CN108707687B - 一种可以区分谷瘟病菌与稻瘟病菌的pcr检测鉴定方法 - Google Patents
一种可以区分谷瘟病菌与稻瘟病菌的pcr检测鉴定方法 Download PDFInfo
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Abstract
一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法,属于植物真菌分子生物学检测技术领域,包括以下步骤:A、DNA提取:分别提取谷瘟病菌和稻瘟病菌的DNA;B、引物设计:利用谷瘟病菌无毒基因PWL3编码区***849bp的反转录转座子的两端设计一对特异性引物Mol3‑F/Mol3‑F;C、PCR:以提取的谷瘟病菌与稻瘟病菌的总DNA为模板进行PCR扩增,得到扩增产物;D、结果判定:将扩增产物进行电泳检测,若检测到1771bp的目标条带,则判定为谷瘟病菌;若检测到922bp的条带,则判定为稻瘟病菌。通过本发明方法可以将难以区分的谷瘟病菌和稻瘟病菌进行快速、准确的区分,对研究梨孢菌在自然条件下田间传播、繁殖与进化具有重要意义。
Description
技术领域
本发明属于植物真菌分子生物学检测技术领域,具体涉及一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法。通过本发明方法可以将难以区分的谷瘟病菌和稻瘟病菌进行快速、准确的区分,对研究梨孢菌在自然条件下田间传播、繁殖与进化具有重要意义。
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。
谷瘟病菌有性态为Magnaporthe oryzae,属于子囊菌门大角间座壳属;无性态为Pyricularia grise,属有丝***孢子真菌中的梨孢属,由梨孢菌Pyricularia引发的谷瘟病对谷子产量造成了严重威胁,在我国谷子产区大面积发生,造成大面积的减产,甚至颗粒无收。梨孢菌除了侵染谷子外,还能侵染牛筋草(Eleusine indica)、狗尾草(Setariaviridis)、马唐(Digitaria sanguinalis)和水稻(Oryza sativa)等多种禾本科和莎草科杂草。目前关于将寄主为马唐的梨孢菌有性态命名为Magnaporthe grise,而其他寄主的梨孢菌命名为Magnaporthe oryzae。稻瘟病菌有性态也为Magnaporthe oryzae,无性态为Pyricularia grise,在形态学如分生孢子大小及菌落等很难将两者区分,但两者之间存在明显的寄主专化性。
在自然条件下谷子田间常见的狗尾草和牛筋草等植物均为梨孢菌自然寄主,经IGS序列分析表明,寄主为狗尾草的梨孢菌与谷瘟病菌是极为相似的,与稻瘟病菌存在差异。杜新法等证实牛筋草和狗尾草的梨孢菌与来自水稻的梨孢菌可发生交叉侵染。直接从杂草寄主上分离的梨孢菌株对水稻的致病性较弱,接种到水稻感病品种发病后再分离的菌株,对水稻致病性增强,对原寄主的致病性基本保持不变。并推断稻瘟病区稻田周围牛筋草、狗尾草等杂草寄主上的梨孢菌在稻瘟病菌的积累和传播方面具有作用。所以从分子水平上快速区分谷瘟病菌与稻瘟病菌在研究梨孢菌在自然条件下田间传播、繁殖与进化具有重要意义。
发明内容
本发明提供了一种利用PCR技术快速区分谷瘟病菌与稻瘟病菌的方法,该方法具有特异性强、灵敏度高的特点,为研究梨孢菌在田间的传播、繁殖及变异提供支持。
本发明为实现其目的采用的技术方案是:
一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法,包括以下步骤:
A、DNA提取:分别提取谷瘟病菌和稻瘟病菌的DNA;
B、引物设计:利用谷瘟病菌无毒基因PWL3设计特异性引物,即在谷瘟病菌无毒基因PWL3基因编码区***849bp的反转录转座子的两端设计得到一对特异性引物Mol3-F/Mol3-F,序列如下:
Mol3-F:5’-TACAACACCCACGGATATCAC-3’,
Mol3-R:5’-TCGTAGCCTTAGCCACACAC-3’;
C、PCR:以提取的谷瘟病菌与稻瘟病菌的总DNA为模板进行PCR扩增,得到扩增产物;
D、结果判定:将扩增产物进行电泳检测,若检测到1771bp的目标条带,则判定为谷瘟病菌;若检测到922bp的条带,则判定为稻瘟病菌。
步骤C中,控制PCR反应的体系为:25.0μL,其中2×Taq MasterMix12.5μL,上下游引物10μM各1.0μL,DNA模板1.0μL,dH2O 9.5μL。
步骤C中,控制PCR反应程序为:94℃预变性5min,94℃变性30s,55℃退火30s,72℃延伸1min,35个循环,72℃延伸10min。
步骤D中,将扩增产物进行电泳检测时,取8μL PCR扩增产物在1.2%的琼脂糖凝胶上电泳检测,经EB染色后用BioRad凝胶成像***成像,观察扩增出的特异性条带,进行结果判定。
本发明的有益效果是:
