CN106916825B - 番茄抗晚疫病基因SpNBS-LRR及其克隆方法与应用方法 - Google Patents
番茄抗晚疫病基因SpNBS-LRR及其克隆方法与应用方法 Download PDFInfo
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Abstract
本发明提供了一种番茄抗晚疫病基因SpNBS‑LRR，其DNA分子序列如SEQ ID NO.1所示；提供了该基因的克隆方法，以野生型抗晚疫病番茄L3708的cDNA为模板进行PCR扩增；将得到的PCR产物与pMD‑19T克隆载体连接，转化大肠杆菌DH5α，挑取单菌落进行测序；还提供了该基因的应用方法，培育抗晚疫病番茄植株。本发明方法得到的过表达植株中SpNBS‑LRR的表达量高于野生型植株，其抗晚疫病效果更优。本发明通过过表达SpNBS‑LRR基因使番茄对晚疫病的抗性增强，对培育抗晚疫病番茄品种具有重要意义。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一个番茄抗晚疫病基因SpNBS-LRR的克隆及应用。
背景技术
番茄是一种世界范围内广泛种植的经济、园艺作物。在其生长过程中，由各种病原物引起的植物病害，严重影响了番茄的产量，造成了巨大的经济损失。近年来，晚疫病成为最严重的番茄病害之一，对农业生产及经济发展造成了重大损失。虽然化学农药对晚疫病的发生和蔓延有一定的控制作用，但由此引发的环境污染，病原菌抗药性等问题，依旧给农业生产带来了极大的困扰，因此，研究番茄抗病的分子机制，用生物学方法提高番茄对晚疫病的抗性成为了关键问题。
在自然界中，包括番茄在内的植物经常遭受各种病原菌的入侵，严重影响它们的生长发育。为了抵御这些病原菌，植物进化出了一系列的免疫机制。当病原菌入侵植物时，其保守分子被植物的胞外跨膜受体所识别，激活病原物相关分子模式触发免疫(PTI)。尽管PTI成功抵御了大部分病原菌的入侵，少数病原菌进化出了相应的效应因子来突破PTI防线。植物中抗性基因(R基因)所编码的抗性蛋白(R蛋白)可以识别这些效应因子，从而激活高效的免疫反应，效应子触发免疫(ETI)。
NBS-LRR基因是植物基因组中一类高度保守且丰富的R基因。其编码的NBS-LRR蛋白可以识别病原物的效应因子Avr蛋白进而引起过敏反应(HR)。HR反应可以导致初始侵染部位的植物细胞程序性死亡抑制病原菌的生长。目前，研究者们已经从多种植物中克隆得到了大量NBS-LRR基因并对其在植物免疫中的功能进行了探究。如从抗性辣椒品系HDA149中得到的CaRKNR基因，对其进行沉默后辣椒对于线虫的抗性明显下降。小麦Pm3b基因可为其提供白粉病的小种特异抗性。在水稻中，Pi64可以提高其对稻瘟病菌的抵抗能力，另一个基因OsRP1L1可以提高其对于白叶枯病的抗性。同时丝瓜中的NBS-LRR基因与叶卷曲和花叶病抗性有关。
发明内容
本发明的目的在于一种克隆番茄抗晚疫病基因SpNBS-LRR的方法，并提供该基因在番茄抗晚疫病中的应用方法。
为了达到上述目的，本发明提供了一个番茄抗晚疫病基因SpNBS-LRR，其序列如SEQ ID NO.1所示。
本发明还提供了上述番茄抗晚疫病基因SpNBS-LRR的克隆方法，以野生型抗晚疫病番茄L3708的cDNA为模板，以SpNBS-LRR-FP、SpNBS-LRR-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；
克隆所用特异性引物具体为如下：
SpNBS-LRR-FP：CGGGATCCATGTGTGTGGGAAGAGTTACAGATT，
SpNBS-LRR-RP：CGAGCTCTTACTCTAGTCTTTCAAGTTTGGGG；
将得到的PCR产物与pMD-19T克隆载体连接，得到的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。
本发明还提供了番茄抗晚疫病基因SpNBS-LRR的应用方法，用于培育抗晚疫病番茄植株。
具体通过以下技术方案实现：
将含有番茄抗晚疫病基因SpNBS-LRR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；选取阳性工程菌侵染预培养后的番茄子叶，共培养，分化出芽；将抗性芽移至生根培养基中诱导生根后移栽，实时定量PCR验证过表达植株；对过表达SpNBS-LRR的番茄苗进行抗病性检测，得到抗晚疫病番茄植株。
本发明目的植物为双子叶植物番茄。
优选方式下，所述重组载体由将所述番茄抗晚疫病基因SpNBS-LRR插入表达载体得到。
进一步优化，所述重组载体的具体制备方法为：
对所述番茄抗晚疫病基因SpNBS-LRR进行克隆：即上述以野生型抗晚疫病番茄L3708的cDNA为模板，以SpNBS-LRR-FP、SpNBS-LRR-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-19T克隆载体连接，得到的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；
构建了含有番茄抗晚疫病基因SpNBS-LRR的重组表达载体：提取上述含有番茄抗晚疫病基因SpNBS-LRR的菌体的质粒，并用限制性内切酶BamHI及SacI双酶切，回收得到目的片段，将所述目的片段与去除GUS基因的pBI121表达载体连接，得到的连接产物即为重组表达载体。
将上述连接产物转化大肠杆菌DH5α并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：
