CN115011613A - 拟南芥菌核病抗病候选相关基因AtSWEET15及其应用 - Google Patents
拟南芥菌核病抗病候选相关基因AtSWEET15及其应用 Download PDFInfo
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Abstract
本发明提供了拟南芥感病相关基因AtSWEET15及其应用,所述拟南芥AtSWEET15的DNA序列如SEQ ID No.1所示。该基因的T‑DNA插入突变体材料对菌核病表现出比哥伦比亚型野生型拟南芥更强的抗病性;通过农杆菌侵染将带有增强型启动子的AtSWEET15转化至哥伦比亚型拟南芥中,得到AtSWEET15过表达拟南芥株系,结果发现,AtSWEET15的过表达会导致拟南芥对菌核病更易感,这表明拟南芥AtSWEET15与植物抗菌核病关系密切,可将该基因应用于白菜类蔬菜及其他园艺植物育种,具有良好的应用前景。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及拟南芥AtSWEET15、及其在植物抗病过程中的应用。
背景技术
拟南芥(Arabidopsis thaliana)是十字花科模式植物,在植物基础科学中有重要研究意义。核盘菌(Sclerotinia sclerotiorum(Lib.)de Bary)是一种兼性真菌,归属于真菌界、子囊菌门、锤舌菌纲、柔膜菌目、核盘菌科、核盘菌属,由其引起的菌核病(S.sclerotiorumstem rot,SSR)对十字花科作物的生长发育造成严重威胁,可导致油菜产量和品质损失惨重,且其发病率极高,位列油菜三大病害之首。核盘菌侵染十字花科植物时最初是在花瓣、叶片上出现针尖大小的褐色侵染斑点,随后病斑扩展形成暗青色、水渍状圆形病斑,最后病斑处组织坏死干枯。至病重时植株萎蔫,甚至导致死亡。
植物和病原菌在几亿年间的共同进化使得这两个物种之间出现相互作用,这些相互作用依赖于植物以光合作用形式提供的碳水化合物,其中包括单糖、双糖和多糖。有研究表明,糖分不仅可以从植物的源器官运输到其库器官,也可以运输到非植物的库中。异养生物在植物上寄生会使库容量增加,因此病原菌的寄生过程存在寄主与宿主之间的糖分竞争。植物及病原菌对糖的吸收、交换和竞争是由膜转运蛋白控制的,其调控模式对于植物与病原菌的相互作用至关重要。
目前植物中主要有3种糖转运蛋白:蔗糖转运蛋白(SUTs),单糖转运蛋白(MSTs)以及SWEET蛋白。SWEET是一种存在于原核和真核生物中进行细胞内和细胞间糖易位的转运蛋白,能够在溶质势驱动下顺浓度梯度对糖分进行跨膜双向运输。同时,SWEET基因家族可以分成四个分化支,不同分支上具有不同的功能,分化支Ⅰ和Ⅱ运输己糖,而Ⅲ、Ⅳ蛋白优先转运蔗糖和果糖。目前越来越多的研究集中在糖转运蛋白如SWEET基因家族及其在植物-微生物相互作用中的作用。有研究表明,在植物抗病方面,病原体可引起感病植物病原响应基因的表达并分泌相应蛋白与之结合,进而激活相关SWEET基因表达,为病原菌自身的生长和繁殖提供能量。
发明内容
本发明的目的是提供拟南芥AtSWEET15及其应用。
本发明提供了一种根肿病感病基因AtSWEET15,该基因为:从哥伦比亚型野生拟南芥中克隆得到的基因,其具有:
1)SEQ ID No.1所示的核苷酸序列;或
2)SEQ ID No.1所示的核苷酸序列经取代、缺失和/或增加一个或几个核苷酸;或
3)在严格条件下与1)限定的DNA序列杂交的核苷酸序列。
本发明提供了含有上述拟南芥AtSWEET15的生物材料,所述生物材料为表达载体,表达盒,宿主细胞或工程菌。
本发明提供了上述拟南芥AtSWEET15或其相应的生物材料在植物抗菌核病中的应用。
进一步地,所述应用具体为:
通过上表达植物的AtSWEET15使植物易感菌核病,通过下表达或敲除植物的AtSWEET15使植物抗菌核病。
通过筛选不表达或弱表达AtSWEET15的植物,获得抗菌核病的植株。
本发明提供了上述拟南芥AtSWEET15或其相应的生物材料在制备转基因拟南芥中的应用。
本发明提供的拟南芥AtSWEET15序列如SEQ ID No.1所示。通过菌丝琼脂块接种结果发现,拟南芥AtSWEET15的T-DNA插入突变体材料中AtSWEET15的表达变化可以抑制核盘菌在拟南芥叶片上形成病斑,从而显著提升植株对菌核病的抗性;通过农杆菌侵染将带有增强型启动子的AtSWEET15转化至哥伦比亚型拟南芥中,得到AtSWEET15过表达拟南芥株系,抗病性分析实验表明,拟南芥AtSWEET15的过量表达可以促进核盘菌侵染拟南芥叶片引起的病斑增大。这表明拟南芥AtSWEET15与植物抗菌核病关系密切,将该基因应用于白菜或其他十字花科蔬菜育种,具有良好的应用前景。
