CN103981265B - 谷子锈菌巢式pcr高效检测方法 - Google Patents
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Abstract
本发明涉及一种谷子锈菌巢式PCR高效检测方法,利用谷子锈菌IGS区与其它真菌的IGS区的差异位点,设计两对特异性引物Urs1‑F/Urs1‑R和Urs2‑F/Urs2‑R,分别作为巢式PCR扩增的外侧引物对和内侧引物对,以样品DNA为模板进行两轮PCR扩增后对扩增产物进行电泳检测。通过目标片段判定待测病原菌是否为谷子锈菌,或所测谷子叶片是否被谷子锈菌侵染。本发明的方法对病原基因组DNA的检测灵敏度为100pg/μL,特异性强,可用于快速,准确和灵敏的检测谷子叶片中的锈菌,对谷子锈病的早期诊断及防治具有重要意义。
Description
技术领域
本发明涉及用巢式PCR(nested PCR)技术对谷子锈菌进行检测,属于植物真菌分子生物学检测技术领域。
背景技术
谷子(Setaria italica)起源于我国,是哺育中华民族的主要粮食作物之一,其种植面积和产量均居世界之首。小米营养丰富,随着世界杂粮热的兴起,出口量在稳步增加,已经成为我国的特色创汇杂粮。谷草是牲畜的优质饲草,在耕地紧缺的情况下是发展畜牧养殖业的首选粮草兼用性作物。但是谷子病害的发生,严重影响着谷子的产量和质量。
谷锈病(Uromyces setariae-italicae)是世界谷子产区经常发生的一种气传流行性病害,严重影响着谷子的稳产和高产。谷子锈病病原菌Uromyces setariae-italicae,属担子菌亚门(Basidiomycotina)、柄锈菌科(Pucciniaceae)、单胞锈属(Uromyces)。谷子锈病在谷子的叶片和叶鞘上都可发生,但在叶片上发生更加严重。发病初期,在叶片表面与叶背面,特别是背面产生隆起的夏孢子堆,夏孢子堆成熟后突破寄主表皮而外露,增强了植株的蒸腾作用,使植株丧失大量水分,减少光合作用面积,一般减产10%-30%,严重时植株倒伏,造成绝产。
在谷子锈病发生的初期阶段,由于潜伏侵染症状并不明显,用传统的病害症状、病原形态学等方法很难进行检测,谷锈病为典型气传流行性病害,等症状明显时已很难进行防控,因此初期诊断非常重要。同时谷锈菌存在许多其他寄主,如青狗尾草、莠狗尾草、倒刺狗尾草、谷莠子等。因此急需找到一种谷锈菌检测方法,以便于对谷锈菌寄主范围进行检测,更加清楚了解其初侵染源。近年来,基于PCR原理的分子生物学技术的发展为植物体内病原菌的快速、准确诊断提供了有效的工具。在真核生物的核糖体DNA(rDNA)中,编码区保守性较强而非编码区具有较大的可变性,是进行真菌分类的有效序列。在非编码区,相对于核糖体基因内转录间隔区( rDNA-ITS)而言,rDNA基因间隔区IGS( intergenic spacer )变异性更高,是真菌内进行种间比较的和分类的重要DNA片段。巢式PCR是一种变异的聚合酶链反应(PCR),使用两对PCR引物扩增完整的片段。第一对PCR引物扩增片段和普通PCR相似。第二对引物称为巢式引物结合在第一次PCR产物内部,使得第二次PCR扩增片段短于第一次扩增。梁宏等根据小麦光腥黑穗病IGS区与小麦矮腥黑穗病菌及小麦网腥黑穗病菌的差异设计特异性引物,对小麦光腥黑穗病菌进行了分子检测;王永强以中国不同地理来源的稻曲病菌菌株为材料,对其IGS序列进行了初步的比较分析,为稻曲病菌种下群体的鉴定奠定了基础。本研究基于真菌rDNA-IGS区域,设计了检测谷子锈菌的巢式PCR特异性引物,为快速、准确的检测谷子锈菌提供了技术支持,能够更早、更及时的发现病原菌,对谷子锈病的早期检测和防治具有重要意义。
发明内容
本发明提供了一种利用巢式PCR技术检测谷子中锈菌的方法,通过该方法可快速诊断出谷子植株中是否携带锈菌,具有特异性强、灵敏度高的特点,为锈病的早期诊断、科学预防和病情的控制提供了技术保障。
本发明的具体步骤是:
1、提取基因组DNA
