WO2021138799A1 - Isolated or purified trichoderma antagonistic representative gene and application, and gene sequence-based trichoderma antagonistic evaluation amplification primer and application - Google Patents

Isolated or purified trichoderma antagonistic representative gene and application, and gene sequence-based trichoderma antagonistic evaluation amplification primer and application Download PDF

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WO2021138799A1
WO2021138799A1 PCT/CN2020/070655 CN2020070655W WO2021138799A1 WO 2021138799 A1 WO2021138799 A1 WO 2021138799A1 CN 2020070655 W CN2020070655 W CN 2020070655W WO 2021138799 A1 WO2021138799 A1 WO 2021138799A1
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trichoderma
antagonistic
gene
sequence
seq
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Chinese (zh)
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窦恺
陈捷
陆志翔
王少青
李雅乾
王新华
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上海交通大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

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  • the invention belongs to the field of microbial Trichoderma antagonism, and in particular relates to an isolated or purified Trichoderma antagonistic characterizing gene and application, and a gene sequence-based Trichoderma antagonistic evaluation amplification primer and application.
  • Agricultural production activities are the basis for human survival and development, and plant diseases and insect pests have always been one of the biggest threats to agricultural production activities.
  • the control of crop diseases and insect pests mainly relied on chemical control methods.
  • the application of biological control in agricultural production has attracted more and more attention.
  • Trichoderma as an important biocontrol fungus, has achieved good control effects on a variety of plant diseases.
  • a variety of Trichoderma has been developed and applied commercially, and the evaluation of the antagonism of the Trichoderma strain is the basis for determining whether the strain has biocontrol application value.
  • Trichoderma strains The current antagonistic evaluation of Trichoderma strains is mainly based on the culture characteristics of the strain itself.
  • the Trichoderma and the pathogen are cultured face-to-face on the culture medium, and then observe the growth advantage of Trichoderma. After the confrontation culture was completed, the colony radius of the pathogenic fungus and the growth of the fungus covered by Trichoderma were measured, and they were divided into 5-level antagonistic standards.
  • Level 1 means that Trichoderma has completely grown over the bacteria and covers the entire petri dish;
  • Level 2 is that Trichoderma occupies more than 2/3 of the entire petri dish area, and
  • Level 3 is that Trichoderma occupies 1/2 to 2/3 of the entire petri dish area.
  • the evaluation criteria for antagonistic Trichoderma strains is that its antagonistic degree reaches level 1 or level 2.
  • the prior art is a method for evaluating the antagonism of Trichoderma on the basis of observing the culture characteristics, and there is subjective judgment of people, and the judgment of the standard by personnel with different operating experience will be different.
  • Trichoderma strains Trichoderma and pathogenic fungi need to be cultured and transferred many times. If there are too many Trichoderma strains to be evaluated, it will consume a lot of manpower and time resources.
  • the first objective of the present invention is to provide an isolated or purified Trichoderma antagonistic characterizing gene and its application, which are based on the genome nucleotide sequence to evaluate the Trichoderma antagonistic, and the evaluation result is not affected by human subjective judgment. It is more objective and more accurate.
  • the second object of the present invention is to provide an amplification primer for evaluating Trichoderma antagonism based on gene sequence and its application.
  • the evaluation of Trichoderma antagonistic based on genomic nucleotide sequence is not affected by human subjective judgment. It is more objective and more accurate.
  • Trichoderma antagonistic gene including the nucleotide sequence shown in SEQ ID No. 1.
  • the Trichoderma antagonistic characteristic gene has a conservative segment 1, such as the nucleotide sequence shown in SEQ ID No. 3.
  • the Trichoderma antagonistic characteristic gene has a conservative segment 2, such as the nucleotide sequence shown in SEQ ID No.4.
  • the Trichoderma antagonistic characterization gene has a conservative segment 3, such as the nucleotide sequence shown in SEQ ID No.5.
  • Trichoderma antagonistic gene An amino acid sequence encoded by an isolated or purified Trichoderma antagonistic gene, such as the amino acid sequence shown in SEQ ID No.2.
  • the application of an isolated or purified Trichoderma antagonistic characterization gene in the evaluation of Trichoderma antagonism includes: constructing the genomic sequence of the Trichoderma strain to be tested into a local nucleotide database; and then setting it as SEQ ID No.
  • the nucleotide sequence shown in 1 is used as a search sequence for comparison with the local nucleotide database. If the similarity between the two nucleotide sequences is greater than 90%, the tested Trichoderma is antagonistic.
  • the present invention also provides a Trichoderma antagonistic evaluation amplification primer based on gene sequence, including a forward primer designed according to the conserved segment 2 of the Trichoderma antagonistic gene, as shown in SEQ ID No. 6, reverse The primer is shown in SEQ ID No.7.
  • An application of amplification primers for evaluating the antagonism of Trichoderma based on gene sequence including the following steps:
  • step S2 Use the genomic DNA prepared in step S1 as a template, and perform PCR amplification using the forward amplification primer shown in SEQ ID No. 6 and the reverse amplification primer shown in SEQ ID No. 7 to prepare PCR amplification primers;
  • step S3 The length of the PCR amplification primer prepared in step S2 is detected by electrophoresis in a 1% agarose gel. If a nucleotide fragment with a length of 1000-2000bp (base pair) is amplified, the Trichoderma bacterium to be detected It is evaluated as antagonistic.
  • the step S1 is specifically inoculating the Trichoderma strain to be tested into the culture medium aseptically on an ultra-clean bench, culturing at 25-28°C for 2-3 days, and collecting the Trichoderma mycelium, Then, the Trichoderma mycelium was ground, and the genomic DNA was extracted using a plant genome extraction kit.
  • the PCR reaction system in step S2 is as follows: the total system is 10 ⁇ L, including: Premix taq enzyme solution 5 ⁇ l, DNA template 90-100ng, and the final concentration of the reaction solution of forward primer and reverse primer is 0.2-1.0 ⁇ mol. /L, sterile water makes up 10 ⁇ L;
  • the PCR reaction conditions were: 94°C pre-denaturation for 10 minutes; 94°C for 10 seconds, 55°C for 30 seconds, 72°C for 3 minutes, 30 cycles, and 68°C for 10 minutes.
  • the invention also provides the application of the amplification primers for evaluating the antagonism of Trichoderma based on the gene sequence in preparing products for evaluating the antagonism of Trichoderma.
  • the present invention has the following advantages and positive effects due to the adoption of the above technical solutions:
  • the method for evaluating the antagonism of Trichoderma proposed in the present invention is based on the genomic nucleic acid sequence, and the evaluation result is not affected by human subjective judgment, and is more objective and accurate.
  • This evaluation method does not require the cultivation of pathogenic bacteria, nor does it need to be transferred to a plate for confrontation. It can save manpower and time when there are too many Trichoderma strains to be evaluated.
  • Figure 1 is the comparison result of Trichoderma asperellum CBS 433.97 genome in Example 1 of the present invention.
  • Figure 2 is an electrophoretic detection diagram of PCR products in Example 2 of the present invention.
