-lncRNA40787;构建了含有番茄抗晚疫病长链非编码RNA-lncRNA40787的重组表达载体;证明了长链非编码RNA-lncRNA40787对miR394的沉默作用。
2、本发明明确了长链非编码RNA-lncRNA40787可以通过沉默致病相关的非编码RNA-miR394来增强番茄对晚疫病的抗性。过表达本发明得到的过表达lncRNA40787(OE-lncRNA40787)后,番茄miR394的表达量显著下降,植株抗晚疫病效果更优,对培育抗晚疫病番茄品种具有重要意义。
附图说明
图1为瞬时过表达lncRNA40787与对照组叶片中lncRNA40787的表达量;
图2为瞬时过表达lncRNA40787与对照组叶片中miR394的表达量;
图3为晚疫病菌处理5天后瞬时过表达lncRNA40787与对照组叶片表型。
具体实施方式
以下结合具体实施例,进一步阐述本发明。实施例中未注明的实验条件及方法,均为常规方法。
实施例一:番茄抗晚疫病长链非编码RNA-lncRNA40787的克隆
1.番茄总RNA的提取
(1)将样品放入研钵中,加入液氮充分研磨至粉末。
(2)取适量粉末,置于1.5mL RNase/DNase Free离心管中,同时迅速加入1mL预冷的Trizol,摇匀,室温静置5min。
(3)向离心管中加入200μL氯仿,摇匀,室温静置5min,4℃12000r/min离心15min。
(4)将上清转移至新离心管中,加入等体积的异丙醇,轻轻颠倒混匀,-20℃静置20min后,4℃12000r/min离心10min。
(5)弃去上清,加入1mL预冷的75%乙醇,清洗沉淀,4℃12000r/min离心5min。
(6)小心弃去上清,在室温下开盖放置,待乙醇完全挥发后,加入20μL RNase FreeddH2O溶解沉淀。
(7)取5μLRNA,1%琼脂糖凝胶电泳检测。
2.番茄cDNA的合成
以总RNA作为模板进行反转录,应用Reverse Transcriptase M-MLV(RNase H-)(购自Takara)参照说明书完成操作。
3.长链非编码RNA-lncRNA40787的PCR扩增
以番茄早粉2号的cDNA为模板,应用特异性引物,进行PCR扩增。
克隆所用特异性引物如下:
lncRNA40787-FP:CGCGG GATCACAAACAAGAAAATGCTTC
lncRNA40787-RP:CGAGCTCGCGAAGGGATCTGCTGGTTCTGTG
反应条件如下:
4.PCR产物的回收纯化
1%琼脂糖凝胶电泳检测上述PCR产物后,用切胶回收试剂盒(购自Takara)回收符合目标的片段。
5.目标片段与克隆载体连接
将上述回收得到的片段与克隆载体pMD-18T(购自Takara)连接,反应体系如下:
16℃连接8h得到克隆载体连接产物pMD-18T-lncRNA40787。
6.克隆载体连接产物转化大肠杆菌
(1)将10μL的克隆载体连接产物加入至100μL的大肠杆菌感受态细胞中,吹打混匀后,冰水浴30min;
(2)将上述冰浴后的混合液立即转移至42℃水浴锅中,90s后再次冰水浴2min;
(3)向混合液中加入1mL新鲜的LB培养基,于37℃恒温振荡培养(180rpm)1.5h;
(4)4000r/min离心10min上述菌液,吸去1mL上清,留下100μL菌液,将其充分吹打悬浮后,均匀涂布于LB平板上(含100mg/L氨苄青霉素、24mg/L IPTG以及20mg/L X-Gal),于37℃恒温培养箱中过夜;
(5)挑取白色单菌落,接至LB液体培养基(含100mg/L氨苄青霉素)中,37℃恒温振荡培养(180rpm)过夜。
7.pMD-18T-lncRNA40787质粒的提取
依据质粒小提试剂盒(购自Vazyme)的说明,提取上述菌液中所含有的pMD-18T-lncRNA40787质粒。取5μL的样品,用1%的琼脂糖凝胶进行电泳检测。
8.测序
将得到的质粒送至华大基因(北京)公司测序,分析测序结果。
实施例二:番茄抗晚疫病长链非编码RNA-lncRNA40787重组表达载体的构建
1.pMD-18T-lncRNA40787质粒的酶切
将pMD-18T-lncRNA40787质粒用BamHI和SacI(购自Takara)进行双酶切,回收目标片段即小片段。酶切反应体系及方法如下:
37℃酶切6h,1%琼脂糖凝胶电泳检测酶切产物。
2.pBI121质粒双酶切
用BamHI和SacI对pBI121质粒进行双酶切,回收pBI121片段即大片段。反应体系如下:
37℃酶切6h,l%琼脂糖凝胶电泳检测酶切产物。
3.目标片段与pBI121载体连接
利用T4DNA连接酶(购自Takara),将上述切胶回收得到的目标片段和双酶切后的pBI121载体连接。反应体系如下:
