CN109022449B - 黄瓜CsMLO1基因及其沉默表达载体构建方法、应用 - Google Patents

黄瓜CsMLO1基因及其沉默表达载体构建方法、应用 Download PDF

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CN109022449B
CN109022449B CN201810824315.6A CN201810824315A CN109022449B CN 109022449 B CN109022449 B CN 109022449B CN 201810824315 A CN201810824315 A CN 201810824315A CN 109022449 B CN109022449 B CN 109022449B
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范海延
于广超
崔娜
于洋
王翔宇
孟祥南
陈秋敏
赵珺玥
杨云
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Abstract

本发明属于分子生物学和生物技术领域，具体涉及一种黄瓜CsMLO1基因及其沉默表达载体构建方法、应用，黄瓜响应棒孢叶斑病调控途径中的CsMLO1基因的全长编码区序列如序列表SEQ ID NO.1所示；氨基酸序列如序列表SEQ ID NO.2所示。本发明还提供将黄瓜CsMLO1基因特异片段***沉默表达载体pTRV2的构建方法，将构建的植物沉默表达载体经农杆菌转化到黄瓜子叶，获得转基因黄瓜植株并开展抗病性鉴定，结果表明该基因是黄瓜与棒孢叶斑病互作中的负调控因子。本发明为利用基因工程技术，为全面揭示黄瓜CsMLO1基因功能研究提供了技术手段与理论依据，具有很大的应用价值。

Description

黄瓜CsMLO1基因及其沉默表达载体构建方法、应用
技术领域
本发明属于分子生物学和生物技术领域，具体涉及一种黄瓜CsMLO1基因(Cucsa.207280)及其沉默表达载体构建方法、应用。
背景技术
黄瓜(Cucumis sativus)是我国北方设施栽培的主要蔬菜作物之一，其产量和品质严重受到非生物胁迫的影响。黄瓜棒孢叶斑病是一种世界性的真菌病害，由致病真菌多主棒孢(Corynespora cassiicola)引起；目前，该病害的研究主要集中在生物防治和化学防治等方面，在黄瓜对棒孢叶斑病的抗性机制研究甚少。
MLO(Mildew Resistance Locus O)蛋白是植物中特有的蛋白，其研究表明MLO家族成员作为感病基因，易受白粉菌的侵染，而隐性突变基因mlo却被赋予广谱抗性(Shirasuet al.,1999；Chen et al.,2006；Kim et al.,2002)。目前，mlo抗病路径与白粉菌以外的其它病原菌的关系也受到越来越多的关注。条形柄锈菌(Puccinia striiformisf.sp.tritici)是引起小麦条锈病的专化真菌，主要危害小麦叶部。研究通过病毒诱导基因沉默(virus-induced gene silencing)技术，对可能参与过敏性细胞死亡信号通路的三个跨膜蛋白基因TaMLO1，TaMLO2，TaMLO5进行基因沉默，试验接种条锈病植株表现不同程度的抗病能力，TaMLO1/2/5可能作为细胞程序性死亡的负调控因子在一定程度上参与调节了小麦与条锈菌的互作过程(史雪霞，2013)。野生型大麦突变基因mlo3、mlo4、mlo5与大麦叶枯病分析比较发现，三突变体对病原菌的抗性增强(罗臻等2009)。因此，根据系列事实认为，MLO这种抗病蛋白可能作为一种调节器，不仅是白粉病的抗性抑制子，而且在其他病原菌中也体现出相互抑制的防卫反应。
综上所述，关于黄瓜与棒孢叶斑病之间相互作用的研究甚少。因此，选取黄瓜CsMLO1基因402bp片段***pTRV2，将构建重组载体pTRV2-CsMLO1和pTRV1等体积导入植物体中，通过表型鉴定分析TRV病毒已成功在黄瓜子叶中大量繁殖，并结合qRT-PCR技术检测CsMLO1基因在转基因黄瓜中的拷贝数明显降低。从而才能深层次了解黄瓜CsMLO1基因作为黄瓜与棒孢叶斑病互作中的负调控因子，为全面揭示黄瓜CsMLO1基因功能奠定基础。
发明内容
为了解决上述技术问题，本发明提供一种黄瓜CsMLO1基因及其沉默表达载体构建方法、应用。
本发明是这样实现的，提供一种黄瓜CsMLO1基因，其编码区序列如SEQ ID NO.1所示。
提供一种用于扩增上述黄瓜CsMLO1基因的引物，其核苷酸序列如下：
CsMLO1-F：5’-ATGGCGGGGGCAGCCGGTGG-3’
CsMLO1-R：5’-TTCAACTCTATCAAATGAAA-3’。
利用上述引物对黄瓜CsMLO1基因进行扩增的方法，以黄瓜叶片为材料，利用RNAprep pure植物总RNA提取试剂盒的方法提取总RNA，反转录合成cDNA，设计如权利要求2所述的引物；
以反转录的cDNA为模版，进行聚合链式PCR反应，回收PCR产物，获得1749bp目的片段，将纯化产物进行测序，得到序列如SEQ ID NO.1所示的黄瓜CsMLO1基因。