  • Figure 3 is a picture of the evaluation of antagonism of the has32-2 Trichoderma strain plate confrontation method in Example 2 of the present invention.
  • Example 4 is a picture of the plate confrontation method for evaluating the antagonism of the has33-1 Trichoderma strain in Example 2 of the present invention
  • Figure 5 is a picture of the plate confrontation method for evaluating the antagonism of the has37-6 Trichoderma strain in Example 2 of the present invention
  • Figure 6 is an electrophoretic detection diagram of PCR products in Example 3 of the present invention.
  • Fig. 7 is a picture of the plate confrontation method for evaluating the antagonism of the hen212s Trichoderma strain in Example 3 of the present invention.
  • Figure 8 is a picture of the plate confrontation method for evaluating the antagonism of the Trichoderma hen214y strain in Example 3 of the present invention.
  • Figure 9 is a picture of the plate confrontation method for evaluating the antagonism of the hen311s Trichoderma strain in Example 3 of the present invention.
  • the present invention provides an isolated or purified Trichoderma antagonistic characterization gene, including the nucleotide sequence shown in SEQ ID No. 1, and an isolated or purified Trichoderma antagonistic characterization gene
  • the encoded amino acid sequence includes the amino acid sequence shown in SEQ ID No.2.
  • the Trichoderma antagonistic characteristic gene has a conservative segment 1, such as the nucleotide sequence of SEQ ID No. 3.
  • the Trichoderma antagonistic characteristic gene has a conservative segment 2, such as the nucleotide sequence shown in SEQ ID No.4.
  • the Trichoderma antagonistic characterization gene has a conservative segment 3, such as the nucleotide sequence shown in SEQ ID No.5.
  • the present invention conducts comparative genomics analysis on Trichoderma with different antagonism, and finds that the genome of the antagonistic Trichoderma has the homologous sequence of the above-mentioned nucleotide sequence, and the homologous sequence is similar to the above-mentioned nucleotide sequence More than 90%, the homologous sequence of the above sequence does not exist in the genome of non-antagonistic Trichoderma. Therefore, the primers are used for amplification and the kit is used to purify the amplified products to obtain the nucleotide sequence shown in SEQ ID No. 1 to meet the needs of the evaluation of the antagonism of Trichoderma. By detecting whether the genome of a Trichoderma strain has the homologous sequence of the above-mentioned nucleotide sequence, it is possible to quickly and objectively evaluate whether the Trichoderma is antagonistic.
  • the present invention also designs a gene sequence-based Trichoderma antagonistic evaluation amplification primer.
  • the primer is designed based on the conservative segment 2, including forward primers such as SEQ ID No. As shown in .6, the reverse primer is shown in SEQ ID No.7.
  • the conserved region 1 or the conserved region 3 can also be designed with corresponding primers, and there is no restriction here. This primer can be used to quickly and objectively evaluate whether Trichoderma is antagonistic. The specific steps are as follows:
  • the Trichoderma strain to be tested is aseptically inoculated into the culture medium, cultured at 25-28°C for 2-3 days, the Trichoderma mycelium is collected, and then the Trichoderma mycelium is ground Body, using plant genome extraction kit to extract genomic DNA;
  • PCR amplification is performed using the genomic DNA prepared in step S1 as a template, and PCR amplification primers include the forward primer shown in SEQ ID No. 6 and the reverse primer shown in SEQ ID No. 6;
  • the PCR reaction system is as follows, the total system is 10 ⁇ L: Premix taq enzyme solution 5ul, DNA template 90-100ng, the final concentration of the reaction solution of forward primer and reverse primer is 0.2-1.0 ⁇ mol/L, sterilized water makes up 10 ⁇ L;
  • the PCR reaction conditions were: 94°C pre-denaturation for 10 minutes, 94°C for 10 seconds, 55°C for 30 seconds, 72°C for 3 minutes, 30 cycles, and 68°C for 10 minutes;
  • step S3 The length of the PCR amplification primer prepared in step S2 is detected by electrophoresis in a 1% agarose gel. If a nucleotide fragment with a length of 1000-2000bp (base pair) is amplified, the Trichoderma bacterium to be detected It is evaluated as antagonistic.
  • Trichoderma strain is Trichoderma asperellum CBS 433.97, and this strain has undergone genome sequencing. Its genome is stored on the website https://genome.jgi.doe.gov/portal/ .
  • the strain used in this example is known to have antagonism, and the method provided in this example is used to obtain an antagonistic result, which proves the effectiveness and applicability of the method.
  • the Trichoderma strains used were three Trichoderma has32-2, has33-1, and has37-6 isolated from Anhui City, China. The three strains have not undergone genome sequencing.
  • the preparation method of PD medium is as follows: Weigh 200g of potatoes that have been peeled and diced, add them to 1000ml of water and boil them for 20-30 minutes. Filter with 2 layers of gauze to remove the potato pieces, and filter the filtrate into a beaker. Add 20 g of glucose to the filtrate and stir to dissolve. Add water to make up to 1000ml.
  • the prepared PD medium was sterilized by autoclaving at 121°C for later use.
  • Trichoderma genomic extraction uses TIANGEN Biotech (Beijing) Co. Ltd.'s Genome Extraction Kit Plant Genomic DNA Kit (DP305).
  • the taq enzyme used in PCR is Premix Taq (Code No. R004Q) from TaKaRa, and the DNA length reference standard for electrophoresis is DL2000 DNA Marker from TaKaRa.
  • Trichoderma has32-2, has33-1, and has37-6 as templates to perform PCR amplification.
  • the PCR amplification primers are shown in SEQ ID No.6.
  • the PCR reaction solution consists of 5ul Premix taq enzyme solution, 90-100ng DNA template, and the final concentration of the reaction solution of the forward primer and reverse primer is 0.2-1.0 ⁇ mol/L, and sterilized water is sufficient.
  • the reaction solution is to 10ul; the reaction conditions are 94°C pre-denaturation for 10min; 94°C for 10s, 55°C for annealing for 30s, 72°C for 3min, 30 cycles; 68°C for 10min;
  • the length of the PCR product is detected by electrophoresis in a 1% agarose gel, as shown in Figure 2.
  • fragments can be amplified from the genome of Trichoderma strains has32-2, has33-1, and has37-6, so these three strains were evaluated as antagonistic Trichoderma.
  • the authorization announcement number is CN102660627B
  • the name of the invention is a Trichoderma biocontrol germplasm evaluation method, and the evaluation method provided is used to verify the judgment made in this embodiment.
  • the verification method is:
  • Fusarium graminearum and three Trichoderma strains to be evaluated were cultured on a PDA plate medium at 25°C for 5 days. After the end of the confrontation culture, the 3 strains of Trichoderma were evaluated, as shown in Figure 3, Figure 4, and Figure 5 (the left side of the figure is Fusarium graminearum and the right side is Trichoderma). The area reached more than two-thirds of the entire culture medium area. Therefore, the antagonistic degree of the three Trichoderma strains to pathogenic fungi was level 2, and all belonged to the antagonistic Trichoderma strains.