采用CTAB法提取谷子锈菌、其它供试菌株以及接种锈菌的谷子叶片基因组DNA。以IGS通用引物扩增锈菌基因组DNA,其上游引物和下游引物碱基序列如序列表中SEQ IDNo.1和SEQ ID No.2所示。PCR扩增获得720bp的特异性片段,片段回收纯化后与PMD19-T载体连接过夜,利用热激法转入大肠杆菌JM109感受态细胞中,通过菌落PCR法检测阳性克隆,把阳性克隆送上海生工进行测序,测序分析后获得谷锈菌IGS序列,其核苷酸序列如序列表中SEQ ID No.3所示。
2、第一轮PCR扩增
以第1步提取的基因组DNA为模板,采用谷子锈菌IGS区外侧特异性引物对Urs1-F/Urs1-R,经PCR扩增得到第一轮PCR扩增产物;
引物序列:SEQ ID No.4 Urs1-F:5'-GCCTCTAAGTCAGAATCCGTGC-3';
SEQ ID No.5 Urs1-R:5'-GACGGGATGCGGTAAGTTCA-3';
PCR反应的体系为:25.0μL,其中2×Taq MasterMix 12.5μL,上下游引物10μM各1.0μL,DNA模板1.0μL,dH2O(灭菌蒸馏水)9.5μL;
PCR反应程序为: 94℃ 5 min;94℃ 30S,57℃ 30s,72℃ 1 min,35个循环;72℃10 min。
3、第二轮PCR扩增
将第一轮扩增产物用水稀释20倍后作为第二轮扩增模板,以谷子锈菌IGS区内侧特异性引物对Urs2-F/Urs2-R为引物进行PCR扩增,得到第二轮PCR扩增产物;
引物序列:SEQ ID No.6 Urs2-F:5'-CGTGCATCTTATAAATGTGTCA-3';
SEQ ID No.7 Urs2-R:5'-AGTGGATCGTAGCAACAAGG-3';
PCR反应的体系为:25.0μL,其中2×Taq MasterMix 12.5μL,上下游引物10μM各1.0μL,第一轮PCR扩增产物的稀释物1.0μL,dH2O(灭菌蒸馏水)9.5μL;
PCR反应程序为: 94℃ 5 min;94℃ 30S,53℃ 30s,72℃ 1 min,35个循环;72℃10 min。
4、PCR扩增产物的检测
取5μL PCR产物在1.2%的琼脂糖凝胶上电泳检测,经EB染色后用BioRad凝胶成像系统成像。若扩增出一条203bp的特异性条带,则确定所测样本为谷子锈菌或被谷子锈菌侵染的谷子植株。谷锈菌巢式扩增的203bp的特异性条带核苷酸序列如序列表中SEQ ID No.8所示。
本发明的优点在于:应用分子生物学手段研究植物体内病原菌,建立了快速、简便的鉴定方法,在谷子感染锈菌24h就能够检测到锈菌。利用此方法能够在病害发生初期还未显现出明显症状时就对病害进行准确的监控,在病害发生早期做出相应的防治措施,将病害的危害程度降到最低。
本发明的有益效果主要体现在:
(1) 灵敏度高:通过倍比稀释DNA标准品进行灵敏度检测,可检测到最低浓度为100pg/μL,较常规PCR提高了1000倍;
(2) 特异性强:结果显示,谷子上常见的7种病菌均未见扩增,只有锈菌产生扩增;
(3) 实用性好:本发明可用于谷子锈病潜伏期或发病初期病害的检测,对谷子锈病的早期诊断和及时预防具有重要意义。
附图说明
图1:谷子锈菌内引物Urs2-F/Urs2-R特异性电泳检测图,其中M: DL2000 DNAmarker;1-5:谷子锈菌;6:谷子白发病菌;7:谷瘟病菌;8:谷子纹枯病菌;9:粟粒黑穗病菌;10:粟胡麻斑病菌;11:粟弯孢霉病菌;12:粟灰斑病菌;13:阴性对照。
图2:谷子锈菌内引物Urs2-F/Urs2-R灵敏性电泳检测图,其中M:DL2000 DNAmarker;1-7:谷子锈菌基因组DNA浓度分别为10μg/μL、1μg/μL、100ng/μL、10ng/μL、1ng/μL、100pg/μL、10pg/μL。
图3:谷子锈菌巢式PCR灵敏性电泳检测图,其中M:DL2000 DNA marker;1~7:谷子锈菌基因组DNA浓度分别为10μg/μL、1μg/μL、100ng/μL、10ng/μL、1ng/μL、100pg/μL、10pg/μL。
图4:人工接种谷子锈菌的谷子植株叶片电泳检测图,其中,M: DL2000 DNAmarker;1~3:接种1天的植株叶片DNA;4~6:接种2天的植株叶片DNA;7~9:接种3天的植株叶片DNA; 10~12:接种4天的植株叶片DNA;13~15:接种5天的植株叶片DNA;16:健康植株叶片DNA;17:阴性对照。