16℃连接14h得到重组表达载体pBI121-lncRNA40787,将上述表达载体连接产物转化大肠杆菌DH5α,涂布在含有卡那霉素的LB固体培养基上,挑取单菌落进行PCR及双酶切验证,结果显示表达载体构建成功。
实施例三:番茄抗晚疫病长链非编码RNA-lncRNA40787的应用
l.pBI121-lncRNA40787农杆菌工程菌的制备
⑴将2.0μL的pBI121-lncRNA40787质粒加入至100μL的农杆菌感受态细胞中,充分混匀后,冰水浴10min,迅速移至液氮中冷冻5min;
(2)将冷冻后的混合液于37℃水浴5min,加入1mL新鲜的YEB培养基,置于28℃恒温摇床中振荡(180rpm)培养3h;
(3)将上述菌液离心后,吸去1mL上清,充分吹打混匀余下的100μL菌液,均匀涂布于YEB固体培养基(含100mg/L链霉素、100mg/L利福平和50mg/L卡那霉素)上,28℃培养36h;
(4)农杆菌工程菌的PCR检测
挑取上述平板中经抗生素筛选得到的菌落,置于5mL YEB中,28℃,180rpm振荡培养16-17h。
吸取上述菌液2μL,加入18μL的ddH2O稀释,于-20℃冷冻30-40min后,99℃加热10min,使菌体充分裂解释放DNA;
以上述DNA为模板,利用lncRNA40787特异性引物进行PCR,反应条件及反应体系同“实施例一中的3”。
2.农杆菌介导瞬时转化番茄早粉2号
(1)番茄种子的萌发
用水浸泡番茄种子12h,均匀摆放于湿润的滤纸上,表面再覆盖一层湿润的滤纸,保持90%以上的湿度,于28℃下暗培养至萌发。
(2)番茄植株的培养
播种刚萌发的种子至土壤中,在25-28℃,16h光照的条件下培养至5叶期。
(3)农杆菌菌液的制备
挑取含有空载体pBI121的农杆菌GV3101和经菌液PCR验证的阳性克隆,分别接种到含50mg/L卡那霉素和100mg/L利福平的5mL YEB液体培养基中,于28℃,180rpm振荡培养18h;
按1:50的比例吸取上述菌液,加到含有l00 mg/L利福平、50mg/L卡那霉素、10mmol/L MES、20μmol/L AS和2mmol/L MgSO4的YEB液体培养基中,于28℃、180rpm振荡培养18h;
4℃、4000r/min离心l0 min,收集农杆菌菌体,重悬菌体于含10mmol/L MES、10mmol/L MgCl2,20μmol/L AS的MMA溶液中,调节OD600为1.0,于28℃、180rpm培养3h以上,以活化农杆菌,得到活化后含有空载和pBI121-lncRNA40787的菌液。
(4)瞬时侵染番茄叶片
转化前将5叶期番茄植株置于弱光下l-2h,并浇足水;
使用去除针头的1mL无菌注射器,在实验组和对照组的番茄叶背分别注射200μL上述活化的含有空载和pBI121-lncRNA40787的菌液;
注射完毕后,将番茄植株于25℃、16/8h的光/暗周期下培养。
3.瞬时转化番茄的表达特性及抗病性分析
(1)瞬时表达番茄的表达特性分析
取瞬时侵染3天的番茄叶片,提取总RNA,应用Reverse Transcriptase M-MLV(RNase H-)(购自Takara)和TransmiRNA First-Strand cDNA Synthesis SuperMix(购自TransGen Biotech)参照说明书完成反转录。以得到的cDNA为模板,利用lncRNA40787和miR394的特异性引物(以qlncRNA40787-FP、qlncRNA40787-RP和qmiR394为特异性引物,特异性引物qlncRNA40787-FP、qlncRNA40787-RP和qmiR394的序列分别如SEQ ID NO.4、SEQID NO.5和SEQ ID NO.6所示)进行实时定量PCR检测,结果如图1和图2所示,实验组的叶片中lncRNA40787的表达量明显高于对照组,而miR394的表达量显著低于对照组。RNA提取同“实施例一,1”。实时定量所用试剂盒为/>Premix Ex TaqTM II(Tli RNaseH Plus)(购自Takara)和Trans/>Green miRNA Two-Step qRT-PCR SuperMix(购自TransGenBiotech),反应体系及条件参照说明书。
实时定量所用特异性引物如下:
qlncRNA40787-FP:AAAATCAAGCACCACGCAGG
qlncRNA40787-RP:CAGGTCCACCAAAGGTCGAG
qmiR394:TTGGCATTCTGTCCACCTCC
(2)瞬时表达番茄的抗病性分析
选取瞬时表达3天的完好番茄植株叶片,于叶片注射处接种10μl浓度为1×106个孢子/mL的晚疫病菌抱子悬浮液;