本发明克隆得到了一个番茄抗晚疫病基因SpNBS-LRR；构建了含有番茄抗晚疫病基因SpNBS-LRR的表达载体；获得了过表达SpNBS-LRR的抗晚疫病番茄植株。本发明方法得到的过表达植株中SpNBS-LRR的表达量高于野生型植株，其抗晚疫病效果更优。本发明通过过表达SpNBS-LRR基因使番茄对晚疫病的抗性增强，对培育抗晚疫病番茄品种具有重要意义。
附图说明
图1为过表达与野生型植株中SpNBS-LRR的表达量；
图2为晚疫病菌处理5天后过表达与野生型植株表型。
具体实施方式
以下结合具体实施例，进一步阐明本发明。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：番茄抗晚疫病基因SpNBS-LRR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，并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 FreedH2O溶解沉淀。
(7)取5μL RNA，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，操作应用Reverse Transcriptase M-MLV(RNaseH-)(购自Takara)参照说明书完成。
3.SpNBS-LRR基因的PCR扩增
以番茄L3708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：
SpNBS-LRR-FP：CGGGATCCATGTGTGTGGGAAGAGTTACAGATT
SpNBS-LRR-RP：CGAGCTCTTACTCTAGTCTTTCAAGTTTGGGG
反应条件如下：
4.PCR扩增产物的回收纯化
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)回收符合目标片段大小的PCR产物。
5.目标片段与克隆载体连接
将上述回收得到的目标片段与克隆载体pMD-19T(购自Takara)连接，反应体系如下：
16℃连接8h，即得到连接产物pMD-19T-SpNBS-LRR。
6.连接产物转化大肠杆菌
(1)将10μL的连接产物加入至上述200μL的感受态细胞中，吹打混匀后，冰水浴30min；
(2)将上述冰水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)向上述混合液中加入1mL新鲜的LB培养基，于37℃恒温摇床中，180rpm振荡培养1.5h；
(4)将上述菌液于4000r/min离心10min，吸去1mL上清，留下200μL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℃恒温培养箱中培养过夜；
(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，于37℃恒温摇床中180rpm振荡培养过夜。
7.pMD-19T-SpNBS-LRR质粒的提取
依据质粒小提试剂盒(购自TIANGEN)的说明，提取上述菌液中所含有的pMD-19T-SpNBS-LRR质粒。取5μL的质粒样品进行1％琼脂糖凝胶电泳检测。
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：番茄抗晚疫病基因SpNBS-LRR表达载体的构建
1.pMD-19T-SpNBS-LRR质粒的酶切
将pMD-19T-SpNBS-LRR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，切除GUS基因，回收pBI121片段即大片段。反应体系如下：
37℃酶切6h，1％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切掉GUS基因的pBI121载体连接。反应体系如下：
16℃连接过夜，连接产物即为表达载体pBI121-SpNBS-LRR。
实施例三：番茄抗晚疫病基因SpNBS-LRR的应用
1.pBI121-SpNBS-LRR农杆菌工程菌的制备
(1)将2μL的pBI121-SpNBS-LRR质粒加入到200μL的农杆菌感受态细胞中，充分吹打混匀后，冰水浴10min，之后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，180rpm振荡培养3h；
(3)将上述得到的菌液离心后，吸去1mL上清，充分吹打悬浮余下的200μL菌液，将其均匀涂布于YEB固体培养基上(含100mg/L链霉素、100mg/L利福平和50mg/L卡那霉素)，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SpNBS-LRR特异性引物进行PCR，反应条件及反应体系同“实施例一，3”。
2.农杆菌介导的叶盘法转化番茄早粉2号
(1)番茄种子的萌发
用水将番茄种子冲洗干净后转移至75％的乙醇中，浸泡30s，中间不断晃动，再将种子移至2.5％的次氯酸钠中表面除菌30min，最后用无菌水冲洗4次，每次2min。将上述表面已除菌的种子均匀摆放于预先湿润的滤纸上，并于其表面覆盖一层湿润的滤纸，以保持较高湿度。将处理后的种子置于无菌环境中，于25-28℃暗培养，直至种子萌发。
(2)番茄子叶的获得
当番茄种子长出胚芽后，将其移至于1/2MS培养基中，待1周后长出子叶。
(3)番茄子叶外植体的制备
待番茄的两片子叶完全舒展后，剪除其两端，作为后续农杆菌侵染的外植体，于含有1.5mg/L 6-BA和0.5mg/L IAA的MS培养基上预培养2d。
(4)农杆菌工程菌液的制备
