WO2020155730A1 - 一种甘蔗白叶病植原体不同亚组的pcr-rflp鉴别方法 - Google Patents

一种甘蔗白叶病植原体不同亚组的pcr-rflp鉴别方法 Download PDF

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WO2020155730A1
WO2020155730A1 PCT/CN2019/116072 CN2019116072W WO2020155730A1 WO 2020155730 A1 WO2020155730 A1 WO 2020155730A1 CN 2019116072 W CN2019116072 W CN 2019116072W WO 2020155730 A1 WO2020155730 A1 WO 2020155730A1
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tuf
sugarcane
pcr
leaf disease
white leaf
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张荣跃
黄应昆
李文凤
李婕
单红丽
王晓燕
仓晓燕
罗志明
尹炯
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云南省农业科学院甘蔗研究所
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  • the invention belongs to the technical field of plant protection, and specifically relates to a PCR-RFLP identification method for the 16SrXI-B subgroup and the 16SrXI-D subgroup of the sugarcane white leaf disease phytoplasma.
  • Sugarcane white leaf disease is a devastating disease of sugarcane caused by phytoplasma, which can cause huge economic losses in sugarcane and sugar industry.
  • the disease was first discovered in Thailand in 1954 and has now widely occurred in sugarcane-growing countries such as India, Pakistan, Sri Lanka, Laos, Sri Lanka, Laos, Sri Lanka, Laos, Sri Lanka, Laos, Sri Lanka, Laos, Vietnamese, Vietnam, the Philippines and Australia.
  • the sugarcane white leaf disease phytoplasma was detected for the first time in the sugarcane area of Baoshan, Yunnan, my country. After that, the white leaf disease of sugarcane spread rapidly in the sugarcane areas of Yunnan and has now spread to the sugarcane areas of Lincang and Pu'er, posing a serious threat to the sugar industry in Yunnan.
  • the 16SrXI-B subgroup and the 16SrXI-D subgroup are distinguished by using universal primers (P1/P7 and R16F2n/R16R2) designed by the phytoplasma 16S rRNA gene to perform nested PCR, and then perform virtual RFLP on nested PCR sequencing Restriction analysis is used for identification.
  • This method requires sequencing and analysis of the product, which is time-consuming and laborious. In addition, errors in the sequencing process will also affect the accuracy of the virtual RFLP analysis results.
  • no method has been established for the identification of 16SrXI-B and 16SrXI-D subgroups of phytoplasma using actual enzyme digestion. The establishment of a rapid, sensitive and accurate method for distinguishing different subgroups of sugarcane white leaf disease phytoplasma is of great significance to the pathogen identification, prevention and control of sugarcane white leaf disease.
  • the present invention provides a fast, accurate and simple method for identifying the 16SrXI-B subgroup and 16SrXI- Method of subgroup D.
  • a PCR-RFLP identification method for different subgroups of sugarcane white leaf disease phytoplasma including the following steps:
  • tuf gene-specific primers are composed of tuf-F primers and tuf-R primers.
  • the nucleotide sequence of the nucleotide sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the tuf-R primer is shown in SEQ ID NO: 2;
  • the digested product was subjected to agarose gel electrophoresis.
  • the digested electrophoresis pattern showed that the digested enzyme was cut into two bands, one of 124 bp and the other of 344 bp.
  • the pathogen of the sugarcane material to be identified was sugarcane white leaves.
