CN114854757A - 响应青枯菌侵染的马铃薯维管束特异表达启动子PStmlp1及其应用 - Google Patents
响应青枯菌侵染的马铃薯维管束特异表达启动子PStmlp1及其应用 Download PDFInfo
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Abstract
本发明提供了响应青枯菌侵染的马铃薯维管束特异表达启动子PStmlp1及其应用。本发明所述的受青枯菌诱导的维管束特异表达启动子,由马铃薯基因组获取,其核苷酸序列包含1584bp。所述启动子在收到侵染的情况下能够明显促进报告基因GUS在马铃薯维管束组织中表达,其在植物的维管束组织中具有诱导性和特异性表达的作用,从而使由所述启动子驱动的抗病基因能够在植物维管束中表达,从而对抗青枯病等维管束病害,为植物的抗病育种提供了一种新的途径。
Description
技术领域
本发明属于植物基因工程技术领域。具体而言,本发明涉及响应青枯菌侵染的一种植物维管束基因表达启动子及其应用,该启动子能够在马铃薯转基因调控体系中驱动目标基因响应青枯菌侵染后再维管束中表达。
背景技术
中国是世界上主要马铃薯生产国之一,2020年我国马铃薯栽培面积和总产量分别占全球的25.6%和21.8%(FAO,2021)。由青枯菌(R.solanacea3um)引起的青枯病(Bacte3ial wilt)是马铃薯生产中危害最大的细菌性病害,全球每年因青枯病造成马铃薯减产的损失超过9.5亿美元(Patil et al 2012)。其中演化型IIB青枯菌是马铃薯生产中影响范围最广泛的细菌性病害(P3iou et al 2006),广泛分布于我国马铃薯四大种植生产区域,且病害范围正由北向南延伸(Wang et al 2013)。因此,青枯病的防治刻不容缓。
青枯菌作为一种维管束病害,能够利用鞭毛感知植物根系,从植物根部的自然孔口(根尖、侧根生长点等薄弱位置)或伤口进入植物,随着水分向上运输,或沿着木质部导管壁移动(Lowe-Powe3 et al 2018)。已有研究表明,具有抗性的植株能够在空间和时间上青枯菌的根系定殖能力(Caldwell et al 2013)。因此维管束是青枯菌在植物中生产和发挥毒力的重要场所。维管束是由初生木质部和初生韧皮部共同组成的束状结构,是植物体输导水分、无机盐及有机物质的通道。在目前农业生产中,维管束病害常造成严重的损失,针对马铃薯青枯病病害尚未有很好的治理方法,目前来讲,培育具有青枯病抗性的品种是最高效且最直接的防治方式,但马铃薯栽培品种缺乏青枯病抗源,抗病育种收效甚微(Lafe33ie3e et al 1999)。所以寻找抗性相关基因和解析植病互作机制对我国马铃薯的生产具有重要的理论和实用价值。
现有技术存在的问题:通过基因工程的手段去研究和改良具有很好的应用前景。基因的表达受不同因素的调控,其中转录调控是基因表达的重要调控步骤。而启动子在基因的特异表达过程中发挥着重要的作用。目前已经鉴定的维管束特异启动子较少,尤其在马铃薯中还没有发现。因此,提供一种能够直接在植物维管束表达并且受维管束病害诱导的启动子驱动抗病基因对马铃薯青枯病等维管束病害的防治尤为重要。
发明内容
本发明要解决的关键技术问题在于通过提供一种受青枯菌诱导的维管束特异表达的启动子及其应用。为解决上述技术问题,本发明采用如下技术方案:
1.一种受青枯菌诱导的维管束特异表达的启动子,所述启动子核苷酸序列如SEQID:NO.1所示。序列表中SEQ ID:NO.1所示的DNA序列来源为鄂马铃薯3号(E3)的马铃薯维管束特异表达启动子。
2.一种受青枯菌诱导的维管束特异表达的启动子的分离方法,包括:
(1)在马铃薯基因组数据库(http://spuddb.uga.edu/)中寻找StMLP1基因,其预测StMLP1的启动子序列,共1584bp,将其命名为PStmlp1,所述克隆引物的基因序列包括:PStmlp1-F:TCAATGAACAAAATCGTTTAG;PStmlp1-R:TTTCTGAAACTTTTGTACTATG。