将上述得到的农杆菌阳性菌接种于新鲜的YEB培养基中(含100mg/L链霉素、100mg/L利福平和50mg/L卡那霉素)，28℃，180rpm振荡培养16h左右。按照1:50的比例将菌液接种于不含任何抗生素的YEB液体培养基中，28℃，180rpm振荡培养至菌液OD600为0.5-0.6之间。
(5)农杆菌对番茄外植体的侵染
将预培养后的番茄外植体转移至上述菌液中，28℃，180rpm振荡培养7-10min，室温静止2min，用无菌滤纸吸干叶片表面多余的菌液，将叶片均匀放置于MS培养基中(含1.5mg/L 6-BA和0.5mg/L IAA)共培养2d(远轴面朝上)。
(6)番茄抗性芽的获得及生根
将共培养后的番茄外植体转移至MS筛选培养基中(含1.5mg/L 6-BA，0.5mg/LIAA，50mg/L卡那霉素和200mg/L羧苄青霉素)，培养至产生抗性芽。待抗性芽长至1-2cm时，将其剪下移至1/2MS生根培养基(含50mg/L卡那霉素和200mg/L羧苄青霉素)中，进行生根培养。
(7)番茄过表达植株的验证
挑选长势一致的过表达及野生型植株，提取叶片总RNA，反转录成cDNA并以此为模板，利用SpNBS-LRR的特异性引物进行实时定量PCR检测，结果如图1所示，过表达植株中SpNBS-LRR的表达量明显高于野生型植株。RNA提取同“实施例一，1”。反转录同“实施例一，2”。实时定量所用试剂盒为
Premix Ex TaqTMII(Tli RNaseH Plus)(购自Takara)，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：
qSpNBS-LRR-FP：GGTATGGGTGGTGTAGGTAAGA
qSpNBS-LRR-RP：GACTGACAGTGACCATGACAAC
3.过表达SpNBS-LRR番茄植株的抗病性检测
(1)晚疫病菌孢子悬浮液的制备
将保存的晚疫病菌菌种接种于燕麦培养基上，于18℃的黑暗环境下培养15d，用无菌水将菌体全部洗下，并用三层纱布过滤菌液。调节孢子终浓度至1×l06spores/mL，于4℃低温保存1h。
(2)晚疫病菌接种番茄材料
选取长势一致的过表达和野生型番茄植株，对其均匀喷洒上述晚疫病菌孢子悬浮液。将处理后的番茄植株首先置于20±2℃，100％湿度的黑暗条件下培养24h，恢复正常光照后，置于温度为20±2℃，75％湿度的条件下培养4d。观察并拍照记录发病情况，如图2所示，过表达植株的病情明显轻于野生型植株，说明过表达SpNBS-LRR的番茄植株对晚疫病具有更高的抗性。
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。
SEQUENCE LISTING
<110> 大连理工大学
<120> 番茄抗晚疫病基因SpNBS-LRR及其克隆方法与应用方法
<130> 2017
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 2316
<212> DNA
<213> 人工序列
<400> 1
atgtgtgtgg gaagagttac agattgcttg atgcagccac ctgcgcaaca gattgggtat 60
ttcttttact acaacagcaa catcacatct ttggataatg gatctcacaa gctggacaat 120
attacaagtg gggtgcagca aagagcggag gctgcacgga gaaacttgca agtcatttca 180
caagatgttg aagcttggtt aaatagtgtt accaagatta attcagatgt ggaaggtgta 240
atgcatggta gagttgaggt tgaaagaggt tgtttttatg gttggtgtcc aaatttgaag 300
tcacggtact cgttgagcaa gagagctaag agaattacac tagcggtgat tgaacttcga 360
aatgatggaa aagattatgt tgatttctcc tatcctgcac cacctgtagt tgaaattcaa 420
gctatgagtg aggagtttga ctccagaaaa ttgaaagagg aagaggtcat ggcagctttg 480
agagatgagg atgtctctgt tattgggatt tgtggtatgg gtggtgtagg taagacgaca 540
ctagctgaga aaataagagt aagggcgaaa aaagaaaggt tttttgatga agttgtcatg 600
gtcactgtca gtcaacaacc agacttgaaa acaattcaag ctgagatagc tggaggagtt 660
ggcctaacat tccaaggcga caatttctgg aatcgtggag atcagttgcg ttcaaggtta 720
atgggtcagg acagcatcct tataatcttg gatgatgtct gggaggctct tgatctgaac 780
aaacttggaa ttcctagttg tagcaatcac aaccatcagt gcaaagtaac attgacaacg 840
cgactccgag atgtttgtga aacaatggag gctcgaaaga tcatagaagt tggaatctta 900
cctgaaaagg aagcatgggt ccttttcagg cagaaagccg gtaattcggt agctgatctt 960
tctcttcatg acacagcaaa agatgttgtg aaagaatgca aggggttgcc acttgcaatt 1020