谷瘟病菌和稻瘟病菌在培养基菌落形态和分生孢子形态均无明显差异,很难实现对两者的区分,而本方法具有快速简便对两者进行区分。本发明应用分子生物学手段,建立了快速、简便的鉴定区分谷瘟病菌与稻瘟病菌的方法,对研究梨孢菌在田间不同寄主之间的传播侵染具有重要作用。
本领域技术人员普遍认为不可能利用谷瘟病菌无毒基因PWL3设计引物,然后利用PCR实现对谷瘟病菌和稻瘟病菌的区分,因为一般研究人员认为谷瘟病菌和稻瘟病菌无毒基因PWL3基因仅仅会是单碱基变异(SNP),而SNP必须通过测序分析方法才可以进行区分,普通PCR检测很难区分。本发明克服技术偏见,利用谷瘟病菌无毒基因PWL3设计一对特异性引物Mol3-F/Mol3-F。作为PCR扩增的特异性引物,分别以提取的谷瘟病菌与稻瘟病菌的总DNA为模板进行PCR扩增,实现了快速、准确区分谷瘟病菌和稻瘟病菌。
本研究的谷瘟病菌无毒基因PWL3为首次从谷瘟中克隆,还没有任何人对谷瘟这个基因进行克隆研究。原来有人通过研究微管蛋白基因和钙调素基因进行梨孢属下面种的区分,但是基因序列变异只能通过测序分析来研究,无法通过PCR扩增电泳检测分析,因为扩增条带大小一致,而测序分析会使试验成本增加及检测周期加长。本领域中无毒基因如PWL3通常只是用来研究病原菌致病和毒力变异,未发现可用于种之间区分,这是本发明的创新之处。本领域技术人员并不知道谷瘟和稻瘟无毒基因会有显著的差异,对谷瘟病菌无毒基因PWL3测序结果经NCBI在线比对,发现与稻瘟病菌相比谷瘟病菌无毒基因PWL3基因编码区***849bp的核苷酸片段,与non-LTR retrotransposon:MGL的相似度达到96.7%。所有谷瘟病菌无毒基因PWL3中均有non-LTR retrotransposon:MGL转座子的部分序列,这是本发明可以区分谷瘟和稻瘟病菌的理论基础。
附图说明
图1是谷瘟病菌引物Mol3-F/Mol3-F电泳检测图。
其中,M表示DL2000DNA marker,CK表示空白对照,1-11表示稻瘟不同分离物基因组DNA,12-22表示谷瘟病菌不同分离物基因组DNA。
具体实施方式
很多学者包括谷子领域权威专家都认为谷瘟是由稻瘟病菌侵染,从菌落形态及分生孢子形态无法将两者区分,因此许多人认为两者无区别,认为谷瘟病就是稻瘟菌侵染后发病,但是许多地区没有水稻,谷瘟病依旧发生严重,对谷瘟病初侵染来源存在很大争议,本研究从分子生物学领域明确了谷瘟病并不是由稻瘟病菌侵染而发病,其侵染来源为谷瘟病菌。为今后研究谷瘟和稻瘟病菌田间潜在寄主和初侵染来源,及研究病害循环及流行有非常重要作用。所以快速区分不同寄主之间的梨孢菌对于研究该菌在不同作物间传播及分布,及其阐明瘟病初侵染源及其寄主分布具有一定的意义。下面结合具体实施例对本发明作进一步的说明。
本发明谷瘟病菌PWL3基因的获取方式如下:
利用特异性引物PWL3-3和PWL3-4对谷瘟病菌基因组DNA进行PCR扩增,回收目的条带并切胶纯化目的片段,通过克隆测序方法获得谷瘟病菌PWL3基因,其中PWL3-3和PWL3-4序列如下,
PWL3-3:5’-CCTGCGAGTAAA AG CCTGAATCTGA-3’
PWL3-4:5’-CCTTCTGCCTCTGTAGAGACTAGCC-3’。
实施例1
(1)谷瘟病菌与稻瘟病菌基因组DNA的提取
a)将菌体加入到1.5mL离心管中,加入500μL提取缓冲液(50mM Tris–HCl,pH 8.0,150mM NaCl,100mM EDTA),充分混匀,得到混合液;
b)向混合液中加入5μL蛋白酶K(1mg/mL),再加入提取缓冲液至1mL,65℃水浴30min,得到混合液II;
c)将混合液II分装到两个1.5mL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1.5mL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1.5mL离心管中,加入等体积的冰冻异丙醇,-20℃冷冻1小时;
f)12,000rpm,4℃离心20min,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0.1mL TE缓冲液(10mMTris–HCl,1mM EDTA,,pH 8.0)使其溶解;
h)加入1μL 10mg/mL RNA酶(终浓度为20μg/mL),37℃酶解2小时,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50μL TE中。
(2)特异性引物的PCR扩增
利用谷瘟病菌和稻瘟病菌无毒基因PWL3的差异,在谷瘟病菌无毒基因PWL3基因编码区***849bp的反转录转座子的两端设计了一对特异性引物Mol3-F/Mol3-F;