提供一种利用上述黄瓜CsMLO1基因编码得到的蛋白质，其氨基酸序列如SEQ IDNO.2所示。
提供上述的黄瓜CsMLO1基因的特异性区域引物，比对黄瓜MLO家族基因序列，选取特异性区域402bp设计引物,其核苷酸序列如下：
TRV-CsMLO1-F:5’-gtgagtaaggttaccgaattcGTGGCAGAGGCCCTTCGCAAC-3’
TRV-CsMLO1-R:5’-ggcctcgagacgcgtgagctcTCATTCAACTCTATCAAATGA-3’。
注：TRV-CsMLO1-F和TRV-CsMLO1-R引物序列前21bp碱基gtgagtaaggttaccgaattc和ggcctcgagacgcgtgagctc，是构建载体需要，人为加入的不属于目的基因序列的表达载体部分序列。
对黄瓜CsMLO1基因特异区域进行聚合链式PCR反应，回收PCR产物，获得402bp目的片段，将纯化产物进行测序，得到序列如SEQ ID NO.3所示的黄瓜CsMLO1基因特异区域。纯化后的胶回收产物CsMLO1特异区域和线性化末端同源序列的pTRV2载体，应用In-FusionHD Cloning kit将CsMLO1特异区域连入pTRV2线性大片段中，反应体系为：2μL 5×In-Fusion HD Enzyme Premix，5μL pTRV2线性大片段，3μL CsMLO1目的片段，50℃反应15min；将连接产物转至大肠杆菌感受态DH5α中，经菌落PCR及测序鉴定，摇菌、提质粒获得带有目的基因pTRV2-CsMLO1植物沉默表达载体。
提供一种利用上述的方法制备的黄瓜CsMLO1基因沉默表达载体的植物转化体。
进一步地，所述的植物转化体中的受体植物为黄瓜“新泰密刺”。
提供上述黄瓜CsMLO1基因沉默在提高黄瓜对棒孢叶斑病菌抗性中的应用。
与现有技术相比，本发明的优点在于：
1、本发明利用现有的植物基因工程技术，利用基因表达分析、基因克隆及序列分析技术，分离鉴定了黄瓜相关基因序列信息；
2、本发明构建的含有黄瓜CsMLO1基因沉默表达载体，为首次报道，可直接进行遗传转化试验；
3、本发明提供的黄瓜CsMLO1基因是一个新的黄瓜响应棒孢叶斑病的编码基因。通过农杆菌介导转化法将该沉默载体pTRV2-CsMLO1和pTRV1瞬时转入黄瓜子叶后沉默CsMLO1基因的表达，接种棒孢叶斑病后与对照相比其病斑面积减少，说明其提高了黄瓜对棒孢叶斑病菌胁迫的抗性，为黄瓜对棒孢叶斑病抗病关键基因的研究提供新的理论依据。
附图说明
图1为CsMLO1基因编码区序列的扩增结果；
图2(a)为重组载体pTRV2-CsMLO1构建方法示意图；
图2(b)为大肠杆菌pTRV2-CsMLO1检测电泳结果；
图2(c)为pTRV2-CsMLO1的测序结果；
图3(a)为沉默CsMLO1基因后黄瓜子叶呈现病毒斑点示意图；
图3(b)为沉默黄瓜子叶CsMLO1基因的表达情况示意图；
图4对照组与转基因黄瓜子叶病原菌处理比较，转基因黄瓜子叶提高了对棒孢叶斑病菌胁迫的抗性。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，下面结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用于解释本发明，并不用于限定本发明。
实施例1
黄瓜CsMLO1基因的克隆
1)黄瓜CsMLO1基因的cDNA序列如SEQ ID NO.1所示。调控黄瓜响应棒孢叶斑病CsMLO1基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
2)黄瓜CsMLO1基因的克隆方法如下：以黄瓜叶片为材料，利用RNAprep pure植物总RNA提取试剂盒的方法提取总RNA，反转录合成cDNA，设计引物扩增黄瓜CsMLO1基因，引物核苷酸序列如下：
CsMLO1-F：5’-ATGGCGGGGGCAGCCGGTGG-3’
CsMLO1-R：5’-TTCAACTCTATCAAATGAAA-3’。
以反转录的cDNA为模板，进行聚合链式PCR反应，回收PCR产物，获得1749bp目的片段，参考图1。将纯化产物进行测序，得到序列如SEQ ID NO.1所示的黄瓜CsMLO1基因。
实施例2
植物沉默表达载体构建
参考图2(a)、图2(b)和图2(c)，以纯化后的胶回收产物pTRV2-CsMLO1，与线性化末端同源序列的pTRV2载体，应用In-Fusion HD Cloning kit将CsMLO1特异目的片段连入pTRV2线性大片段中，反应体系为：2μL 5×In-Fusion HD Enzyme Premix，5μL线性大片段，3μL目的片段，50℃反应15min。将连接产物转至大肠杆菌感受态DH5α中，经菌落PCR及测序鉴定，摇菌、提质粒获得带有目的基因pTRV2-CsMLO1植物沉默表达载体。
实施例3
瞬时转化黄瓜子叶进行功能验证，参考图3(a)和图3(b)。
1)重组质粒转化黄瓜子叶
将pTRV1和含有目的基因pTRV2-CsMLO1的农杆菌EHA105阳性克隆，接种于液体YEP培养基(含50μg mL-1Rif和50μg mL-1Kan)，28℃200rpm震荡培养48h，使农杆菌菌液培养至OD600为0.6-1.0。