附图说明
图1为AtSWEET15过表达载体示意图。
图2为AtSWEET15基因T-DNA插入纯合突变体检测电泳图。其中M泳道为DNAmarker,长方形框所在泳道为纯和突变体植株。
图3为AtSWEET15过表达植株三个株系(OE-1、OE-2和OE-3)和转化空载pBI121拟南芥植株qPCR鉴定。
图4为核盘菌侵染AtSWEET15过表达植株和转化空载pBI121拟南芥植株表型观察。
图5为核盘菌侵染T-DNA插入突变体SALK_116181植株和野生型拟南芥表型观察。
图6为核盘菌侵染AtSWEET15过表达植株和转化空载pBI121拟南芥(A)及T-DNA插入突变体SALK_116181植株和野生型拟南芥(B)病斑百分比折线图。
具体实施方式
下面通过具体实施例对本发明进行说明,实施例中未作详细描述的技术手段属于本领域专业技术人员熟知的常规技术。实施例只用于说明本发明,但不限制本发明的范围,任何本领域的技术人员在不付出创造性劳动的情况下,以本发明的实施例为基础所获得的其他实施例均属于本发明的保护范围。
本发明实施例提供了一种抗菌核病基因AtSWEET15,该基因从哥伦比亚型野生拟南芥中克隆到的基因,其基因序列如SEQ ID No.1所示。
本发明实施例还提供了上述拟南芥AtSWEET15在调控十字花科植物菌核病中的应用,下面对其进行具体描述。
实施例1:AtSWEET15过表达载体的构建
1、pBI121载体双酶切
Bam H Ⅰ和Xba Ⅰ双酶切,电泳分离大片段条带,割胶回收;
2、Trizol法提RNA
野生型拟南芥叶片取样,Trizol法提取RNA:计算样品数,准备相应的研钵研棒和小铁勺,锡箔纸包好180℃烘4-5h;1.5ml离心管(RNAFree),RNAFree的枪和枪头,液氮,离心管板,121℃,高压蒸汽灭菌40min。取相应数量离心管,做好编号;通风橱中每管加1mlTrizol,放冰上;从液氮中取样品至预冷的研钵中,加液氮研磨3-5次至粉末状,移入Trizol,充分震荡涡旋;于通风橱加入200ml三氯甲烷,剧烈震荡15s,冰上5min;4℃,12000rpm,离心10min,600μl上清液移入新的1.5ml离心管中,加入1倍体积异丙醇颠倒混匀,-20℃静置30min;4℃,12000rpm,离心10min,去上清;加入1ml预冷的DEPC水溶解的75vol%乙醇,4℃,12000rpm,离心10min,倒掉上清;空离20sec,吸出液体,通风厨中晾干。加入50μl DEPC水溶解RNA沉淀,测定浓度备用。
3、cDNA的制备
Takara反转录试剂盒去基因组DNA:体系包含2.0μl 5xgDNA Eraser Buffer,1.0μl gDNA Eraser,1.0μg/μl Tatal RNA,6.0μl RNAFree ddH2O,42℃金属浴2min;反转录:反应体系包含10μl上述得到的去基因组DNA产物,4.0μl 5xPrimerscript Buffer,1.0μlPrimerscript RT Enzyrne Mix,1.0μl RT Primer Mix,4.0μl RNase Free ddH2O,37℃,20min,85℃反应5sec,-20℃保存备用。
4、设计特异引物(引物序列信息见表1)高保真KOD酶扩增AtSWEET15的CDS序列,PCR产物0.8%琼脂糖凝胶电泳分离,长度符合,进行割胶回收。
5、同源重组连接
上述线性化载体和PCR扩增纯化的片段同源重组连接:用诺唯赞的单片段同源重组试剂盒C112进行,体系为:4μl 5xCE Ⅱ Buffer,2μl Exase Ⅱ,200ng双酶切回收的线性化载体,20ng AtSWEET15的CDS片段,ddH2O补齐至20μl;37℃反应30min。
6、转化大肠杆菌,培养所得菌液PCR验证,并提取目的质粒测序验证,验证比对成功的质粒电转法转化农杆菌感受态,转化成功的菌液菌液PCR有条带,扩大体积培养;提取质粒测序验证(终载体图谱见图1),验证成功的菌液保存菌种并留母液4℃储藏备用。
表1异源表达载体构建所用引物
实施例2:拟南芥浸花法转化及阳性转化株的筛选
1、浸花法转化拟南芥