attacagttg caggagcgct aaagcgtaaa agcaagcctt catgggagga tgcccttaaa 1080
caattacaaa aatccacacc aaaaaatatc ccaggagtga ttaaaaatgt gtatcaatct 1140
ctcaagctaa gctatgatca gttggaaagt gatgaagtca ggtacctgtt tttgctttgt 1200
tccttgtttg aggaagatag taatatctgg catgaacaat tacttagata tggaatgggg 1260
cttggcatct tctcggaaat tgaaaattta gaagaagcaa gaaagagggt gtgccatctg 1320
ctagaaacat tgaaggatcg tttcttgcta tcccaaggtt caggaaaaaa ttatgtcaaa 1380
atgcatgatg tggtccgaga cgtggctata tatatcgcct ctgagggaag gcatgttttt 1440
atggtaagtc acagtgtgaa ctcagaagag ttcccaagaa gaacctctta cgagccatac 1500
agtcacatgt caattgttgc acaaaaaatt gatgagcttc ctaaaccaat atctttccca 1560
agacttgagt ttctaatgtt aaaactatta gaagagccgt tcaaattaca ggatgatttt 1620
tttattggaa tgagtaaact taatgtctta agcctgagtg gatatgagga ctccatttta 1680
acctttccaa attctgttca gttattgtca aatctgcgca cactgtctct gatgaattta 1740
aagttggatg acatatcaat tattggcgaa cttgtcactt tagagattct cattattaga 1800
gattctacta tagatgtgct tccagtggag attggaaatt tgagcaattt aattttgtta 1860
gagttttgga acgagagagt accacttgaa aatatttcac ctggagtctt atcaagacta 1920
gttcgattag aggaactaac actagtgaaa tgttctggag atgtaattca cagtaacttg 1980
gacatttcct ccaatttgac acggtattat ctaaatatgg gccaacaagt tcatagttac 2040
cacgatagtt cgctcatgga taattacaac aggattatgg tcctcaatgt tattgagacc 2100
accccattgg gtgattggat ctgccgcatg ttgaagaaga gcgaacttgt acattcaaga 2160
ggaaatggtt ccaagaatgt gctgaccgag ttgctgggac atggagttca gaacatgaaa 2220
gatctcctcc tggctgattg tgattcaatg acacatctct tgaatatcca ctgtcagaat 2280
aatattccat tccccaaact tgaaagacta gagtaa 2316
<210> 2
<211> 33
<212> DNA
<213> 人工引物
<400> 2
cgggatccat gtgtgtggga agagttacag att 33
<210> 3
<211> 32
<212> DNA
<213> 人工引物
<400> 3
cgagctctta ctctagtctt tcaagtttgg gg 32
Claims (4)
1.一种番茄抗晚疫病基因SpNBS-LRR的应用方法，其特征在于，用于培育抗晚疫病番茄植株。
2.权利要求1所述番茄抗晚疫病基因SpNBS-LRR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基因SpNBS-LRR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；
选取阳性工程菌侵染预培养后的番茄子叶，共培养，分化出芽；
将抗性芽移至生根培养基中诱导生根后移栽，实时定量PCR验证过表达植株；
对过表达SpNBS-LRR的番茄苗进行抗病性检测，得到抗晚疫病番茄植株。
3.根据权利要求2所述番茄抗晚疫病基因SpNBS-LRR的应用方法，其特征在于，所述重组表达载体由将所述番茄抗晚疫病基因SpNBS-LRR插入表达载体得到。
4.根据权利要求3所述番茄抗晚疫病基因SpNBS-LRR的应用方法，其特征在于，所述重组表达载体的具体制备方法为：
以野生型抗晚疫病番茄L3708的cDNA为模板，以SpNBS-LRR-FP、SpNBS-LRR-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-19T克隆载体连接，得到的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；
提取上述连接番茄抗晚疫病基因SpNBS-LRR的单菌落的质粒，并用限制性内切酶BamHI及SacI双酶切，回收得到目的片段，将所述目的片段与去除GUS基因的pBI121表达载体连接，得到的连接产物即为重组表达载体。
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