(2)马铃薯材料E3的基因组提取使用的新型植物基因组DNA提取试剂盒(DP320-03,TIANGEN),操作步骤严格按照说明书进行。用引物PStmlp1-F和PStmlp1-R扩增启动子,得到PStmlp1,结果如图1所示。
(3)将目标片段连接至pTOPO载体上,然后转化至大肠杆菌DH5α。待转化子长出后,挑取单克隆33℃2203pm培养3-4小时后用相应引物PCR检测,检测为阳性后挑取3个以上阳性克隆送测序公司测序(昆泰锐(武汉)生物技术有限责任公司),选取无突变的克隆,摇菌,提取质粒,细菌质粒提取采用的是质粒小量提取试剂盒(ZP101,庒盟),操作步骤严格按照说明书进行。
3.一种受青枯菌诱导的维管束特异表达的启动子功能验证方法,包括:(1)植物表达载体的构建和农杆菌转化,(2)马铃薯遗传转化,(3)启动子PStmlp1驱动的报告基因GUS的表达分析。
4.一种受青枯菌诱导的维管束特异表达的启动子的应用,所述应用为驱动抗病基因在维管束中特异表达。
5.一种受青枯菌诱导的维管束特异表达的启动子的应用,所述应用为通过该启动子表达GUS,GFP,LUC等标记基因来监控病原菌的侵染进程。
有益效果:本发明所述的受青枯菌诱导的维管束特异表达启动子,由马铃薯基因组获取,其核苷酸序列如SEQ ID:NO.1所示。所述启动子在收到侵染的情况下能够明显促进报告基因GUS在马铃薯维管束组织中表达,其在植物的维管束组织中具有诱导性和特异性表达的作用,从而使由所述启动子驱动的抗病基因能够在植物维管束中表达,从而对抗青枯病等维管束病害,为植物的抗病育种提供了一种新的途径。
附图说明
图1是本发明实施例所述的PStmlp1启动子克隆的PCR电泳图。
图2是本发明实施例所述的包含由所述启动子表达的GUS基因的载体结构示意图。
图3是本发明实施例中pBi121-PStmlp1-GUS转基因植株阳性检测结果图。
图4是本发明实施例中pBi121-PStmlp1-GUS转基因植株GUS基因表达水平检测结果图。
图5是本发明实施例中pBi121-PStmlp1-GUS转基因植株GUS染色的示意图。
具体实施方法
本发明专利下述实施例中使用方法和装置,如无特殊说明,均为常规方法和装置;所用器材、试剂均为试剂公司购买的常规器材和试剂。为使本发明专利的目的、技术方案和优点更加清楚,下面结合具体实施例对本发明专利的具体实施方式进行详细说明。这些优选实施方式的示例在具体实施例中进行了例示。在此,还需要说明的是,为了避免因不必要的细节而模糊了本发明专利的技术方案,在实施例中仅仅示出了与根据本发明专利的方案密切相关的技术方案和/或处理步骤,而省略了关系不大的其他细节。
实施例1
本实施例提供一种受青枯菌诱导的维管束特异表达的启动子,所述启动子由马铃薯基因组数据库(http://spuddb.uga.edu/)获取,其核苷酸序列如SEQ ID:NO.1所示。序列表中SEQ ID:NO.1所示的DNA序列来源为鄂马铃薯3号(E3)的马铃薯维管束特异表达启动子,本文称PStmlp1。
实施例2
本实施例提供一种受青枯菌诱导的维管束特异表达的启动子的分离方法,包括:
1.在马铃薯基因组数据库(http://spuddb.uga.edu/)中寻找StMLP1基因,其预测StMLP1的启动子序列,共1584bp,将其命名为PStmlp1,所述克隆引物的基因序列包括:PStmlp1-F:TCAATGAACAAAATCGTTTAG;PStmlp1-R:TTTCTGAAACTTTTGTACTATG。
2.马铃薯材料E3的基因组提取使用的新型植物基因组DNA提取试剂盒(DP320-03,TIANGEN),操作步骤严格按照说明书进行。用引物PStmlp1-F和PStmlp1-R扩增启动子,得到PStmlp1,结果如图1所示。