经过测序验证的上述菌液以及pBI121空载体质粒的农杆菌GV3101菌液作为母液,浸花法转化拟南芥,步骤如下:含有目的载体的农杆菌菌液500μl,加入200ml含卡那霉素和利福平的液体LB中(50mg/L),28℃,200rpm摇菌30h左右至OD600为1.2;8000rpm离心10min得到农杆菌沉淀,200ml 5wt%蔗糖重悬菌液;加入Silwet-77至终浓度200μl/L,28℃200rpm震荡2min;野生型拟南芥去掉开放花及角果,花蕾浸入菌液30sec,吸干多余菌液,25℃保湿暗培养24h,以后正常培养,一周后重复浸花一次。
2、配制卡那霉素播种培养基
2.22g MS粉,10g蔗糖2M NaOH调pH至5.8获得MS培养基,加入4g琼脂粉121℃高压蒸汽灭菌20min,超净工作台中,冷却到50-60℃,加入卡那霉素至终浓度75mg/L,倒固体平板。
3、阳性转化株的筛选
在卡那霉素播种培养基上播种浸花得到的拟南芥种子T1代,步骤如下:10vol%NaClO消毒2min;75vol%乙醇清洗种子2min;无菌水冲洗5次,每次1min;播种于卡那霉素播种培养基上,封口,置于22℃培养间培养(16h光照,8h黑暗),约两周后,移出长势健壮,叶色深绿的阳性植株,PCR检测验证(引物如表2)。
表2转基因拟南芥PCR检测所用引物
引物名称 | 引物序列(5’-3’) |
pBI121-Oe-F | CCACGTCTTCAAAGCAAGTG(SEQ ID No.4) |
pBI121-Oe-R | TTGTAACGCGCTTTCCCAC(SEQ ID No.5) |
实施例3:T-DNA插入突变体材料的筛选
1、DNA快速提取法提取DNA:装有拟南芥叶片的离心管中加入200μl DNA提取缓冲液及磁珠,破碎仪破碎组织(65Hz,120s);研磨后组织液全部转入1.5ml离心管,13000rpm离心8min;准备新的1.5ml离心管,每管加入100μl异丙醇,取100μl上清转入含等体积异丙醇的离心管中,温和的晃动约50次,室温静置5min;13000rpm离心6min,去上清;1ml 70vol%乙醇洗涤沉淀两次(上下摇动20次,13000rpm离心3min,弃上清,重复一次;空离1min),吸出液体,沉淀晾干5min,加入25-50μl ddH2O,-20℃备用。
2、三引物法PCR检测(引物如表3):1.1x T3 Super Mix PCR反应体系如下:44μl1.1x T3 super Mix,2μl Template,2μl Primer F,2μl Primer R;程序设定为98℃预变性2min 30sec,进入35圈循环扩增(98℃变性10sec,55℃退火10sec,72℃延伸10sec),最后72℃充分延伸2min保证片段完整的扩增;LP+RP引物扩增的PCR产物与RP+BP引物扩增的PCR产物混合均匀,用0.8wt%琼脂糖凝胶电泳分离。只含有单一小片段的纯合个体(图2)即为突变体材料SALK_116181,收种用于后续实验。
表3 T-DNA插入突变体材料筛选所用引物
引物名称 | 引物序列(5’-3’) |
T-DNA-BP | ATTTTGCCGATTTCGGAAC(SEQ ID No.6) |
SALK_116181-LP | CTTGTATTCCTCGCTCCAGTG(SEQ ID No.7) |
SALK_116181-RP | GATGAACGGCTTCAGAGAGTG(SEQ ID No.8) |
实施例4:实时荧光定量PCR
1、实时荧光定量PCR分析AtSWEET15表达量的变化:取拟南芥AtSWEET15 T-DNA插入突变体材料SALK_116181及对照组野生型拟南芥各自至少10株进行混合取材,做好标记后,迅速置入液氮中固定,全部取材完成后,提取总RNA并反转录合成cDNA后,进行qPCR分析。qPCR分析所用引物通过Primer Premier 6设计,如表4所示。反应体系为15μL:7.5μL的SYBR Green Master Mix,正反向引物各0.3μL,模板1μL,5.9μL双蒸水。qPCR反应流程:95℃:30s,40个循环(95℃:5s,55℃:45s)。通过熔融曲线确定反应的特异性,内参基因为Atactin7,基因的相对表达量通过2-ΔΔCt方法计算。(取样时设三次生物学重复)
结果(图3)显示AtSWEET15在过表达植株中与转入pBI121空载的拟南芥植株相比上调表达。
表4拟南芥植株qRT-PCR分析所用引物
引物名称 | 引物序列(5’-3’) |
SALK_116181-F | GAGTCCGTTAGGTGTGTCGG(SEQ ID No.9) |