  • Trichoderma strains used were three Trichoderma hen212s, hen214y, and hen311s isolated from Henan province, China. The three strains had not undergone genome sequencing.
  • the preparation method of PD medium is as follows: Weigh 200g of peeled and diced potatoes, add to 1000ml of water and boil for 20-30 minutes. Filter with 2 layers of gauze to remove the potato pieces, and filter the filtrate into a beaker. Add 20g of glucose to the filtrate and stir until it dissolves, and add water to make up to 1000ml.
  • the prepared PD medium was sterilized by autoclaving at 121°C for later use.
  • Trichoderma genomic extraction uses TIANGEN Biotech (Beijing) Co. Ltd.'s Genome Extraction Kit Plant Genomic DNA Kit (DP305).
  • the taq enzyme used in PCR is Premix Taq (Code No. R004Q) of TaKaRa Company, and the DNA length reference standard for electrophoresis is DL2000 DNA Marker of TaKaRa Company
  • the PCR reaction solution consists of 5ul Premix taq enzyme solution, 90-100ng DNA template, and the final concentration of the reaction solution of the forward primer and reverse primer is 0.2-1.0 ⁇ mol/L, and sterilized water is sufficient.
  • the reaction solution is to 10ul; the reaction conditions are 94°C pre-denaturation for 10min; 94°C for 10s, 55°C for annealing for 30s, 72°C for 3min, 30 cycles; 68°C for 10min;
  • the PCR products are detected by electrophoresis in a 1% agarose gel.
  • the provided primers can be used to amplify the fragments on the genome of Trichoderma strains hen212s, hen214y, and hen311s, so the The strain was evaluated as antagonistic Trichoderma.
  • the authorization announcement number is CN102660627B
  • the name of the invention is a Trichoderma biocontrol germplasm evaluation method, and the evaluation method provided is used to verify the judgment made in this embodiment.
  • the verification method is:
  • the Fusarium graminearum and the three Trichoderma strains to be evaluated were cultured at 25°C for 5 days. After the end of the confrontation culture, observe the growth of Trichoderma and Fusarium graminearum, as shown in Figure 7, Figure 8, Figure 9 (the left side of the figure is Fusarium graminearum, and the right side is Trichoderma), on the medium 3
  • the area of the Trichoderma strains reached more than two-thirds of the area of the entire culture medium, so the antagonistic degree of the three Trichoderma strains to pathogenic fungi was level 2, and they all belonged to the antagonistic Trichoderma strains.
  • the present invention uses gene sequences to evaluate the antagonism of Trichoderma.
  • the method is simple, and the antagonism of Trichoderma can be evaluated objectively, quickly and accurately.

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Abstract

Provided are an isolated or purified Trichoderma antagonistic representative gene and application, and a gene sequence-based Trichoderma antagonistic evaluation amplification primer and application. The isolated or purified Trichoderma antagonistic representative gene comprises the nucleotide sequence represented by SEQ. ID NO. 1. The gene sequence-based Trichoderma antagonistic evaluation amplification primer comprises the forward amplification primer represented by SEQ. ID NO. 6 and the reverse amplification primer represented by SEQ. ID NO. 7. Also provided is a method for quickly evaluating the antagonism of Trichoderma using a genomic nucleic acid sequence.

Description

分离出的或纯化的木霉菌拮抗性表征基因及应用及基于基因序列的木霉菌拮抗性评价扩增引物及应用Isolated or purified Trichoderma antagonistic characterization gene and its application, and Trichoderma antagonistic evaluation based on gene sequence amplification primer and its application 技术领域Technical field
本发明属于微生物木霉菌拮抗性领域，尤其涉及一种分离出的或纯化的木霉菌拮抗性表征基因及应用及基于基因序列的木霉菌拮抗性评价扩增引物及应用。The invention belongs to the field of microbial Trichoderma antagonism, and in particular relates to an isolated or purified Trichoderma antagonistic characterizing gene and application, and a gene sequence-based Trichoderma antagonistic evaluation amplification primer and application.
背景技术Background technique
农业生产活动是人类得以生存发展的基础，而植物病虫害一直是农业生产活动的最大威胁之一。过去，对于农作物病虫害的防治，主要依赖于化学防治方法。近年来，随着人们生态保护意识的增强以及农作物病原菌持续对化学药剂产生抗性，生物防治在农业生产中的应用越来越受到人们的重视。在生物防治的实践活动中，木霉菌作为一种重要的生防真菌，对多种植物病害都取得了良好的防治效果。目前已有多种木霉菌进行了商业化的开发应用，而对木霉菌株的拮抗性评价，是决定该菌株是否具有生防应用价值的基础。Agricultural production activities are the basis for human survival and development, and plant diseases and insect pests have always been one of the biggest threats to agricultural production activities. In the past, the control of crop diseases and insect pests mainly relied on chemical control methods. In recent years, with the enhancement of people's awareness of ecological protection and the continued resistance of crop pathogens to chemical agents, the application of biological control in agricultural production has attracted more and more attention. In the practice of biological control, Trichoderma, as an important biocontrol fungus, has achieved good control effects on a variety of plant diseases. At present, a variety of Trichoderma has been developed and applied commercially, and the evaluation of the antagonism of the Trichoderma strain is the basis for determining whether the strain has biocontrol application value.
目前对木霉菌株的拮抗性评价，主要基于菌株本身的培养性状。如将木霉菌与病原菌在培养基上进行对峙培养，然后观察木霉菌的生长优势。对峙培养完成后测定病原真菌菌落半径和被木霉菌覆盖生长的情况，分为了5级拮抗标准。1级为木霉菌完全长过病菌，并覆盖整个培养皿；2级为木霉菌占整个培养皿面积的2/3以上，3级为木霉菌占整个培养皿面积的1/2至2/3；4级为病原菌占整个培养皿面积的2/3以上；5级为病原菌长过木霉菌，并覆盖整个培养皿。具有拮抗性的木霉菌株评价标准为其拮抗程度达到1级或2级。The current antagonistic evaluation of Trichoderma strains is mainly based on the culture characteristics of the strain itself. For example, the Trichoderma and the pathogen are cultured face-to-face on the culture medium, and then observe the growth advantage of Trichoderma. After the confrontation culture was completed, the colony radius of the pathogenic fungus and the growth of the fungus covered by Trichoderma were measured, and they were divided into 5-level antagonistic standards. Level 1 means that Trichoderma has completely grown over the bacteria and covers the entire petri dish; Level 2 is that Trichoderma occupies more than 2/3 of the entire petri dish area, and Level 3 is that Trichoderma occupies 1/2 to 2/3 of the entire petri dish area. ; Level 4 means that the pathogen occupies more than 2/3 of the area of the entire petri dish; grade 5 means that the pathogen has grown over Trichoderma and covers the entire petri dish. The evaluation criteria for antagonistic Trichoderma strains is that its antagonistic degree reaches level 1 or level 2.