将上述处理的叶片置于18℃光照培养箱,16/8h光周期,保持90%以上相对湿度,培养5天后拍照记录发病情况。如图3所示,实验组叶片的病情程度明显弱于对照组,说明过表达lncRNA40787(OE-lncRNA40787)可以降低番茄中miR394的表达量从而提高番茄对晚疫病的抗性。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
序列表
<110> 大连理工大学
<120> 番茄抗晚疫病长链非编码RNA-lncRNA40787及其克隆方法与应用方法
<160> 6
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 番茄(Lycopersicon esculentum)
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tggagaagtt gaagttgcat ttagtcgatg aacgaatgcc tttgttccaa atcttcatgt 240
atctcctctg cgtttacctg gtatagcaag gctacggtac aacttattac tttgtgataa 300
gacaggctag ttgacaactc cattcacata tttctcagca ttagaggtgt ttgtggaggt 360
aatctcgacc tttggtggac ctgaatgccg tcttgtaatg gctggtgaag gtgagtaaga 420
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ttggcattct gtccacctcc 20
Claims (8)
1.一种番茄抗晚疫病长链非编码RNA-lncRNA40787,其特征在于,其DNA分子序列如SEQID NO.1所示。
2.权利要求1所述番茄抗晚疫病长链非编码RNA-lncRNA40787的克隆方法,其特征在于:以野生型感晚疫病番茄早粉2号的cDNA为模板,以lncRNA40787-FP、lncRNA40787-RP为特异性引物进行PCR扩增;
所述特异性引物的序列分别如SEQ ID NO.2、SEQ ID NO.3所示:
将得到的PCR产物与pMD-18T克隆载体连接,得到的克隆载体连接产物转化大肠杆菌DH5α并涂布在含有氨苄青霉素的LB固体培养基上,挑取单菌落进行测序,所得产物即为番茄抗晚疫病长链非编码RNA-lncRNA40787。
3.权利要求1所述番茄抗晚疫病长链非编码RNA-lncRNA40787的应用方法,其特征在于,用于沉默番茄miR394,抵御病原菌侵染。
4.根据权利要求3所述番茄抗晚疫病长链非编码RNA-lncRNA40787的应用方法,其特征在于,用于提高番茄对晚疫病的抗性。
5.根据权利要求3所述番茄抗晚疫病长链非编码RNA-lncRNA40787的应用方法,其特征在于,通过以下技术方案实现:
将含有番茄抗晚疫病长链非编码RNA-lncRNA40787的重组表达载体转化农杆菌GV3101并涂布在含有卡那霉素、链霉素及利福平的YEB固体培养基上,挑取单菌落进行菌液PCR验证;
选取阳性工程菌瞬时侵染番茄叶片,并检测叶片中miR394的表达量及叶片对晚疫病的抗性。
6.根据权利要求5所述番茄抗晚疫病长链非编码RNA-lncRNA40787的应用方法,其特征在于,还包括检测叶片对晚疫病的抗性。
7.根据权利要求5或6所述番茄抗晚疫病长链非编码RNA-lncRNA40787的应用方法,其特征在于所述重组表达载体由番茄抗晚疫病长链非编码RNA-lncRNA40787插入表达载体得到。
8.根据权利要求7所述番茄抗晚疫病长链非编码RNA-lncRNA40787的应用方法,其特征在于,所述重组表达载体的具体制备过程为:
①克隆番茄抗晚疫病长链非编码RNA-lncRNA40787:以野生型感晚疫病番茄早粉2号的cDNA为模板、以lncRNA40787-FP和lncRNA40787-RP为特异性引物进行PCR扩增;所述特异性引物的序列分别如SEQ ID NO.2和SEQ ID NO.3所示:将得到的PCR产物与pMD-18T克隆载体连接,得到的克隆载体连接产物转化大肠杆菌DH5α并涂布在含有氨苄青霉素的LB固体培养基上,挑取单菌落于LB液体培养基后,提取含有番茄抗晚疫病长链非编码RNA-lncRNA40787菌体中的质粒进行测序;
②构建含有番茄抗晚疫病长链非编码RNA-lncRNA40787的重组表达载体:用限制性内切酶BamHI及SacI对上述测序正确的质粒进行双酶切,回收得到目的片段,将所述目的片段与同样经BamHI及SacI双酶切的pBI121表达载体连接,得到的表达载体连接产物即为重组表达载体。
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