具体实施方式
下面通过实施例并结合附图对本发明作进一步描述,但以下描述对本发明的保护范围不构成任何意义上的限定,仅仅做示例说明。
实施例1:谷子锈菌PCR检测的引物特异性验证
选择谷子上常见的其它7种真菌,谷子白发病菌(Sclerospora graminicola)、谷瘟病菌(Pyricularia setariae)、谷子纹枯病菌(Rhizoctonia solani)、粟粒黑穗病菌(Ustilago crameri)、粟胡麻斑病菌(Bipolaris setariae)、粟弯孢霉病菌(Curvularia lunata)和粟灰斑病菌(Cercospora setariae)进行引物特异性的验证,具体步骤为:
(1)锈菌及其它供试菌株基因组DNA的提取
a) 将菌体加入到1.5 mL离心管中,加入500 μL提取缓冲液(50 mM Tris–HCl,,pH 8.0,150 mM NaCl, 100 mM EDTA),充分混匀;
b) 向混合液中加入5μL 蛋白酶 K (1 mg/mL),再加入提取缓冲液至1 mL,65°C水浴30 min;
c)将混合液分装到两个1.5 mL离心管中,加入等体积的苯酚/氯仿/异戊醇(25:24:1,vol/vol/vol, pH=8.0),离心;
d)取上清,移到一个干净的1.5 mL离心管中,加入等体积的氯仿,离心;
e)取上清,移到一个干净的1.5 mL离心管中,加入等体积的冰冻异丙醇,−20°C冷冻1小时;
f)12,000 rpm ,4°C离心20 min,使DNA沉淀;
g) 弃上清,用70%的冰冻乙醇漂洗两次,自然风干后加入0.1 mL TE 缓冲液 (10mM Tris–HCl,1 mM EDTA,,pH 8.0)使其溶解;
h) 加入1μL 10 mg/ mL RNA酶(终浓度为20μg/ mL),4°C过夜,彻底酶解RNA;
i))将DNA再次沉淀,用70%冰冻乙醇漂洗,自然风干,溶解在50 μL TE中。
(2)以内引物对Urs2-F(5'-CGTGCATCTTATAAATGTGTCA-3')和Urs2-R(5'-AGTGGATCGTAGCAACAAGG-3')对来自山东济南、河南新乡、辽宁朝阳、河北石家庄和承德的锈菌DNA及其它7种真菌DNA进行PCR扩增;
PCR反应的体系为:25.0μL,其中2×Taq MasterMix 12.5μL,上下游引物10μM各1.0μL,DNA模板1.0μL,dH2O(灭菌蒸馏水)9.5μL;PCR反应程序为: 94℃预变性5min;94℃变性30s,53℃退火30s,72℃延伸1min共进行35个循环;72℃延伸10min。
(3)琼脂糖凝胶电泳及成像观察
分别取5μL不同病原菌基因组DNA扩增后的PCR产物在1.2%的琼脂糖凝胶上进行电泳,经EB染色后用BioRad凝胶成像系统成像。结果显示,只有锈菌DNA有扩增产物,其它7种真菌及阴性对照没有相应的扩增产物。结果说明引物的特异性很好,可以用于锈菌的特异性分析。
通过PCR扩增对引物特异性进行检测表明,在5个不同地理来源的锈菌样本中均扩增得到一条203bp的条带,与预期一致,而在其它7种真菌样本及阴性对照中均未扩增出任何条带(图1),表明该引物对谷子锈菌具有较强的特异性,可用于谷子锈菌的鉴定和检测。
实施例2:谷子锈菌常规PCR灵敏性检测
(1)DNA标准品的制备
将谷子锈菌基因组DNA浓度调整为10μg/μL,并按10倍梯度依次稀释为10μg/μL、1μg/μL、100ng/μL、10ng/μL、1ng/μL、100pg/μL、10pg/μL7个浓度梯度。
(2)分别取1μL稀释标准品作为灵敏度检测的模板,以Urs2-F/Urs2-R为引物进行PCR反应;
PCR反应的体系为:25.0μL,其中2×Taq MasterMix 12.5μL,上下游引物10μM各1.0μL,DNA模板1.0μL,dH2O(灭菌蒸馏水)9.5μL;PCR反应程序为: 94℃预变性5min;94℃变性30s,53℃退火30s,72℃延伸1min共进行35个循环;72℃延伸10min。
(3)琼脂糖凝胶电泳及成像观察