现有技术都是基于观察培养性状对木霉菌进行拮抗性评价的方法，存在 人的主观判断性，不同操作经验的人员对标准的判断会存在差异。在木霉菌株的评价过程中，需要对木霉菌及病原真菌进行多次培养及转接，如果需要评价木霉菌株过多，则会耗费大量的人力及时间资源。The prior art is a method for evaluating the antagonism of Trichoderma on the basis of observing the culture characteristics, and there is subjective judgment of people, and the judgment of the standard by personnel with different operating experience will be different. In the evaluation process of Trichoderma strains, Trichoderma and pathogenic fungi need to be cultured and transferred many times. If there are too many Trichoderma strains to be evaluated, it will consume a lot of manpower and time resources.
发明内容Summary of the invention
本发明的第一目的是提供一种分离出的或纯化的木霉菌拮抗性表征基因及其应用，基于基因组核苷酸序列对木霉菌拮抗性进行评价，评价结果不受人的主观判断影响，更具有客观性，且较为准确。The first objective of the present invention is to provide an isolated or purified Trichoderma antagonistic characterizing gene and its application, which are based on the genome nucleotide sequence to evaluate the Trichoderma antagonistic, and the evaluation result is not affected by human subjective judgment. It is more objective and more accurate.
本发明的第二目的是提供一种基于基因序列的木霉菌拮抗性评价扩增引物及其应用，基于基因组核苷酸序列对木霉菌拮抗性进行评价，评价结果不受人的主观判断影响，更具有客观性，且较为准确。The second object of the present invention is to provide an amplification primer for evaluating Trichoderma antagonism based on gene sequence and its application. The evaluation of Trichoderma antagonistic based on genomic nucleotide sequence is not affected by human subjective judgment. It is more objective and more accurate.
为解决上述问题，本发明的技术方案为：In order to solve the above-mentioned problems, the technical scheme of the present invention is as follows:
一种分离出的或纯化的木霉菌拮抗性表征基因，包括如SEQ ID No.1所示的核苷酸序列。An isolated or purified Trichoderma antagonistic gene, including the nucleotide sequence shown in SEQ ID No. 1.
根据本发明优选地，所述木霉菌拮抗性表征基因具有保守区段1，如SEQ ID No.3所示的核苷酸序列。According to the present invention, preferably, the Trichoderma antagonistic characteristic gene has a conservative segment 1, such as the nucleotide sequence shown in SEQ ID No. 3.
根据本发明优选地，所述木霉菌拮抗性表征基因具有保守区段2，如SEQ ID No.4所示的核苷酸序列。According to the present invention, preferably, the Trichoderma antagonistic characteristic gene has a conservative segment 2, such as the nucleotide sequence shown in SEQ ID No.4.
根据本发明优选地，所述木霉菌拮抗性表征基因具有保守区段3，如SEQ ID No.5所示的核苷酸序列。According to the present invention, preferably, the Trichoderma antagonistic characterization gene has a conservative segment 3, such as the nucleotide sequence shown in SEQ ID No.5.
一种根据分离出的或纯化的木霉菌拮抗性表征基因编码的氨基酸序列，如SEQ ID No.2所示的氨基酸序列。An amino acid sequence encoded by an isolated or purified Trichoderma antagonistic gene, such as the amino acid sequence shown in SEQ ID No.2.
一种分离出的或纯化的木霉菌拮抗性表征基因在评价木霉菌拮抗性上的应用，包括：将待检测木霉菌株的基因组序列构建成本地核苷酸数据库；然后将如SEQ ID No.1所示的核苷酸序列作为检索序列与所述本地核苷酸数据库进行比对，若两种核苷酸序列的相似性大于90％，则待测木霉菌具有拮抗 性。The application of an isolated or purified Trichoderma antagonistic characterization gene in the evaluation of Trichoderma antagonism includes: constructing the genomic sequence of the Trichoderma strain to be tested into a local nucleotide database; and then setting it as SEQ ID No. The nucleotide sequence shown in 1 is used as a search sequence for comparison with the local nucleotide database. If the similarity between the two nucleotide sequences is greater than 90%, the tested Trichoderma is antagonistic.
本发明还提供了一种基于基因序列的木霉菌拮抗性评价扩增引物，包括根据所述木霉菌拮抗性基因的保守区段2设计的正向引物如SEQ ID No.6所示，反向引物如SEQ ID No.7所示。The present invention also provides a Trichoderma antagonistic evaluation amplification primer based on gene sequence, including a forward primer designed according to the conserved segment 2 of the Trichoderma antagonistic gene, as shown in SEQ ID No. 6, reverse The primer is shown in SEQ ID No.7.
一种基于基因序列的木霉菌拮抗性评价扩增引物的应用，包括如下步骤：An application of amplification primers for evaluating the antagonism of Trichoderma based on gene sequence, including the following steps:
S1：提取待测的木霉菌真菌样品的DNA，制得基因组DNA；S1: Extract the DNA of the Trichoderma fungus sample to be tested to obtain genomic DNA;
S2：以步骤S1制得的基因组DNA为模板，利用如SEQ ID No.6所示的正向扩增引物和如SEQ ID No.7所示的反向扩增引物进行PCR扩增，制得PCR扩增引物；S2: Use the genomic DNA prepared in step S1 as a template, and perform PCR amplification using the forward amplification primer shown in SEQ ID No. 6 and the reverse amplification primer shown in SEQ ID No. 7 to prepare PCR amplification primers;
S3：对步骤S2制得的PCR扩增引物在1％的琼脂糖凝胶中电泳检测长度，若扩增到长度为1000-2000bp(base pair)的核苷酸片段，则待检测的木霉菌评价为拮抗性。S3: The length of the PCR amplification primer prepared in step S2 is detected by electrophoresis in a 1% agarose gel. If a nucleotide fragment with a length of 1000-2000bp (base pair) is amplified, the Trichoderma bacterium to be detected It is evaluated as antagonistic.
优选地，所述步骤S1具体为在超净台上，将待测的木霉菌株无菌接种到培养基中，在25-28℃条件在培养2-3天，收集木霉菌菌丝体，然后研磨木霉菌菌丝体，采用植物基因组提取试剂盒提取基因组DNA。Preferably, the step S1 is specifically inoculating the Trichoderma strain to be tested into the culture medium aseptically on an ultra-clean bench, culturing at 25-28°C for 2-3 days, and collecting the Trichoderma mycelium, Then, the Trichoderma mycelium was ground, and the genomic DNA was extracted using a plant genome extraction kit.
优选地，所述步骤S2中PCR反应体系如下：总体系为10μL，包括：Premix taq酶液5μl，DNA模板90-100ng，正向引物、反向引物的反应液终浓度均为0.2-1.0μmol/L，灭菌水补足10μL；Preferably, the PCR reaction system in step S2 is as follows: the total system is 10 μL, including: Premix taq enzyme solution 5 μl, DNA template 90-100ng, and the final concentration of the reaction solution of forward primer and reverse primer is 0.2-1.0 μmol. /L, sterile water makes up 10μL;
PCR反应条件为：94℃预变性10min；94℃维持10s，55℃退火30s，72℃维持3min，30个循环；68℃延伸10min。The PCR reaction conditions were: 94°C pre-denaturation for 10 minutes; 94°C for 10 seconds, 55°C for 30 seconds, 72°C for 3 minutes, 30 cycles, and 68°C for 10 minutes.