3.将目标片段连接至pTOPO载体上,然后转化至大肠杆菌DH5α。待转化子长出后,挑取单克隆33℃2203pm培养3-4小时后用相应引物PCR检测,检测为阳性后挑取3个以上阳性克隆送测序公司测序(昆泰锐(武汉)生物技术有限责任公司),选取无突变的克隆,摇菌,提取质粒,细菌质粒提取采用的是质粒小量提取试剂盒(ZP101,庒盟),操作步骤严格按照说明书进行。
实施例3
本实施例提供一种受青枯菌诱导的维管束特异表达的启动子功能验证方法,包括:
1.植物表达载体的构建和农杆菌转化
用分别带有pBi121载体部分片段的引物pBi121-PStmlp1-F(TATGACCATGATTACGCCAAGCTTTCAATGAACAAAATCGTTTAG)和pBi121-PStmlp1-R(AAGGGACTGACCACCCGGGGATCCTTTCTGAAACTTTTGTACTATG)去扩增启动子,利用同源重组方法,采用一步法重组试剂盒(C112-01,Vazyme),将启动子PStmlp1插入载体pBi121-GUS中,然后转化至大肠杆菌DH5α中,步骤同上,进行测序,获得重组质粒pBi121-PStmlp1-GUS,载体图如图2所示。获得正确的质粒后,将该质粒转入农杆菌GV3101中。具体操作如下:
(1)取50μL冰浴上融化的农杆菌GV3101感受态细胞,加入(5-10μL)目的基因质粒pBi121-PStmlp1-GUS,冰浴10分钟。
(2)液氮冷激5分钟,33℃水浴5分钟。
(3)加入400μL YEB,28℃2003pm摇床,复苏3-4小时。
(4)40003pm离心4分钟,根据实验要求,吸取不同体积已转化的感受态细胞加到含相应抗生素的YEB琼脂培养基上将细胞均匀涂开。待液体被吸收后封口,倒置平板,28℃培养2-3天。
(5)挑取单菌落进行PCR菌落检测,获得阳性克隆。。
2.马铃薯遗传转化
将上述转入pBi121-PStmlp1-GUS表达载体的农杆菌GV3101划线于YEB固体培养基上进行活化,挑取单克隆于20ml YEB液体培养基中,28℃2003pm培养24小时;取2ml菌液于40ml YEB液体培养基中,28℃200 3pm进行亚培养,直至OD600约为0.5(约6小时);50003pm,离心6分钟,去上清,10ml 3%MS液体培养基重悬。将生长6-8周的试管薯横切为厚度约1-2mm的薄片;然后在上述重悬液中侵染10分钟,中途轻轻地摇一下;弃菌液,用无菌滤纸吸干表面菌液,然后转入P1共培养培养基(3%MS固体培养基+0.2mg/L GA3+0.2mg/L IAA+0.5mg/L 6-BA+2mg/L ZT,pH 5.8),23℃培养箱黑暗培养48小时。再转至P2分化培养基(3%MS固体培养基+0.2mg/L IAA+0.2mg/L GA3+2mg/L ZT+0.5mg/L 6-BA+35mg/L Kan+400mg/LCef),23℃培养箱培养(16小时光照/8小时暗光周期)。当抗性芽生长至0.5-1cm时将其接种至P3生根培养基(3%MS+50mg/L Kan+400mg/L Cef),抗性芽长大后进行二次生根,进行DNA鉴定,获得转基因阳性植株。鉴定结果如图3所示,选取其中1个阳性株系进行后续实验。
3.启动子PStmlp1驱动的报告基因GUS的表达分析
对上述筛选的转基因阳性马铃薯植株侵染前后植株根部GUS的表达水平检测,提取阳性苗的根部RNA,反转录得到其cDNA,实时荧光定量qRT-PCR检测GUS在收到青枯菌侵染前后的相对表达水平,具体操作如下:实时荧光定量qRT-PCR(BlastqTM 2X qPCRMaste3Mix,Cat.No.G891),反应程序为:95℃,30sec;95℃,1s;60℃,10s,采集荧光;40个循环,65℃,5sec,每次上升0.5℃,至95℃,每次温度上升完毕采集荧光。马铃薯基因Stef1a(NM_001288491)作为内参基因。结果如图4所示,GUS在未受青枯菌侵染的情况下是不表达的,但是在受到侵染后GUS在植株根部中明显表达。