  • the PCR amplification reaction conditions described in step (1) are: 25 ⁇ L PCR reaction system, in which, sterile ddH 2 O 15.3 ⁇ L, 10 ⁇ PCR buffer 2.5 ⁇ L, 25 mmol/L MgCl 2 2.0 ⁇ L, 10 mmol /L dNTPs 2.0 ⁇ L, 20 ⁇ mol/L tuf-F primer 1.0 ⁇ L, 20 ⁇ mol/L tuf-R primer 1.0 ⁇ L, 5U/ ⁇ L Taq DNA polymerase 0.2 ⁇ L, template DNA 1.0 ⁇ L; reaction program: 94°C pre-denaturation 3min ; Denaturation at 94°C for 15s, annealing at 55°C for 30s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 7 min;
  • restriction enzyme digestion reaction conditions in step (2) are: 25 ⁇ L digestion reaction system, including 15 ⁇ L PCR product, 7.5 ⁇ L sterilized ddH 2 O, 1.5 ⁇ L 10 ⁇ buffer buffer, 20000U restriction endonuclease Msp I 1.0 ⁇ L;
  • the reaction procedure is: 37°C30min; 65°C10min;
  • step (3) taking the digested product for agarose gel electrophoresis is to take 10 ⁇ L of the digested product for electrophoresis on a 3% agarose gel.
  • the present invention also provides a kit for identifying different subgroups of sugarcane white leaf disease phytoplasma based on the PCR-RFLP method.
  • the kit contains tuf gene-specific primers, restriction endonuclease Msp I or its isoschizomer, and The tuf gene-specific primers are composed of tuf-F primers and tuf-R primers.
  • the nucleotide sequence of tuf-F primer is shown in SEQ ID NO: 1
  • the nucleotide sequence of tuf-R primer is shown in SEQ ID NO: 2 shown.
  • the present invention also provides the above-mentioned kit for identifying different subgroups of sugarcane white leaf disease phytoplasma based on the PCR-RFLP method for identifying the sugarcane white leaf disease phytoplasma 16SrXI-B subgroup and/or sugarcane white leaf disease phytoplasma 16SrXI-D Applications in subgroups.
  • the present invention has the following beneficial effects:
  • the inventors of the present invention designed specific primers to amplify the tuf gene fragment size to 468bp. On this basis, a simple method was established for the first time , Fast, accurate and efficient method to identify different subgroups of sugarcane white leaf disease phytoplasma.
  • the prior art method for determining the classification of different subgroups of sugarcane white leaf disease phytoplasma is to perform virtual RFLP digestion analysis on the nested PCR product of the 16S rRNA gene.
  • the actual RFLP digestion method has not been established yet.
  • the present invention establishes an actual RFLP identification method to distinguish different subgroups of sugarcane white leaf disease phytoplasma, according to the enzyme digestion reaction band type of PCR amplified products
  • the type of sugarcane white leaf disease phytoplasma subgroup can be judged simply, quickly, accurately and efficiently without sequencing analysis, which saves time, reduces costs, and improves the accuracy of identification.
  • the method of the present invention can quickly, simply and accurately distinguish the 16SrXI-B subgroup and the 16SrXI-D subgroup of the sugarcane white leaf disease phytoplasma, and is of great significance to the pathogen identification and prevention and control of sugarcane white leaf disease.
  • the pathogenicity, epidemic law and prevention and control of different subgroups of pathogenic phytoplasma provide technical support.
  • SEQ ID NO: 1 in the sequence table shows the nucleotide sequence of the tuf-F primer.
  • SEQ ID NO: 2 in the sequence table shows the nucleotide sequence of the tuf-R primer.
  • Figure 1 is a PCR electrophoresis diagram of the tuf gene of the sugarcane white leaf disease phytoplasma, where M is DNA Marker E, 1 is a sugarcane leaf sample infected with the sugarcane white leaf disease phytoplasma 16SrXI-B subgroup, showing one band; 2 is The sugarcane leaf samples infected with the sugarcane white leaf disease phytoplasma 16SrXI-D subgroup showed one band; 3 was a healthy sugarcane leaf sample, and 4 was ddH 2 O.
  • Figure 2 is an electrophoresis diagram of enzyme digestion using the method of the present invention, where M is DNA Marker E, 1 is a sugarcane leaf sample infected with the sugarcane white leaf disease phytoplasma 16SrXI-B subgroup, showing two bands; 2 is infection The sugarcane leaf samples of the 16SrXI-D subgroup of the sugarcane white leaf disease phytoplasma showed one band.