本发明还提供了基于基因序列的木霉菌拮抗性评价扩增引物在制备评价木霉菌拮抗性的产品中的应用。The invention also provides the application of the amplification primers for evaluating the antagonism of Trichoderma based on the gene sequence in preparing products for evaluating the antagonism of Trichoderma.
本发明由于采用以上技术方案，使其与现有技术相比具有以下的优点和积极效果：Compared with the prior art, the present invention has the following advantages and positive effects due to the adoption of the above technical solutions:
本发明提出的木霉菌拮抗性评价方法是基于基因组核酸序列，评价结果不受人的主观判断影响，更具有客观性且较准确。此种评价方法不需要培养 病原菌，也不需要转接到平板进行对峙，当需要评价的木霉菌株数量过多时可以节省人力及时间。The method for evaluating the antagonism of Trichoderma proposed in the present invention is based on the genomic nucleic acid sequence, and the evaluation result is not affected by human subjective judgment, and is more objective and accurate. This evaluation method does not require the cultivation of pathogenic bacteria, nor does it need to be transferred to a plate for confrontation. It can save manpower and time when there are too many Trichoderma strains to be evaluated.
附图说明Description of the drawings
图1为本发明实施例1中Trichoderma asperellum CBS 433.97基因组中的比对结果；Figure 1 is the comparison result of Trichoderma asperellum CBS 433.97 genome in Example 1 of the present invention;
图2为本发明实施例2中PCR产物电泳检测图；Figure 2 is an electrophoretic detection diagram of PCR products in Example 2 of the present invention;
图3为本发明实施例2中has32-2木霉菌株平板对峙方法评价拮抗性的图片；Figure 3 is a picture of the evaluation of antagonism of the has32-2 Trichoderma strain plate confrontation method in Example 2 of the present invention;
图4为本发明实施例2中has33-1木霉菌株平板对峙方法评价拮抗性的图片；4 is a picture of the plate confrontation method for evaluating the antagonism of the has33-1 Trichoderma strain in Example 2 of the present invention;
图5为本发明实施例2中has37-6木霉菌株平板对峙方法评价拮抗性的图片；Figure 5 is a picture of the plate confrontation method for evaluating the antagonism of the has37-6 Trichoderma strain in Example 2 of the present invention;
图6为本发明实施例3中PCR产物电泳检测图；Figure 6 is an electrophoretic detection diagram of PCR products in Example 3 of the present invention;
图7为本发明实施例3中hen212s木霉菌株平板对峙方法评价拮抗性的图片；Fig. 7 is a picture of the plate confrontation method for evaluating the antagonism of the hen212s Trichoderma strain in Example 3 of the present invention;
图8为本发明实施例3中hen214y木霉菌株平板对峙方法评价拮抗性的图片；Figure 8 is a picture of the plate confrontation method for evaluating the antagonism of the Trichoderma hen214y strain in Example 3 of the present invention;
图9为本发明实施例3中hen311s木霉菌株平板对峙方法评价拮抗性的图片。Figure 9 is a picture of the plate confrontation method for evaluating the antagonism of the hen311s Trichoderma strain in Example 3 of the present invention.
具体实施方式Detailed ways
以下结合附图和具体实施例对本发明提出的一种分离出的或纯化的木霉菌拮抗性表征基因及应用及基于基因序列的木霉菌拮抗性评价扩增引物及应用作进一步详细说明。根据下面说明和权利要求书，本发明的优点和特征将更清楚。The following describes in further detail an isolated or purified Trichoderma antagonistic characterizing gene and its application, and the amplification primer and its application based on the gene sequence-based Trichoderma antagonistic evaluation of the present invention with reference to the accompanying drawings and specific examples. According to the following description and claims, the advantages and features of the present invention will be clearer.
本发明提供了一种分离出的或纯化的木霉菌拮抗性表征基因，包括如SEQ ID No.1所示的核苷酸序列，以及一种根据分离出的或纯化的木霉菌拮抗性表征基因编码的氨基酸序列，包括，如SEQ ID No.2所示的氨基酸序列。The present invention provides an isolated or purified Trichoderma antagonistic characterization gene, including the nucleotide sequence shown in SEQ ID No. 1, and an isolated or purified Trichoderma antagonistic characterization gene The encoded amino acid sequence includes the amino acid sequence shown in SEQ ID No.2.
根据本发明优选地，所述木霉菌拮抗性表征基因具有保守区段1，如SEQ ID No.3的核苷酸序列。According to the present invention, preferably, the Trichoderma antagonistic characteristic gene has a conservative segment 1, such as the nucleotide sequence of SEQ ID No. 3.
根据本发明优选地，所述木霉菌拮抗性表征基因具有保守区段2，如SEQ ID No.4所示的核苷酸序列。According to the present invention, preferably, the Trichoderma antagonistic characteristic gene has a conservative segment 2, such as the nucleotide sequence shown in SEQ ID No.4.
根据本发明优选地，所述木霉菌拮抗性表征基因具有保守区段3，如SEQ ID No.5所示的核苷酸序列。According to the present invention, preferably, the Trichoderma antagonistic characterization gene has a conservative segment 3, such as the nucleotide sequence shown in SEQ ID No.5.
本发明通过对具有不同拮抗性的木霉菌进行比较基因组学分析，发现拮抗性木霉菌的基因组中都具有上述核苷酸序列的同源序列，且同源序列与上述核苷酸序列的相似性大于90％，而非拮抗性木霉菌的基因组中则不存在上述序列的同源序列。因此，利用引物进行扩增和利用试剂盒对扩增产物进行提纯得到如SEQ ID No.1所示的核苷酸序列，以满足对木霉菌拮抗性的评价的需要。通过检测一株木霉菌的基因组中是否具有上述核苷酸序列的同源序列可以快速客观的评价该木霉菌是否具有拮抗性。The present invention conducts comparative genomics analysis on Trichoderma with different antagonism, and finds that the genome of the antagonistic Trichoderma has the homologous sequence of the above-mentioned nucleotide sequence, and the homologous sequence is similar to the above-mentioned nucleotide sequence More than 90%, the homologous sequence of the above sequence does not exist in the genome of non-antagonistic Trichoderma. Therefore, the primers are used for amplification and the kit is used to purify the amplified products to obtain the nucleotide sequence shown in SEQ ID No. 1 to meet the needs of the evaluation of the antagonism of Trichoderma. By detecting whether the genome of a Trichoderma strain has the homologous sequence of the above-mentioned nucleotide sequence, it is possible to quickly and objectively evaluate whether the Trichoderma is antagonistic.