SALK_116181-R | TTCAGGACGAGTAGCCTCCA(SEQ ID No.10) |
AtActin7-F | GGAACTGGAATGGTGAAGGCTG(SEQ ID No.11) |
AtActin7-R | CGATTGGATACTTCAGAGTGAGGA(SEQ ID No.12) |
实施例5:转基因拟南芥植株的抗病性鉴定
1、核盘菌菌种培养
配制PDA固体培养基:将200g的马铃薯洗净后,加入1L纯净水煮沸30min。用纱布过滤后,向滤液中加入17g琼脂和20g葡萄糖,溶解后再次过滤,将液体装至三角瓶中。置于121℃,20min高压灭菌锅内,灭菌后取出,冷却至温热后,分装至培养皿中,每个培养皿倒入液体约20mL,彻底冷却后封口保存备用。取出保存在培养基中的核盘菌,将菌丝接种在新的PDA培养基中央,确保菌丝均匀生长。28℃培养箱暗培养4d左右,待菌丝长满整个培养基,即可用来进行下一步侵染实验。
2、侵染发病及表型观察
将检测为纯合的T-DNA插入突变体植株SALK_116181与野生型拟南芥同时播种,在成长到两片真叶时进行移栽,于光照培养箱培养约20d。使用打孔器在长满菌丝的PDA培养基上打孔,孔径6mm,将含有菌丝的圆柱形培养基长满菌丝的一面倒扣在叶片背面上。在空培养皿中放入滤纸,加入1mL的ddH2O润湿,再将叶片放于滤纸上,封口后放置在28℃培养箱进行暗培养。在侵染后24h、48h和72h记录叶片的褐变情况。使用软件Image J对叶片上发生褐变的部位面积进行计算,根据计算结果,分析各组发病情况的差异显著性。
结果显示:在野生型拟南芥中过表达该基因后,过表达株系比pBI121空载更加感病(图4);在T-DNA插入拟南芥突变体植株的菌核病发病实验中发现,SALK_116181拟南芥植株相比于对照组出现了对菌核病抗性提升的表型(图5),用Image J和SPSS的统计分析结果表明,过表达株系叶片上病斑面积显著大于转化pBI121空载的拟南芥植株,而SALK_116181拟南芥株系的叶片上病斑面积显著小于野生型拟南芥(图6)。
以上所述为本发明较佳的具体实施方式,但在其基础上可以进行一些改进或修改,这对任何熟悉本领域的技术人员而言是显而易见的。因此,在本发明基础上所作的这些修改和改进,均属于本发明要求保护的范围。
序列表
<110> 浙江大学
<120> 拟南芥菌核病抗病候选相关基因AtSWEET15及其应用
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 879
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 1
atgggagtca tgatcaatca ccatttcctc gcttttatct tcggcatctt aggaaacgtg 60
atatccttcc ttgtattcct cgctccagtg ccaacttttt atagaatata caagagaaaa 120
tcgacggaaa gtttccagtc gctaccgtac caagtgtcgc tatttagctg catgctatgg 180
ctctactacg cattgattaa gaaagacgct tttctcctaa ttaccatcaa ctcctttggc 240
tgcgtcgtgg agactctcta catagccatg ttcttcgctt acgccaccag ggagaaaagg 300
atatcggcta tgaagttgtt catagcaatg aacgttgcct tcttctcgtt gattctaatg 360
gtaacacatt tcgtggttaa aactcctccc ctccaagtct ctgtactcgg ctggatttgt 420
gttgccattt ctgtttctgt tttcgctgcc cctctaatga tcgtggctcg tgtgataaag 480
acaaagagtg tggagtacat gcccttcacg ctttctttct tcctcactat aagcgccgtt 540
atgtggttcg cttatggttt attcctcaat gacatatgca tagcgattcc aaacgtggtg 600
ggattcgtac tagggctgtt gcaaatggtt ttgtacttgg tttacaggaa ctcaaatgag 660