  • M DNA Marker E
  • 1 is a sugarcane leaf sample infected with the sugarcane white leaf disease phytoplasma 16SrXI-B subgroup, showing two bands
  • 2 is infection
  • the sugarcane leaf samples of the 16SrXI-D subgroup of the sugarcane white leaf disease phytoplasma showed one band.
  • the sugarcane leaves of the 16SrXI-B and 16SrXI-D subgroups are each 0.2g, using a plant genomic DNA extraction kit (take Beijing Quanshijin Biotechnology Company's Easy Pure plant Genomic DNA Kit as an example) Separately extract the total DNA of leaves, and the specific steps follow the instructions.
  • a plant genomic DNA extraction kit take Beijing Quanshijin Biotechnology Company's Easy Pure plant Genomic DNA Kit as an example
  • tuf-F primer/tuf-R primer to PCR amplify the extracted total DNA, and add sterile double distilled water (ddH 2 O) 15.3 to the 25 ⁇ L PCR reaction system ⁇ L, 10 ⁇ PCR buffer 2.5 ⁇ L, MgCl 2 (25mmol/L) 2.0 ⁇ L, dNTPs (10mmol/L) 2.0 ⁇ L, tuf-F primer (20 ⁇ mol/L) 1.0 ⁇ L, tuf-R primer (20 ⁇ mol/L) 1.0 ⁇ L, Taq DNA polymerase (5U/ ⁇ L) 0.2 ⁇ L, template DNA 1.0 ⁇ L.
  • ddH 2 O sterile double distilled water
  • the reaction procedure is: pre-denaturation at 94°C for 3min; denaturation at 94°C for 15s, annealing at 55°C for 30s, extension at 72°C for 1 min, 35 cycles; finally, extension at 72°C for 7 minutes.
  • the amplified products were detected by 1.5% agarose gel electrophoresis.
  • Figure 1 Both the 16SrXI-B subgroup and 16SrXI-D subgroup samples of the sugarcane white leaf disease phytoplasma can only amplify the specificity of about 468bp. Sexual banding.
  • Figure 1 shows that when there is no restriction enzyme digestion, whether the sugarcane material infected with the sugarcane white leaf disease phytoplasma 16SrXI-B subgroup or the sugarcane material infected with the sugarcane white leaf disease phytoplasma 16SrXI-D subgroup, its PCR amplification