利用引物Mol3-F:5’-TACAACACCCACGGATATCAC-3’和Mol3-R:5’-TCGTAGCCTTAGCCACACAC-3’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,55℃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8μL谷瘟病菌与稻瘟病菌基因组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***成像。结果显示,谷瘟病菌扩增目的条带为1771bp,稻瘟病菌扩增目的条带为922bp(图1)。谷瘟病菌PWL3无毒基因1771bp的特异性条带与稻瘟病菌毒PWL3基因922bp的特异性条带的核苷酸序列如序列表所示。表明本发明中的特异性引物可以区分谷瘟病菌与稻瘟病菌。
SEQUENCE LISTING
<110> 河北省农林科学院谷子研究所
<120> 一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 922
<212> DNA
<213> 人工序列
<400> 1
tacaacaccc acggaatcac gtcggcgatg gaccagccga atatggaaat tatggaggtg 60
ggcattgggg cgatggatac tatggtcctc caggggagtt tgtacagacc agcgaatacg 120
aatagtgaat aggatggata ctatggtcat caagggcagt ctacaaacaa cagatgacac 180
ggacatggtg gaagcaattg cacctttatg tgaatggtat tgctccgtca agttgagaca 240
gctgtcaaag gcgcgagggt gccgttggaa ataaaagaga gaggggtgca gacaggggtt 300
agaggtgcga aggcgttggc agaggttgca gggagagaaa tgaaggaagc tatgtcgata 360
tattatagga ctttgatttt gataaatttc ttttggacca gcatagaaac ctcttgttgc 420
ctcgcgcatc ccgtcgattg ccgctggaac cgcaaagcag ccctagtacc gtcaaccaca 480
atgcttgtcg cttccttgcc gagcttgtca tattctgaat attacagccc tgcgctccag 540
gccaaaattc cataatcatt tgataaacat ttcatttgga ccagctgcca tttcttaatt 600
gtgcacgcag gtgcccggct aataaagccc gacctctttg agagagaaga cacgaatgaa 660
attcgtatat ggttcgccgc cgtctttggg cctgtttcac ttgtttggaa ctcgtgtctg 720
ctaatttgtt gatagattta aagtcgtata tgatccgcgt tccaacgacc aggcacgtat 780
ggagataaac tatacttaac atgagaatat agtaataaaa cctgtaacct tagttttgga 840
ccctccagtt gcgtgaacat ctatatatat cacagcagat cttgaatcga aactacaaga 900
cagtgtgtgg ctaaggctac ga 922
<210> 2
<211> 1771
<212> DNA
<213> 人工合成
<400> 2
tacaacaccc acggatatca cgtcggcgat ggaccagccg aatatggaaa ttatggaggt 60
gggcattggg gcgatggata ctatggtcct ccaggggagt ttgtacagac cagcgaatac 120
gaatagtgaa taggatggat actatggtca tcaagggcag tctacaaaca acagatgaca 180
cggacatggt ggaagcaatt gcacctttat gtgaatggta ttgctccgtc aagttgagac 240
agctgtcaaa ggcgcgaggg tgccgttgga aataaaagag agaggggtgc agacaggggt 300
tagaggtgcg aaggcgttgg cagaggttgc agggagagaa atgaaggaag ctatgtcgat 360
atattatagg actttgattt tgataaattt cttttggacc agcatagaaa cctcttgttg 420
cctcgcgcat cccgtcgatt gccgctggaa ctgcaaagca gccctagtac cgtcaaccac 480
aatgcttgtc gcttccttgc cgagcttgtc atattctgaa tattacagcc ctgcgctcca 540
ggccaagtag tagtagtagt agtagtagta gtattagtta acgccgggct cggcccggct 600