对上述转基因的幼苗进行GUS染色,具体操作如下:把植株洗净,用GUS染色液(1mM磷酸钠缓冲液(pH=3),10mM EDTA,0.1%T3iton-X,100μg/mL氯霉素,2mM亚铁氰化钾,2mM铁氰化钾,0.5mg/mL X-glucu3onide)浸泡,真空渗透整个植株15min,33℃培养过夜,然后用30%乙醇脱色,中间一般至少换3次以上的30%乙醇。结果如图5所示,在未受侵染的情况下GUS没有表达,但是在侵染的情况下转基因植株的根部,茎部的维管束均有表达。上述结果表明植株在受到青枯菌侵染的情况下启动子PStmlp1能够驱动GUS基因在马铃薯维管束中特异性高水平表达。
实施例4
本实施例提供一种受青枯菌诱导的维管束特异表达的启动子的应用,包括:
由于其具有维管束特异性表达并且被病原菌诱导的特征,通过该启动子对马铃薯等植物通过该启动子驱动抗病基因在维管束中特异表达,从而在维管束中直接对抗病原菌。同时,也可通过该启动子表达GUS,GFP,LUC等标记基因来监控病原菌的侵染进程,将有助于鉴定病原菌的侵染机制。为马铃薯维管束病害的抗病研究提供基础。
以上所述仅是本申请的具体实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本申请原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本申请的保护范围。
参考文献:
1.Caldwell D,Kim BS,Iye3-Pascuzzi AS.Ralstonia solanacearumDiffe3entially Colonizes Roots of Resistant and Susceptible TomatoPlants.Phytopathology,2013,1035:528-536.
2.Lafe33ie3e LT,Helgeson JP,Allen C.Fe3tile Solanum tube3osum plus S-commersonii somatic hyb3ids as sou3ces of 3esistance to bacte3ial wilt causedby Ralstonia solanacearum.Theo3Appl Genet,1999,98:1232-1238.
3.Lowe-Powe3 TM,Khokhani D,Allen C.How Ralstonia solanacearumExploits and Th3ives in the Flowing Plant Xylem Envi3onment.T3ends Mic3obiol,2018,2611:929-942
4.Patil VU,Gopal J,Singh BP.Imp3ovement fo3 Bacte3ial Wilt Resistancein Potato By Conventional and Biotechnological App3oaches.Ag3ic Res,2012,14:299-316.
5.P3iou S,Guta33a L,Aley P.An imp3oved en3ichment b3oth fo3 thesensitive detection of Ralstonia solanacea3um(biova3s 1 and 2A)in soil usingDAS–ELISA.Plant Pathology,2006,55:36-45.
6.Wang L,Wang B,Zhao G,Cai X,Jabaji S,Seguin P,Chen H.Genetic andPathogenic Dive3sity of Ralstonia solanacearum Causing Potato B3own Rot inChina.Ame3ican Jou3nal of Potato Resea3ch,2013,94:403–416.