aaaccagaga agattaattc gtcagaacaa caacttaaga gtattgtcgt gatgagtccg 720
ttaggtgtgt cggaagtgca cccagttgtg acggaatcgg tggacccact ctctgaagcc 780
gttcatcatg aggatctgtc caaagttact aaagtggagg agccgtcaat tgaaaacggc 840
aagtgctacg tggaggctac tcgtcctgaa accgtttga 879
<210> 2
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaacacgggg gactctagag atgggagtca tgatcaatca 40
<210> 3
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggactgacca cccggggatc ctcaaacggt ttcaggacga g 41
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccacgtcttc aaagcaagtg 20
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttgtaacgcg ctttcccac 19
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
attttgccga tttcggaac 19
<210> 7
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cttgtattcc tcgctccagt g 21
<210> 8
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<213> 人工序列(Artificial Sequence)
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gatgaacggc ttcagagagt g 21
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<213> 人工序列(Artificial Sequence)
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gagtccgtta ggtgtgtcgg 20
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<213> 人工序列(Artificial Sequence)
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ttcaggacga gtagcctcca 20
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<213> 人工序列(Artificial Sequence)
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ggaactggaa tggtgaaggc tg 22
<210> 12
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cgattggata cttcagagtg agga 24
Claims (7)
1.一种拟南芥菌核病抗病候选基因AtSWEET15,其特征在于,所述候选基因AtSWEET15的核苷酸序列如SEQ ID No.1所示。
2.一种权利要求1所述拟南芥菌核病抗病候选基因AtSWEET15在筛选抗菌核病植物和/或调控植物抗菌核病功能中的应用。
3.一种权利要求1所述拟南芥菌核病抗病候选基因AtSWEET15在制备转基因植物中的应用。
4.一种含有权利要求1所述拟南芥菌核病抗病候选基因AtSWEET15的生物材料。
5.根据权利要求4所述生物材料,其特征在于,所述生物材料为表达载体、表达盒、宿主细胞或工程菌。
6.根据权利要求4所述生物材料在调控植物抗菌核病功能中的应用。
7.根据权利要求4所述生物材料在制备转基因植物中的应用。
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