tgttagccgg ccgttagcaa cctcccgagg ggtatctcgg cgcgcgttct atcttcttta 660
tttacacagg gggaaaacaa ggagggcaga ggcggatcgt gcctgaccgg gttcgggccc 720
ggtcctgctt agggggttag ttcactgacg cgaccccgtg cctaaggtgc aaaaagggtt 780
cgtctgacgg cttgtaccgt gaaatgtggg tgaaaaggca ataaatctat tcctcgtcaa 840
agttcgaatc gtttacgatc ggtgtcccta aacgtaaaat gcgttcgtgg cgcgcggggc 900
gctggcgggc cgctctgggg cgggcgctga agtaattggt ggctatcgaa aacgcctcaa 960
agcttttcgg ttgcccgaaa cttgtctgga agaatttgcg gcgttgggcg cggtctggcg 1020
gcccgaccgg ccgctcgtta tcaaaccacg gccagttttt ccacaccgcc cgcgaaaagc 1080
ggcagtgcac cgggtattta ggggaggtcc gcttccagca ccaggtacac gaggtgtttg 1140
catcctggtg gttgaaacgg tcatggtagg ctttaaaatc gccgtggccg gtcctcatgg 1200
ccaaataatg gcccagtagg ggtcttggca aacgcagttc ctcgggctcc tttctcggtg 1260
tgtattggaa tttccattcc ctatatgcgg gggaccgttc acaaagttct ttacgccacc 1320
agtccttctc tatattcgaa agaatggctc tgaggaccgt gccggcaccg ctatacaagc 1380
gcgctccagg ccaaaattcc ataatcattt gataaacatt tcatttggac cagctgccat 1440
ttcttaattg tgcacgcagg tgcccggcta ataaagcccg acctctttga gagagaagac 1500
acgaatgaaa ttcgtatatg gttcgccgcc gtctttgggc ctgtttcact tgtttggaac 1560
tcgtgtttgc taatttgttg atagatttaa agtcgtatat gatccgcgtt ccaacgacca 1620
ggcacgtatg gagataaact atacttaaca tgagaatata gtaataaaac ctgtaacctt 1680
agttttggac cctccagttg cgtgaacatc tatatatatc acagcagatc ttgaatcgaa 1740
actacaagac agtgtgtggc taaggctacg a 1771
Claims (4)
1.一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法,其特征在于,包括以下步骤:
A、DNA提取:分别提取谷瘟病菌和稻瘟病菌的DNA;
B、引物设计:在谷瘟病菌无毒基因PWL3基因编码区***849bp的反转录转座子的两端设计一对特异性引物Mol3-F/Mol3-F,序列如下:
Mol3-F:5’-TACAACACCCACGGATATCAC-3’,
Mol3-R:5’-TCGTAGCCTTAGCCACACAC-3’;
C、PCR:以提取的谷瘟病菌与稻瘟病菌的总DNA为模板进行PCR扩增,得到扩增产物;
D、结果判定:将扩增产物进行电泳检测,若检测到1771bp的目标条带,则判定为谷瘟病菌;若检测到922bp的条带,则判定为稻瘟病菌。
2.根据权利要求1所述的一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法,其特征在于,步骤C中,控制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 9.5μL。
3.根据权利要求1所述的一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法,其特征在于,步骤C中,控制PCR反应程序为:94℃预变性5min,94℃变性30s,55℃退火30s,72℃延伸1min,35个循环,72℃延伸10min。
4.根据权利要求1所述的一种可以区分谷瘟病菌与稻瘟病菌的PCR检测鉴定方法,其特征在于,步骤D中,将扩增产物进行电泳检测时,取8μL PCR扩增产物在1.2%的琼脂糖凝胶上电泳检测,经EB染色后用BioRad凝胶成像***成像,观察扩增出的特异性条带,进行结果判定。
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