如果待检测木霉菌株的基因组序列已经测序并拼接，具体实行步骤如下：If the genome sequence of the Trichoderma strain to be tested has been sequenced and assembled, the specific steps are as follows:
1、用bioedit软件，将待检测木霉菌株基因组序列构建成本地核苷酸blast数据库；1. Use the bioedit software to construct the genomic sequence of the Trichoderma strain to be detected into a local nucleotide blast database;
2、在bioedit软件中，将如SEQ ID No.1所示的核苷酸序列作为检索序列对刚建立的数据库进行blast比对；2. In the bioedit software, use the nucleotide sequence shown in SEQ ID No. 1 as the search sequence to perform blast comparison on the newly established database;
3、查看比对结果。如果在基因组中比对到与上述序列相似性大于90％的序列，则该木霉菌株被评价为具有拮抗性，否则被评价为不具有拮抗性。3. View the comparison result. If a sequence with the above sequence similarity greater than 90% is found in the genome, the Trichoderma strain is evaluated as having antagonism, otherwise it is evaluated as not having antagonism.
而对于待检测木霉菌株没有基因组序列，本发明还设计了一种基于基因序列的木霉菌拮抗性评价扩增引物，该引物是根据保守区段2设计的，包括正向引物如SEQ ID No.6所示，反向引物如SEQ ID No.7所示。当然保守区 段1或保守区段3也可以设计相应的引物，在此不做限制。采用该引物可以快速客观评价木霉菌是否具有拮抗性，具体实行步骤如下：As for the Trichoderma strain to be detected, there is no genome sequence. The present invention also designs a gene sequence-based Trichoderma antagonistic evaluation amplification primer. The primer is designed based on the conservative segment 2, including forward primers such as SEQ ID No. As shown in .6, the reverse primer is shown in SEQ ID No.7. Of course, the conserved region 1 or the conserved region 3 can also be designed with corresponding primers, and there is no restriction here. This primer can be used to quickly and objectively evaluate whether Trichoderma is antagonistic. The specific steps are as follows:
S1：提取待测的木霉菌真菌样品的DNA，制得基因组DNA；S1: Extract the DNA of the Trichoderma fungus sample to be tested to obtain genomic DNA;
具体地，在超净台上，将待测的木霉菌株无菌接种到培养基中，在25-28℃条件在培养2-3天，收集木霉菌菌丝体，然后研磨木霉菌菌丝体，采用植物基因组提取试剂盒提取基因组DNA；Specifically, on a clean bench, the Trichoderma strain to be tested is aseptically inoculated into the culture medium, cultured at 25-28°C for 2-3 days, the Trichoderma mycelium is collected, and then the Trichoderma mycelium is ground Body, using plant genome extraction kit to extract genomic DNA;
S2：以步骤S1制得的基因组DNA为模板，进行PCR扩增，PCR扩增引物包括如SEQ ID No.6所示的正向引物和SEQ ID No.6所示的反向引物；S2: PCR amplification is performed using the genomic DNA prepared in step S1 as a template, and PCR amplification primers include the forward primer shown in SEQ ID No. 6 and the reverse primer shown in SEQ ID No. 6;
PCR反应体系如下，总体系为10μL：Premix taq酶液5ul，DNA模板90-100ng，正向引物、反向引物的反应液终浓度均为0.2-1.0μmol/L，灭菌水补足10μL；The PCR reaction system is as follows, the total system is 10μL: Premix taq enzyme solution 5ul, DNA template 90-100ng, the final concentration of the reaction solution of forward primer and reverse primer is 0.2-1.0μmol/L, sterilized water makes up 10μL;
PCR反应条件为：94℃预变性10min，94℃维持10s，55℃退火30s，72℃维持3min，30个循环，68℃延伸10min；The PCR reaction conditions were: 94°C pre-denaturation for 10 minutes, 94°C for 10 seconds, 55°C for 30 seconds, 72°C for 3 minutes, 30 cycles, and 68°C for 10 minutes;
S3：对步骤S2制得的PCR扩增引物在1％的琼脂糖凝胶中电泳检测长度，若扩增到长度为1000-2000bp(base pair)的核苷酸片段，则待检测的木霉菌评价为拮抗性。S3: The length of the PCR amplification primer prepared in step S2 is detected by electrophoresis in a 1% agarose gel. If a nucleotide fragment with a length of 1000-2000bp (base pair) is amplified, the Trichoderma bacterium to be detected It is evaluated as antagonistic.
实施例1Example 1
木霉菌株为Trichoderma asperellum CBS 433.97，该菌株已经进行了基因组测序。其基因组保存在网站 https://genome.jgi.doe.gov/portal/。 The Trichoderma strain is Trichoderma asperellum CBS 433.97, and this strain has undergone genome sequencing. Its genome is stored on the website https://genome.jgi.doe.gov/portal/ .
1、用bioedit软件，将基因组序列构建成本地核苷酸blast数据库；1. Use bioedit software to construct the genome sequence into a local nucleotide blast database;
2、在bioedit软件中，将如SEQ ID No.1所示的核苷酸序列作为检索序列对刚刚建立的数据库进行blast比对；2. In the bioedit software, use the nucleotide sequence shown in SEQ ID No. 1 as the search sequence to perform blast comparison on the newly established database;
3、查看比对结果，如图1所示。在基因组中比对到的序列与本发明提供的序列相似性为97％的序列，即Trichoderma asperellum CBS 433.97被评价为具有拮抗性。3. Check the comparison result, as shown in Figure 1. The sequence aligned in the genome is 97% similar to the sequence provided by the present invention, that is, Trichoderma asperellum CBS 433.97 is evaluated as having antagonism.
本实施例采用的菌株已知具有拮抗性，采用本实施例提供的方法得到具 有拮抗性的结果，证明该方法的有效性、适用性。The strain used in this example is known to have antagonism, and the method provided in this example is used to obtain an antagonistic result, which proves the effectiveness and applicability of the method.
实施例2Example 2
所用木霉菌株为分离自中国安徽省的3株木霉菌has32-2，has33-1,has37-6,该3株菌株没有经过基因组测序。The Trichoderma strains used were three Trichoderma has32-2, has33-1, and has37-6 isolated from Anhui Province, China. The three strains have not undergone genome sequencing.
PD培养基配制方法如下：称取去皮切块后的马铃薯200g，加入到1000m l水中煮沸后维持20-30分钟。用2层纱布过滤除去马铃薯块，将滤液过滤至烧杯中。在滤液中加入20g葡萄糖搅拌至溶解。加水补足至1000ml。配制的PD培养基121℃高压蒸汽灭菌后备用。The preparation method of PD medium is as follows: Weigh 200g of potatoes that have been peeled and diced, add them to 1000ml of water and boil them for 20-30 minutes. Filter with 2 layers of gauze to remove the potato pieces, and filter the filtrate into a beaker. Add 20 g of glucose to the filtrate and stir to dissolve. Add water to make up to 1000ml. The prepared PD medium was sterilized by autoclaving at 121°C for later use.
木霉菌基因组提取采用TIANGEN Biotech(Beijing)Co.Ltd.公司的基因组提取试剂盒Plant Genomic DNA Kit(DP305)。Trichoderma genomic extraction uses TIANGEN Biotech (Beijing) Co. Ltd.'s Genome Extraction Kit Plant Genomic DNA Kit (DP305).