  • the product bands were all the same, and all showed only a 468bp band (sugarcane white leaf disease phytoplasma tuf gene band).

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Abstract

本发明公开了一种甘蔗白叶病植原体不同亚组的PCR-RFLP鉴别方法。以甘蔗白叶病植原体tuf基因为对象设计特异性引物，通过PCR扩增得到tuf基因片段，然后采用限制性核酸内切酶Msp I对扩增得到的tuf基因片段进行酶切，通过差异性的RFLP酶切图谱对甘蔗白叶病植原体进行不同亚组的区分，无需测序即可鉴别甘蔗白叶病植原体16SrXI-B亚组和甘蔗白叶病植原体16SrXI-D亚组。

Description

一种甘蔗白叶病植原体不同亚组的PCR-RFLP鉴别方法 技术领域
本发明属于植物保护技术领域，具体涉及一种甘蔗白叶病植原体16SrXI-B亚组和16SrXI-D亚组的PCR-RFLP鉴别方法。
背景技术
甘蔗白叶病是由植原体引起的甘蔗毁灭性病害，可造成甘蔗和制糖产业巨大的经济损失。该病于1954年首次在泰国发现，现已广泛发生于印度、巴基斯坦、斯里兰卡、老挝、缅甸、越南、菲律宾和澳大利亚等植蔗国家。2012年我国云南保山蔗区首次检测发现甘蔗白叶病植原体，之后甘蔗白叶病在云南蔗区迅速扩散蔓延，现已扩展到临沧、普洱蔗区，对云南蔗糖产业产生了严重威胁。先前对病原的多样性研究表明引起甘蔗白叶病的植原体可以分为2个亚组，即16SrXI-B亚组和16SrXI-D亚组，其中16SrXI-D亚组为新发现的亚组。云南临沧蔗区和保山蔗区发现的甘蔗白叶病植原体分别属于16SrXI-B亚组和16SrXI-D亚组。
目前，16SrXI-B亚组和16SrXI-D亚组的区分是通过使用植原体16S rRNA基因设计的通用引物(P1/P7和R16F2n/R16R2)进行巢式PCR，然后对巢式PCR测序进行虚拟RFLP酶切分析来鉴定，该方法需要对产物进行测序分析，费时费力，另外，测序过程中产生的错误对虚拟RFLP酶切分析结果的准确性也会产生影响。目前还尚未建立使用实际酶切进行植原体16SrXI-B亚组和16SrXI-D亚组鉴定的方法。建立一种快速、灵敏、准确的区分甘蔗白叶病植原体不同亚组的方法对甘蔗白叶病的病原鉴定和防控具有重要意义。
发明内容
为解决现有技术缺乏简便、快速地鉴别甘蔗白叶病植原体不同亚组的技术问题，本发明提供一种快速、准确、简便的鉴别甘蔗白叶病植原体16SrXI-B亚组和16SrXI-D亚组的方法。
本发明采取的技术方案为：
一种甘蔗白叶病植原体不同亚组的PCR-RFLP鉴别方法，包括下述步骤：
(1)以待鉴别甘蔗材料的基因组DNA为模板，采用tuf基因特异性引物进行PCR扩增，所述tuf基因特异性引物由tuf-F引物和tuf-R引物组成，tuf-F引物的核苷酸序列如SEQ ID NO：1所示，tuf-R引物的核苷酸序列如SEQ ID NO：2所示；
(2)使用限制性核酸内切酶Msp I对步骤(1)的PCR扩增产物酶切；
(3)结果判别，取酶切产物进行琼脂糖凝胶电泳，酶切电泳图谱显示被Msp I酶切为两 条带，一条为124bp，另一条为344bp的待鉴别甘蔗材料病原为甘蔗白叶病植原体16SrXI-B亚组；没有被酶切的，即只有1条468bp大小条带的待鉴别甘蔗材料病原为甘蔗白叶病植原体16SrXI-D亚组。
进一步，步骤(1)中所述的PCR扩增的反应条件为：25μL PCR反应体系，其中，灭菌ddH 2O 15.3μL，10×PCR缓冲液2.5μL，25mmol/L MgCl 2 2.0μL，10mmol/L dNTPs 2.0μL，20μmol/L tuf-F引物1.0μL，20μmol/L tuf-R引物1.0μL，5U/μL Taq DNA聚合酶0.2μL，模板DNA 1.0μL；反应程序为：94℃预变性3min；94℃变性15s，55℃退火30s，72℃延伸1min，35个循环；最后72℃延伸7min；