分别取5μL不同病原菌基因组DNA扩增后的PCR产物在1.2%的琼脂糖凝胶上进行电泳,经EB染色后用BioRad凝胶成像系统成像。结果显示,单独使用Urs2-F/Urs2-R为引物进行PCR反应,泳道1-3均扩增出203bp的条带(图2),表明能检测到的锈菌基因组DNA最低浓度为100ng/μL。
实施例3:谷子锈菌巢式PCR灵敏性检测
(1)DNA标准品的制备
与实施例2相同,将谷子锈菌基因组DNA浓度调整为10μg/μL,并按10倍梯度依次稀释为10μg/μL、1μg/μL、100ng/μL、10ng/μL、1ng/μL、100pg/μL、10pg/μL7个浓度梯度。
(2)第一轮PCR扩增
分别取1μL稀释标准品作为灵敏度检测的模板,以Urs1-F(5'-GCCTCTAAGTCAGAATCCGTGC-3')和Urs2-R(5'-GACGGGATGCGGTAAGTTCA-3')为引物进行PCR反应;
PCR反应的体系为:25.0μL,其中2×Taq MasterMix 12.5μL,上下游引物10μM各1.0μL,DNA模板1.0μL,dH2O(灭菌蒸馏水)9.5μL;PCR反应程序为: 94℃ 5 min;94℃ 30S,57℃ 30s,72℃ 1 min,35个循环;72℃ 10 min。
(3)第二轮PCR扩增
第一轮扩增产物用水稀释20倍后作为第二轮扩增模板,以Urs2-F/Urs2-R为引物进行第二轮PCR扩增;
PCR反应的体系为:25.0μL,其中2×Taq MasterMix 12.5μL,上下游引物10μM各1.0μL,第一轮PCR扩增产物的稀释物1.0μL,dH2O(灭菌蒸馏水)9.5μL;PCR反应程序为: 94℃ 5 min;94℃ 30S,53℃ 30s,72℃ 1 min,35个循环;72℃ 10 min。
(4)琼脂糖凝胶电泳及成像观察
分别取5μL不同病原菌基因组DNA扩增后的PCR产物在1.2%的琼脂糖凝胶上进行电泳,经EB染色后用BioRad凝胶成像系统成像。结果显示,经过两轮PCR扩增反应,泳道1-6均扩增出203bp的条带(图3),表明能检测到的锈菌基因组DNA最低浓度为100pg/μL。
与实施例2的结果相比,本发明的巢式PCR灵敏性是单独使用特异性引物Urs2-F/Urs2-R进行一次PCR的1000倍。
实施例4:人工接种谷子锈菌的谷子植株叶片检测
(1)叶片样品的采集
无菌栽培15盆谷子感病品种豫谷1号,每盆5株苗,在培养箱中培养至谷子幼苗长到6~7叶期时用于实验。将谷子锈菌新鲜的夏孢子配成5×106个孢子/mL的孢子悬浮液,均匀的喷洒在谷子叶片上,黑暗条件下保湿48h后置于培养箱中继续培养。在谷子接种锈菌1d,2d,3d,4d,5d后分别采集谷子叶片样本,每盆采集1片叶子,共15片,作为1个重复,总共设置3个重复。采集到的叶片迅速放到液氮中,然后保存到-80℃。以相同方法提取的健康植株叶片样本为阴性对照,ddH2O为空白对照。
(2)将各时间点采集的叶片样本提取总DNA
a)取谷子叶片1-1.5g,加液氮研磨成粉末;
b)将粉末转移到1.5mL离心管中,加入650μL CTAB提取缓冲液,65℃水浴40min;
c)冷却至室温后,加入等体积的氯仿/异戊醇混合液(v:v=24:1),颠倒混匀,4℃,12000rpm离心15 min;
d)取上清于新的1.5mL离心管中,加入等体积的氯仿/异戊醇混合液(v:v=24:1),颠倒混匀,4℃,12000rpm离心15 min;
e)取上清于新的1.5mL离心管中,加入等体积的冰冻异丙醇,颠倒混匀,置于-20℃冷冻30min;
f)4℃,12000rpm 离心15 min;
g)弃上清,使用质量浓度75%的乙醇漂洗沉淀1-2次;
h)待沉淀自然风干后,溶解于200μL TE中,利用NanoDrop 1000测定接种后各时间点叶片总DNA浓度,4℃保存备用。
(3)按照实施例3的步骤2和步骤3,依次进行第一轮和第二轮PCR扩增反应。
(4)琼脂糖凝胶电泳及成像观察
分别取5μL不同病原菌基因组DNA扩增后的PCR产物在1.2%的琼脂糖凝胶上进行电泳,经EB染色后用BioRad凝胶成像系统成像。结果显示,泳道1-15即接种谷锈菌Uromyces setariae-italicae 1d,2d,3d,4d,5d的谷子植株叶片基因组DNA均扩增出203bp的特异性条带,而健康植株叶片及阴性ddH2O对照均未扩增出任何条带(图4)。表明本发明所设计的引物可用于检测谷子叶片中的锈菌。