PCR使用的taq酶为TaKaRa公司的Premix Taq(Code No.R004Q)，电泳的DNA长度参照标准采用TaKaRa公司的DL2000DNA Marker。The taq enzyme used in PCR is Premix Taq (Code No. R004Q) from TaKaRa, and the DNA length reference standard for electrophoresis is DL2000 DNA Marker from TaKaRa.
1、在超净台中，以无菌操作分别接种木霉菌has32-2、has33-1、has37-6于20ml PD培养基中，在25-28℃条件下震荡培养2-3天，使用纱布对培养的木霉菌has32-2、has33-1、has37-6过滤出去培养基，收集菌丝体，研磨木霉菌菌丝体，然后用天根公司的植物基因组提取试剂盒提取研磨后的木霉菌has32-2、has33-1、has37-6的基因组DNA；1. In an ultra-clean table, inoculate Trichoderma has32-2, has33-1, and has37-6 in 20ml PD medium by aseptic operation, shake culture at 25-28℃ for 2-3 days, use gauze to The cultured Trichoderma has32-2, has33-1, and has37-6 are filtered out of the medium, the mycelium is collected, the Trichoderma mycelium is ground, and then the ground Trichoderma has32 is extracted with the plant genome extraction kit of Tiangen Company -2. Genomic DNA of has33-1 and has37-6;
2、分别以木霉菌has32-2、has33-1、has37-6的基因组DNA为模板，进行PCR扩增，PCR扩增引物如SEQ ID No.6所示。2. Use the genomic DNA of Trichoderma has32-2, has33-1, and has37-6 as templates to perform PCR amplification. The PCR amplification primers are shown in SEQ ID No.6.
3、使用TaKaRa Premix Taq进行PCR反应，PCR反应液组成为Premix taq酶液5ul，DNA模板90-100ng，正引物和反引物的反应液终浓度均为0.2-1.0μmol/L，灭菌水补足反应液至10ul；反应条件为94℃预变性10min；94℃维持10s，55℃退火30s，72℃维持3min，30个循环；68℃延伸10min；3. Use TaKaRa Premix Taq for PCR reaction. The PCR reaction solution consists of 5ul Premix taq enzyme solution, 90-100ng DNA template, and the final concentration of the reaction solution of the forward primer and reverse primer is 0.2-1.0 μmol/L, and sterilized water is sufficient. The reaction solution is to 10ul; the reaction conditions are 94°C pre-denaturation for 10min; 94°C for 10s, 55°C for annealing for 30s, 72°C for 3min, 30 cycles; 68°C for 10min;
4、反应完成后在1％的琼脂糖凝胶中电泳检测PCR产物长度，如图2所示。利用所提供引物，可以在木霉菌株has32-2、has33-1、has37-6的基因组上扩增得到片段，所以该3株菌株评价为拮抗性木霉菌。4. After the reaction is completed, the length of the PCR product is detected by electrophoresis in a 1% agarose gel, as shown in Figure 2. Using the provided primers, fragments can be amplified from the genome of Trichoderma strains has32-2, has33-1, and has37-6, so these three strains were evaluated as antagonistic Trichoderma.
本实施例方法的有效性验证：采用授权公告号为CN102660627B，发明名称为一种木霉生防菌种质评价方法，提供的评价方法对本实施例做出的判断进行验证。验证方法为：Verification of the effectiveness of the method in this embodiment: the authorization announcement number is CN102660627B, and the name of the invention is a Trichoderma biocontrol germplasm evaluation method, and the evaluation method provided is used to verify the judgment made in this embodiment. The verification method is:
在PDA平板培养基上，在25℃对峙培养禾谷镰孢菌与待评价的三株木霉菌，培养时间5天。对峙培养结束后对3株木霉菌进行评价，如图3、图4、图5所示(图中左侧为禾谷镰孢菌，右侧为木霉菌)，培养基上3株木霉菌的面积均达到整个培养基面积的三分之二以上，因此3株木霉菌对病原真菌的拮抗程度均为2级，均属于拮抗性木霉菌株。Fusarium graminearum and three Trichoderma strains to be evaluated were cultured on a PDA plate medium at 25°C for 5 days. After the end of the confrontation culture, the 3 strains of Trichoderma were evaluated, as shown in Figure 3, Figure 4, and Figure 5 (the left side of the figure is Fusarium graminearum and the right side is Trichoderma). The area reached more than two-thirds of the entire culture medium area. Therefore, the antagonistic degree of the three Trichoderma strains to pathogenic fungi was level 2, and all belonged to the antagonistic Trichoderma strains.
因此，本实施例提供的方法具有准确性。Therefore, the method provided in this embodiment is accurate.
实施例3Example 3
所用木霉菌株为分离自中国河南省的3株木霉菌hen212s,hen214y,hen311s，该3菌株没有经过基因组测序。The Trichoderma strains used were three Trichoderma hen212s, hen214y, and hen311s isolated from Henan Province, China. The three strains had not undergone genome sequencing.
PD培养基配制方法如下：称取去皮切块后的马铃薯200g，加入到1000ml水中煮沸后维持20-30分钟。用2层纱布过滤除去马铃薯块，将滤液过滤至烧杯中。在滤液中加入20g葡萄糖搅拌至溶解，加水补足至1000ml。配制的PD培养基121℃高压蒸汽灭菌后备用。The preparation method of PD medium is as follows: Weigh 200g of peeled and diced potatoes, add to 1000ml of water and boil for 20-30 minutes. Filter with 2 layers of gauze to remove the potato pieces, and filter the filtrate into a beaker. Add 20g of glucose to the filtrate and stir until it dissolves, and add water to make up to 1000ml. The prepared PD medium was sterilized by autoclaving at 121°C for later use.
木霉菌基因组提取采用TIANGEN Biotech(Beijing)Co.Ltd.公司的基因组提取试剂盒Plant Genomic DNA Kit(DP305)。Trichoderma genomic extraction uses TIANGEN Biotech (Beijing) Co. Ltd.'s Genome Extraction Kit Plant Genomic DNA Kit (DP305).