进一步，步骤(2)中的酶切反应条件为：25μL酶切反应体系，其中，PCR产物15μL，灭菌ddH 2O 7.5μL，10×buffer缓冲液1.5μL，20000U限制性核酸内切酶Msp I 1.0μL；反应程序为：37℃30min；65℃10min；
进一步，步骤(3)中取酶切产物进行琼脂糖凝胶电泳为取10μL酶切产物用3％琼脂糖凝胶进行电泳。
本发明还提供一种基于PCR-RFLP方法鉴别甘蔗白叶病植原体不同亚组的试剂盒，该试剂盒含有tuf基因特异性引物、限制性核酸内切酶Msp I或其同裂酶，所述tuf基因特异性引物由tuf-F引物和tuf-R引物组成，tuf-F引物的核苷酸序列如SEQ ID NO：1所示，tuf-R引物的核苷酸序列如SEQ ID NO：2所示。
本发明还提供上述一种基于PCR-RFLP方法鉴别甘蔗白叶病植原体不同亚组的试剂盒在鉴别甘蔗白叶病植原体16SrXI-B亚组和/或甘蔗白叶病植原体16SrXI-D亚组中的应用。
与现有技术相比，本发明的有益效果：
本发明的本发明人在前期研究获得的甘蔗白叶病植原体的tuf基因序列的基础上，设计特异性引物，扩增tuf基因片段大小为468bp，在此基础上，首次建立了一种简便、快速、准确、高效的鉴别甘蔗白叶病植原体不同亚组的方法。
现有技术确定甘蔗白叶病植原体不同亚组分类的方法是对16S rRNA基因的巢式PCR产物进行虚拟RFLP酶切分析来确定，实际的RFLP酶切鉴别方法尚未建立。以之相比，本发明基于不同的基因(tuf基因)，建立了一种实际的RFLP酶切鉴别方法来区分甘蔗白叶病植原体不同亚组，根据PCR扩增产物的酶切反应带型即可简便、快速、准确、高效的判断甘蔗白叶病植原体亚组的类型，不需要进行测序分析，节约了时间，降低了成本，提高了鉴别的准确性。
本发明方法对甘蔗白叶病植原体16SrXI-B亚组和16SrXI-D亚组的快速、简便、准确的鉴别，对甘蔗白叶病的病原鉴定和防控具有重要意义，为研究甘蔗白叶病植原体不同亚组的 致病力、流行规律及其防控提供了技术支持。
序列表中SEQ ID NO：1所示的是tuf-F引物的核苷酸序列。
序列表中SEQ ID NO：2所示的是tuf-R引物的核苷酸序列。
附图说明
图1为甘蔗白叶病植原体tuf基因PCR电泳图，其中，M为DNA Marker E，1为感染了甘蔗白叶病植原体16SrXI-B亚组的甘蔗叶片样品，显示一条条带；2为感染了甘蔗白叶病植原体16SrXI-D亚组的甘蔗叶片样品，显示一条条带；3为健康甘蔗叶片样品，4为ddH 2O。
图2为采用本发明方法的酶切电泳图，其中，M为DNA Marker E，1为感染了甘蔗白叶病植原体16SrXI-B亚组的甘蔗叶片样品，显示两条条带；2为感染了甘蔗白叶病植原体16SrXI-D亚组的甘蔗叶片样品，显示一条条带。
具体实施方式
以下结合实施例对本发明进行详细说明，实施例中无特殊说明的为常规方法。
1.总DNA提取
按文献“Zhang R Y,Li W F,Huang Y K,et al.Group 16SrXI phytoplasma strains,including subgroup 16SrXI-B and a new subgroup,16SrXI-D,are associated with sugar cane white leaf.International journal of systematic and evolutionary microbiology,2016,66(1):487-491.”方法检测得到感染了甘蔗白叶病植原体16SrXI-B亚组和16SrXI-D亚组的甘蔗叶片，分别取感染了甘蔗白叶病植原体16SrXI-B亚组和16SrXI-D亚组的甘蔗叶片各0.2g，使用植物基因组DNA提取试剂盒(以北京全式金生物技术公司的Easy Pure plant Genomic DNA Kit植物DNA提取试剂盒为例)分别提取叶片总DNA，具体步骤按照该说明书操作。
2.PCR扩增