将菌液转入无菌的离心管中，5000rpm离心10min收集菌体；用10mmol·L-1MES+10mmol·L-1MgCl2+200μmol·L-1As的水溶液悬浮菌体，至OD600为0.4，室温放置3h；将菌体悬浮液用无针头注射器注入苗龄为9d黄瓜子叶。
采用植物材料为“新泰密刺”黄瓜；
2)黄瓜基因瞬时过表达株系鉴定
a)表型鉴定：采用注射法对苗龄为9d的黄瓜子叶接种重组质粒pTRV2-CsMLO1+EHA105和pTRV1+EHA105的1:1(v/v)混合液，23℃条件下培养。接种10d在注射TRV:00+EHA105(pTRV1+EHA105和pTRV2+EHA105的1:1混合液)和TRV:CsMLO1+EHA105子叶上均可以观察到黄色病毒斑点，而在Control的子叶上均未见病毒斑点，说明TRV病毒已成功在黄瓜子叶中大量繁殖。
b)RT-PCR检测：进一步分析CsMLO1基因的瞬时沉默表达水平，以注射TRV:00+EHA105植株cDNA为对照，检测注射TRV:CsMLO1+EHA105的黄瓜中CsMLO1基因的表达水平。注射TRV:CsMLO1的黄瓜中CsMLO1基因的表达量显著降低至对照的0.03-0.1倍；结果表明，在转基因黄瓜子叶中成功瞬时沉默CsMLO1基因表达。
实施例4
瞬时沉默CsMLO1基因黄瓜的棒孢叶斑病抗性鉴定
参考图4，为了进一步验证CsMLO1基因在黄瓜-棒孢叶斑病互作过程中负调控因子功能，本试验对TRV:CsMLO1注射10d的黄瓜子叶进行棒孢叶斑病菌接种处理以观察其发病情况。接种棒孢叶斑病菌10d与Control和注射TRV:00植株相比，注射TRV:CsMLO1的黄瓜子叶未显现出棒孢叶斑病症状。同时，CsMLO1沉默植株的病情指数是24.3，而Control和注射TRV:00植株的病情指数分别是52.44和58.30。因此，CsMLO1基因的沉默表达提高了黄瓜对棒孢叶斑病菌胁迫的抗性。
SEQUENCE LISTING
<110> 沈阳农业大学
<120> 黄瓜CsMLO1基因及其沉默表达载体构建方法、应用
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1749
<212> DNA
<213> 人工序列
<400> 1
atggcggggg cagccggtgg caagtcgctg gagcaaacac cgacatgggc cgttgccgtt 60
gtttgctttg ttttgctcgt catctctatt ttcatcgaat atagtctcca tcttatcgga 120
cattggctaa agaagagaca caaacgggcg ttgtttgaag cattagagaa gatcaaatca 180
gagcttatgt tattggggtt tatatcattg ctactaacgg tggggcaagg accaataacg 240
gagatatgta ttccacaaca tgtagctgca acgtggcatc catgtacaaa ggaaagagaa 300
gatgagatga acaaagaggt ggagaaatct gtggaacatt tgggtcttaa tcgccggaga 360
ctccttcatc tcctcggaaa tggtgaaagt ttccggcgga gtttggccgc tgcgggagga 420
gaggataaat gtgccgccaa gggtaaagct tcctttattt cagcagatgg aattcatcaa 480
cttcatatct tcatttttgt gttggctgtt tttcatgttt tgtattgtgt tctaacttat 540
gcgttggcta gagctaagat gaggagttgg aaaacatggg aaaaagagac caaaactgct 600
gaataccaat tctcacatga tccagagagg tttaggtttg caagagacac ctcatttggg 660
agaagacatt tgagcttttg gaccaaaaat cctgccttga tgtggatcgt ttgtttcttc 720
agacaatttg taagatctgt tccaaaagtt gattacttga cattaagaca tgggtttata 780
atggcacatt tagcacctca aagtcataca caatttgatt ttcaaaaata cattaataga 840
tcccttgaag aagacttcaa agttgttgtg ggaatcagcc caccaatttg gttctttgct 900
gttctatttc tcctctcaaa cactcacggt tggagggcgt atctatggct gccattcatc 960
ccactaatca ttttgctgtt gattggaaca aaattgcaag tgatcataac gaaaatggca 1020
ctaagaatac aagaaagagg tgaagtagtg aagggcgtgc cggtggtgga gcctggcgat 1080