<110> 华中农业大学
<120> 响应青枯菌侵染的马铃薯维管束特异表达启动子PStmlp1及其应用
<160> 1
<210> 1
<211> 1584
<212> DNA
<213> 马铃薯(Solanum tuberosum)
<400> 1
1 tcaatgaaca aaatcgttta gcactcatca acgatgttcc tctcgtgttc aacttcaaaa
61 gagaattcaa ttcttgaaat taaaaagaca gtctggtgca ctaagcttcc gttatgtgca
121 gagtcgggtg aagaaagtat ctatatgtta atgtaattga acaaattact taaataagat
181 aaatctacca aatgaagaag aagataactt gtccttaatt caaataatag tgatgtactt
241 cataaggatc gagtgccctt tgctaataaa cttgtgtgtg ttttgttttg caaaagctcc
301 acgtgagatt tttggttgta taaaatgtta tttcgtgaga ataaatttaa attgaataac
361 tttattgata taaaaataaa gtaggtattt ttactatatg attttaattg gattgatcct
421 ttgaaaattt aatcgagtta caattttttt tcattgttct ttatttctta taatttactc
481 tctaatctat catgttcttt tccaacactt tatttatgac tatatatatc atataaataa
541 aggaataata gtagtaaaac attttactac caaaaaacgt ataatttctt aaagcactaa
601 ttataaagaa aaaaggtgta gttattggat ctggctcctc ttcatttttc tctctctctt
661 cattttccta aaattcttac ttggtttgga gacacatgtc acagttctaa cttaatggct
721 gaaatttaat taactagaat aaagctctaa tcataattaa aataattaat tacttatata
781 tatatatgct cccaaaatgt ttttacaatt tgttacaccg catcacgata ttatgatatt
841 tgacatgact tttgatatgt gttttaatta tttttgtttt atatttggta caatatgcag
901 catttatgaa gaaaaaaaat gaaatacaga ttatcatatc gaaatatata attatatata
961 caactcatat attttaatta ataatatgat actaacaaat ttgtaaatta gtattagtat
1021 gaaaaaaaat attagacctt caaattttgt ctactctatt ttaattaaat tatctttttg
1081 tgtcattgat taatgaaatt aattacattt acttttcttt ttttaaaatt tctacatatg
1141 taagatgtta gtatgatata attgagagac gtttcgtaaa aagtaaaaat cataattatc
1201 tattacatat ataattatat ataagtccaa gtcgaacaaa ctattaagta atatatatat
1261 attaaaaaat ttcaaatacc atgcacacca agtgacagac ctccctcaga aataatatcc
1321 atcaaacctc ccaaaattgc tcattggatt agaaaagtga aagtcaaaca aaagccacac
1381 aaaaatattt gatttaatgt caaagtcaaa caccgtcgac ctatttaggt acgtgcccaa
1441 gttttaaaca aaaaatccac aaccaatgtg ctatattttc tttatgaaaa aacagaatcc
1501 acacctttaa atgtccatac aaaaattctc catcacaaaa ccaaaataaa aaaaatacca
1561 atcatagtac aaaagtttca gaaa
Claims (6)
1.一种受青枯菌诱导的维管束特异表达的启动子,其特征在于所述启动子核苷酸序列如SEQ ID:NO.1所示。
2.根据权利要求1所述的一种受青枯菌诱导的维管束特异表达的启动子,其特征在于所述SEQ ID:NO.1所示的DNA序列来源为鄂马铃薯3号。
3.一种受青枯菌诱导的维管束特异表达的启动子的应用,其特征在于所述应用为驱动抗病基因在维管束中特异表达,所述启动子核苷酸序列如SEQ ID:NO.1所示。
4.一种受青枯菌诱导的维管束特异表达的启动子的应用,其特征在于所述应用为通过该启动子表达GUS,GFP,LUC标记基因来监控病原菌的侵染进程,所述启动子核苷酸序列如SEQ ID:NO.1所示。
5.一种受青枯菌诱导的维管束特异表达的启动子的分离方法,包括:
(1)在马铃薯基因组数据库中寻找StMLP1基因,其预测StMLP1的启动子序列,共1584bp,将其命名为PStmlp1,所述克隆引物的基因序列包括:PStmlp1-F:TCAATGAACAAAATCGTTTAG;PStmlp1-R:TTTCTGAAACTTTTGTACTATG;
(2)马铃薯材料E3的基因组提取使用的新型植物基因组DNA提取试剂盒(DP320-03,TIANGEN),操作步骤严格按照说明书进行,用引物PStmlp1-F和PStmlp1-R扩增启动子,得到PStmlp1,结果如图1所示;
(3)将目标片段连接至pTOPO载体上,然后转化至大肠杆菌DH5α,待转化子长出后,挑取单克隆33℃2203pm培养3-4小时后用相应引物PCR检测,检测为阳性后挑取3个以上阳性克隆送测序公司测序,选取无突变的克隆,摇菌,提取质粒,细菌质粒提取采用的是质粒小量提取试剂盒,操作步骤严格按照说明书进行。
6.一种受青枯菌诱导的维管束特异表达的启动子功能验证方法,包括:(1)植物表达载体的构建和农杆菌转化,(2)马铃薯遗传转化,(3)启动子PStmlp1驱动的报告基因GUS的表达分析。
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