使用甘蔗白叶病植原体tuf基因特异性引物：tuf-F引物/tuf-R引物分别对提取的总DNA进行PCR扩增，25μL PCR反应体系中加入灭菌双蒸水(ddH 2O)15.3μL，10×PCR缓冲液2.5μL，MgCl 2(25mmol/L)2.0μL，dNTPs(10mmol/L)2.0μL，tuf-F引物(20μmol/L)1.0μL，tuf-R引物(20μmol/L)1.0μL，Taq DNA聚合酶(5U/μL)0.2μL，模板DNA 1.0μL。反应程序为：94℃预变性3min；94℃变性15s，55℃退火30s，72℃延伸1min，35个循环；最后72℃延伸7min。扩增产物用1.5％的琼脂糖凝胶电泳检测，结果如图1所示，甘蔗白叶病植原体16SrXI-B亚组和16SrXI-D亚组样品均只可扩增出1条约468bp的特异性条带。图1表明：在没有进行酶切时，无论感染了甘蔗白叶病植原体16SrXI-B亚组的甘蔗材料或感染了甘蔗白 叶病植原体16SrXI-D亚组的甘蔗材料，其PCR扩增产物条带均相同，均只显示一条大小为468bp的条带(甘蔗白叶病植原体tuf基因条带)。
3.RFLP分析
PCR反应结束后，取15μL PCR产物进行酶切反应。25μL酶切反应体系中加入PCR产物15μL，灭菌双蒸水(ddH 2O)7.5μL，10×buffer缓冲液1.5μL，限制性核酸内切酶Msp I(20000U)1.0μL。反应程序为：37℃30min；65℃10min。酶切结束后取10μL酶切产物进行3％琼脂糖凝胶电泳分析，酶切结果如图2所示，被Msp I酶切为两条带，一条带约为124bp，另一条带约为344bp的为感染了甘蔗白叶病植原体16SrXI-B亚组的甘蔗材料(即甘蔗材料病原为甘蔗白叶病植原体16SrXI-B亚组)，而没有被酶切只有1条约468bp大小条带的为感染了甘蔗白叶病植原体16SrXI-D亚组的甘蔗材料(即甘蔗材料病原为甘蔗白叶病植原体16SrXI-D亚组)。

Claims (4)

  1. 一种甘蔗白叶病植原体不同亚组的PCR-RFLP鉴别方法，其特征在于，包括下述步骤：
    (1)以待鉴别甘蔗材料的基因组DNA为模板，采用tuf基因特异性引物进行PCR扩增，所述tuf基因特异性引物由tuf-F引物和tuf-R引物组成，tuf-F引物的核苷酸序列如SEQ ID NO：1所示，tuf-R引物的核苷酸序列如SEQ ID NO：2所示；
    (2)使用限制性核酸内切酶Msp I对步骤(1)的PCR扩增产物酶切；
    (3)结果判别，取酶切产物进行琼脂糖凝胶电泳，酶切电泳图谱显示被Msp I酶切为两条带，一条为124bp，另一条为344bp的待鉴别甘蔗材料病原为甘蔗白叶病植原体16SrXI-B亚组；没有被酶切的只有1条468bp大小条带的待鉴别甘蔗材料病原为甘蔗白叶病植原体16SrXI-D亚组。
  2. 根据权利要求1所述的甘蔗白叶病植原体不同亚组的PCR-RFLP鉴别方法，其特征在于：步骤(1)所述的PCR扩增的反应条件为：25μL PCR反应体系，其中，灭菌ddH 2O 15.3μL，10×PCR缓冲液2.5μL，25mmol/L MgCl 2 2.0μL，10mmol/L dNTPs 2.0μL，20μmol/L tuf-F引物1.0μL，20μmol/L tuf-R引物1.0μL，5U/μL Taq DNA聚合酶0.2μL，模板DNA 1.0μL；反应程序为：94℃预变性3min；94℃变性15s，55℃退火30s，72℃延伸1min，35个循环；最后72℃延伸7min；
    步骤(2)中的酶切反应条件为：25μL酶切反应体系，其中，PCR产物15μL，灭菌ddH 2O 7.5μL，10×buffer缓冲液1.5μL，20000U限制性核酸内切酶Msp I 1.0μL；反应程序为：37℃ 30min；65℃ 10min；
    步骤(3)中取酶切产物进行琼脂糖凝胶电泳为取10μL酶切产物用3％琼脂糖凝胶进行电泳。
  3. 一种基于PCR-RFLP方法鉴别甘蔗白叶病植原体不同亚组的试剂盒，含有tuf基因特异性引物、限制性核酸内切酶Msp I或其同裂酶，所述tuf基因特异性引物由tuf-F引物和tuf-R引物组成，tuf-F引物的核苷酸序列如SEQ ID NO：1所示，tuf-R引物的核苷酸序列如SEQ ID NO：2所示。
  4. 权利要求3所述的基于PCR-RFLP方法鉴别甘蔗白叶病植原体不同亚组的试剂盒在鉴别甘蔗白叶病植原体16SrXI-B亚组和/或甘蔗白叶病植原体16SrXI-D亚组中的应用。
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