gacctctttt ggtttaatcg acctcgcctt attctttatc tcatcaactt tgttctcttt 1140
caaaatgcct tccaagttgc cttctttgct tggacttggt atgagtttgg gttgaattct 1200
tgcttccatg agcatataga agatgtggtg atcagaattt ctatgggggt gcttgtacaa 1260
atcctttgca gttatgttac tcttcctctt tatgcactag tcactcagat gggttcaaca 1320
atgaagccaa ctatattcaa tgagagagtg gcagaggccc ttcgcaattg gtaccactcg 1380
gctcgaaagc acatcaaaca caaccgcggt tcggtcactc caatgtcgag ccgacccgcc 1440
accccgactc acagcatgtc acctgtccac cttctccgac actacaagag tgaagtcgat 1500
agcttccaca cctcaccgag aaggtcaccg ttcgacaccg atcgttggga caacgattcg 1560
ccctctccat ctcgccatgt tgatggttcg tcttcgtcac aaccccacgt tgagatggga 1620
ggttatgaaa aagatcccgt tgaatcaagt tcgtctcaag ttgatccggt tcaaccatct 1680
cgaaaccgca atcaacatga gattcatatt ggaggcccca aagacttttc atttgataga 1740
gttgaatga 1749
<210> 2
<211> 582
<212> PRT
<213> 人工序列
<400> 2
Met Ala Gly Ala Ala Gly Gly Lys Ser Leu Glu Gln Thr Pro Thr Trp
1 5 10 15
Ala Val Ala Val Val Cys Phe Val Leu Leu Val Ile Ser Ile Phe Ile
20 25 30
Glu Tyr Ser Leu His Leu Ile Gly His Trp Leu Lys Lys Arg His Lys
35 40 45
Arg Ala Leu Phe Glu Ala Leu Glu Lys Ile Lys Ser Glu Leu Met Leu
50 55 60
Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Gly Gln Gly Pro Ile Thr
65 70 75 80
Glu Ile Cys Ile Pro Gln His Val Ala Ala Thr Trp His Pro Cys Thr
85 90 95
Lys Glu Arg Glu Asp Glu Met Asn Lys Glu Val Glu Lys Ser Val Glu
100 105 110
His Leu Gly Leu Asn Arg Arg Arg Leu Leu His Leu Leu Gly Asn Gly
115 120 125
Glu Ser Phe Arg Arg Ser Leu Ala Ala Ala Gly Gly Glu Asp Lys Cys
130 135 140
Ala Ala Lys Gly Lys Ala Ser Phe Ile Ser Ala Asp Gly Ile His Gln
145 150 155 160
Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Leu Tyr Cys
165 170 175
Val Leu Thr Tyr Ala Leu Ala Arg Ala Lys Met Arg Ser Trp Lys Thr
180 185 190
Trp Glu Lys Glu Thr Lys Thr Ala Glu Tyr Gln Phe Ser His Asp Pro
195 200 205
Glu Arg Phe Arg Phe Ala Arg Asp Thr Ser Phe Gly Arg Arg His Leu
210 215 220
Ser Phe Trp Thr Lys Asn Pro Ala Leu Met Trp Ile Val Cys Phe Phe
225 230 235 240
Arg Gln Phe Val Arg Ser Val Pro Lys Val Asp Tyr Leu Thr Leu Arg
245 250 255
His Gly Phe Ile Met Ala His Leu Ala Pro Gln Ser His Thr Gln Phe
260 265 270
Asp Phe Gln Lys Tyr Ile Asn Arg Ser Leu Glu Glu Asp Phe Lys Val
275 280 285