谷子锈菌IGS序列片段扩增通用引物的上游引物:
ATCAGACGGGATGCGGT
谷子锈菌IGS序列片段扩增通用引物的下游引物:
CTGAACGCCTCTAAGTCAGAA
谷子锈菌(Uromyces setariae-italicae)IGS序列通用引物扩增的目标片段:
ATCAGACGGGATGCGGTAAGTTCATTGTGGTATGGCCCCAGATGAAATAAAGAAGAGTTTTGTTGGTATATAACTATAGAATGGCCAGAGTAAGCCCCATTTTGTTACGCCTCCCTAGTGTCCCTAGGAAGAGGTGACCATATCACTTTTGTTATGATGGAAGTTTTTTCCGGGGACTTTCTCCATGTGTCAAAAAATTTAAAAGCTTGTATTGCTGAAAGTATAACACCCAAAAATGGTCCTTGGCGTTTGACTAGCACCCCCAGTGAAAATTTCAAAGTCATGAATTTTTCAACAAAACTTTGAAAAAAAAGTGCACATAATTGGACTGAATTGGTTGAAAAAGAACAAGGGCAAAAAAAGGAAAAGTTTAGATGGGTGGACAGACCCTAAAGCAGAGTCTACTAAAAAAAAAAAAAAAAAAGTTCAAACAAAGTTGAACAAATCTTTAGAACAAGGGCTGAACCTCAGTGGATCGTAGCAACAAGGCTACTCTACCACTTACAGTACCCTGTTCCGATTCAAGTCGTCTGCAAAGGATTTACCCCCGCACATATTTTTGATAATATAAGTTGGATAACTAACCAAGTCTTTCCACTTGATCAGTCAGGACCAACAGATAATGGTTCACGAGTTTAAAAGCTCTATTTTGACACATTTATAAGATGCACGGGACAAAATCATTGTTTCTAGCACGGATTCTGACTTAGAGGCGTTCAG
谷子锈菌IGS区外侧特异性引物Urs1-F:
GCCTCTAAGTCAGAATCCGTGC
谷子锈菌IGS区外侧特异性引物Urs1-R:
GACGGGATGCGGTAAGTTCA
谷子锈菌IGS区内侧特异性引物Urs2-F:
CGTGCATCTTATAAATGTGTCA
谷子锈菌IGS区内侧特异性引物Urs2-R:
AGTGGATCGTAGCAACAAGG
谷子锈菌(Uromyces setariae-italicae)IGS巢式扩增的203bp特异性片段:
AGTGGATCGTAGCAACAAGGCTACTCTACCACTTACAGTACCCTGTTCCGATTCAAGTCGTCTGCAAAGGATTTACCCCCGCACATATTTTTGATAATATAAGTTGGATAACTAACCAAGTCTTTCCACTTGATCAGTCAGGACCAACAGATAATGGTTCACGAGTTTAAAAGCTCTATTTTGACACATTTATAAGATGCACG
Claims (1)
1.一种谷子锈菌巢式PCR高效检测方法,其特征在于:利用谷子锈菌两对特异性引物,该特异性引物对由正向外引物、反向外引物、正向内引物和反向内引物组成:
正向外引物Urs1-F:5'-GCCTCTAAGTCAGAATCCGTGC-3';
反向外引物Urs1-R:5'-GACGGGATGCGGTAAGTTCA-3';
正向内引物Urs2-F:5'-CGTGCATCTTATAAATGTGTCA-3';
反向内引物Urs2-R:5'-AGTGGATCGTAGCAACAAGG-3';
所述引物分别作为巢式PCR扩增的外侧引物对和内侧引物对,以提取的谷子叶片总DNA为模板进行PCR扩增反应,扩增产物进行电泳检测,若检测到203bp的目标片段,则说明所测样本为被锈菌侵染的谷子,该方法可以排除谷子上其它常见病原菌的干扰,准确检测出谷子植株中的锈菌。
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