Val Val Gly Ile Ser Pro Pro Ile Trp Phe Phe Ala Val Leu Phe Leu
290 295 300
Leu Ser Asn Thr His Gly Trp Arg Ala Tyr Leu Trp Leu Pro Phe Ile
305 310 315 320
Pro Leu Ile Ile Leu Leu Leu Ile Gly Thr Lys Leu Gln Val Ile Ile
325 330 335
Thr Lys Met Ala Leu Arg Ile Gln Glu Arg Gly Glu Val Val Lys Gly
340 345 350
Val Pro Val Val Glu Pro Gly Asp Asp Leu Phe Trp Phe Asn Arg Pro
355 360 365
Arg Leu Ile Leu Tyr Leu Ile Asn Phe Val Leu Phe Gln Asn Ala Phe
370 375 380
Gln Val Ala Phe Phe Ala Trp Thr Trp Tyr Glu Phe Gly Leu Asn Ser
385 390 395 400
Cys Phe His Glu His Ile Glu Asp Val Val Ile Arg Ile Ser Met Gly
405 410 415
Val Leu Val Gln Ile Leu Cys Ser Tyr Val Thr Leu Pro Leu Tyr Ala
420 425 430
Leu Val Thr Gln Met Gly Ser Thr Met Lys Pro Thr Ile Phe Asn Glu
435 440 445
Arg Val Ala Glu Ala Leu Arg Asn Trp Tyr His Ser Ala Arg Lys His
450 455 460
Ile Lys His Asn Arg Gly Ser Val Thr Pro Met Ser Ser Arg Pro Ala
465 470 475 480
Thr Pro Thr His Ser Met Ser Pro Val His Leu Leu Arg His Tyr Lys
485 490 495
Ser Glu Val Asp Ser Phe His Thr Ser Pro Arg Arg Ser Pro Phe Asp
500 505 510
Thr Asp Arg Trp Asp Asn Asp Ser Pro Ser Pro Ser Arg His Val Asp
515 520 525
Gly Ser Ser Ser Ser Gln Pro His Val Glu Met Gly Gly Tyr Glu Lys
530 535 540
Asp Pro Val Glu Ser Ser Ser Ser Gln Val Asp Pro Val Gln Pro Ser
545 550 555 560
Arg Asn Arg Asn Gln His Glu Ile His Ile Gly Gly Pro Lys Asp Phe
565 570 575
Ser Phe Asp Arg Val Glu
580
<210> 3
<211> 402
<212> DNA
<213> 人工序列
<400> 3
gtggcagagg cccttcgcaa ttggtaccac tcggctcgaa agcacatcaa acacaaccgc 60
ggttcggtca ctccaatgtc gagccgaccc gccaccccga ctcacagcat gtcacctgtc 120
caccttctcc gacactacaa gagtgaagtc gatagcttcc acacctcacc gagaaggtca 180
ccgttcgaca ccgatcgttg ggacaacgat tcgccctctc catctcgcca tgttgatggt 240
tcgtcttcgt cacaacccca cgttgagatg ggaggttatg aaaaagatcc cgttgaatca 300
agttcgtctc aagttgatcc ggttcaacca tctcgaaacc gcaatcaaca tgagattcat 360
attggaggcc ccaaagactt ttcatttgat agagttgaat ga 402

Claims (1)

1.一种黄瓜CsMLO1基因在负调控黄瓜对棒孢叶斑病菌抗性中的应用，其特征在于，所述黄瓜CsMLO1基因的编码区序列如SEQ ID NO.1所示，所述棒孢叶斑病菌为致病真菌多主棒孢，拉丁文名称为Corynespora cassiicola。
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CN113528538B (zh) * 2021-08-16 2022-04-19 东北农业大学 黄瓜CsSTK基因、蛋白、表达载体及应用
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