PCR使用的taq酶为TaKaRa公司的Premix Taq(Code No.R004Q)，电泳的DNA长度参照标准采用TaKaRa公司的DL2000 DNA MarkerThe taq enzyme used in PCR is Premix Taq (Code No. R004Q) of TaKaRa Company, and the DNA length reference standard for electrophoresis is DL2000 DNA Marker of TaKaRa Company
1、在超净台中，以无菌操作分别接种木霉菌hen212s、hen214y、hen311s于20ml PD培养基中，在25-28℃条件下震荡培养2-3天；1. In an ultra-clean table, inoculate Trichoderma hen212s, hen214y, and hen311s in 20ml PD medium by aseptic operation, and culture at 25-28℃ for 2-3 days with shaking;
2、使用纱布分别对培养的木霉菌hen212s,hen214y,hen311s过滤出去培养基，收集菌丝体；2. Use gauze to filter the cultured Trichoderma hen212s, hen214y, and hen311s to remove the culture medium, and collect the mycelium;
3、分别研磨木霉菌hen212s,hen214y,hen311s的菌丝体；3. Grind the mycelium of Trichoderma hen212s, hen214y, and hen311s respectively;
4、用天根公司的植物基因组提取试剂盒分别提取研磨后的木霉菌hen212 s,hen214y,hen311s的基因组DNA；4. Extract the genomic DNA of the ground Trichoderma hen212 s, hen214y, and hen311s with the plant genome extraction kit of Tiangen Company;
5、分别以木霉菌hen212s,hen214y,hen311s的基因组DNA为模板，进行PCR扩增，PCR扩增引物如SEQ ID No.6所示；5. Use the genomic DNA of Trichoderma hen212s, hen214y, and hen311s as templates to perform PCR amplification. The PCR amplification primers are shown in SEQ ID No. 6;
6、使用TaKaRa Premix Taq进行PCR反应，PCR反应液组成为Premix taq酶液5ul，DNA模板90-100ng，正引物和反引物的反应液终浓度均为0.2-1.0μmol/L，灭菌水补足反应液至10ul；反应条件为94℃预变性10min；94℃维持10s，55℃退火30s，72℃维持3min，30个循环；68℃延伸10min；6. Use TaKaRa Premix Taq to perform PCR reaction. The PCR reaction solution consists of 5ul Premix taq enzyme solution, 90-100ng DNA template, and the final concentration of the reaction solution of the forward primer and reverse primer is 0.2-1.0μmol/L, and sterilized water is sufficient. The reaction solution is to 10ul; the reaction conditions are 94°C pre-denaturation for 10min; 94°C for 10s, 55°C for annealing for 30s, 72°C for 3min, 30 cycles; 68°C for 10min;
7、反应完成后在1％的琼脂糖凝胶中电泳检测PCR产物，如图6所示，利用所提供引物，可以在木霉菌株hen212s,hen214y,hen311s的基因组上扩增得到片段，所以该菌株评价为拮抗性木霉菌。7. After the reaction is completed, the PCR products are detected by electrophoresis in a 1% agarose gel. As shown in Figure 6, the provided primers can be used to amplify the fragments on the genome of Trichoderma strains hen212s, hen214y, and hen311s, so the The strain was evaluated as antagonistic Trichoderma.
本实施例方法的有效性验证：采用授权公告号为CN102660627B，发明名称为一种木霉生防菌种质评价方法，提供的评价方法对本实施例做出的判断进行验证。验证方法为：Verification of the effectiveness of the method in this embodiment: the authorization announcement number is CN102660627B, and the name of the invention is a Trichoderma biocontrol germplasm evaluation method, and the evaluation method provided is used to verify the judgment made in this embodiment. The verification method is:
在PDA平板培养基上，在25℃分别对峙培养禾谷镰孢菌与待评价的三株木霉菌，培养时间5天。对峙培养结束后观察木霉菌与禾谷镰孢菌的长势，如图7、图8、图9所示(图中左侧为禾谷镰孢菌，右侧为木霉菌)，培养基上3株木霉菌的面积均达到整个培养基面积的三分之二以上，因此3株木霉菌对病原真菌的拮抗程度均为2级，均属于拮抗性木霉菌株。On the PDA plate medium, the Fusarium graminearum and the three Trichoderma strains to be evaluated were cultured at 25°C for 5 days. After the end of the confrontation culture, observe the growth of Trichoderma and Fusarium graminearum, as shown in Figure 7, Figure 8, Figure 9 (the left side of the figure is Fusarium graminearum, and the right side is Trichoderma), on the medium 3 The area of the Trichoderma strains reached more than two-thirds of the area of the entire culture medium, so the antagonistic degree of the three Trichoderma strains to pathogenic fungi was level 2, and they all belonged to the antagonistic Trichoderma strains.
因此本实施例提供的评价方法具有准确性。Therefore, the evaluation method provided in this embodiment is accurate.
综合所述，本发明采用基因序列对木霉菌拮抗性进行评价，方法简单，客观、快速、准确地评价木霉菌的拮抗性。In summary, the present invention uses gene sequences to evaluate the antagonism of Trichoderma. The method is simple, and the antagonism of Trichoderma can be evaluated objectively, quickly and accurately.
上面结合附图对本发明的实施方式作了详细说明，但是本发明并不限于上述实施方式。即使对本发明作出各种变化，倘若这些变化属于本发明权利要求及其等同技术的范围之内，则仍落入在本发明的保护范围之中。The embodiments of the present invention have been described in detail above with reference to the drawings, but the present invention is not limited to the above-mentioned embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and equivalent technologies, they still fall into the protection scope of the present invention.
Figure PCTCN2020070655-appb-000001
Figure PCTCN2020070655-appb-000001
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Figure PCTCN2020070655-appb-000005
Figure PCTCN2020070655-appb-000006
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Figure PCTCN2020070655-appb-000010

Claims (11)

  1. 一种分离出的或纯化的木霉菌拮抗性表征基因，其特征在于，包括如SEQ ID No.1所示的核苷酸序列。An isolated or purified Trichoderma antagonistic characterization gene, which is characterized by comprising the nucleotide sequence shown in SEQ ID No. 1.
  2. 根据权利要求1所述的分离出的或纯化的木霉菌拮抗性表征基因，其特征在于，所述木霉菌拮抗性表征基因具有保守区段1，如SEQ ID No.3所示的核苷酸序列。The isolated or purified Trichoderma antagonistic characterizing gene according to claim 1, wherein the Trichoderma antagonistic characterizing gene has a conservative segment 1, such as the nucleotide shown in SEQ ID No. 3. sequence.
  3. 根据权利要求1所述的分离出的或纯化的木霉菌拮抗性表征基因，其特征在于，所述木霉菌拮抗性表征基因具有保守区段2，如SEQ ID No.4所示的核苷酸序列。The isolated or purified Trichoderma antagonistic characterizing gene according to claim 1, wherein the Trichoderma antagonistic characterizing gene has a conservative segment 2, such as the nucleotide shown in SEQ ID No. 4 sequence.
  4. 根据权利要求4所述的分离出的或纯化的木霉菌拮抗性表征基因，其特征在于，所述木霉菌拮抗性表征基因具有保守区段3，如SEQ ID No.5所示的核苷酸序列。The isolated or purified Trichoderma antagonistic characterizing gene according to claim 4, wherein the Trichoderma antagonistic characterizing gene has a conservative segment 3, such as the nucleotide shown in SEQ ID No. 5. sequence.
  5. 一种根据权利要求1所述的分离出的或纯化的木霉菌拮抗性表征基因编码的氨基酸序列，其特征在于，包括如SEQ ID No.2所示的氨基酸序列。An amino acid sequence encoded by an isolated or purified Trichoderma antagonistic characterizing gene according to claim 1, characterized in that it comprises the amino acid sequence shown in SEQ ID No.2.
  6. 一种分离出的或纯化的木霉菌拮抗性表征基因在评价木霉菌拮抗性上的应用，其特征在于，包括：将待检测木霉菌株的基因组序列构建成本地核苷酸数据库；然后将权利要求1所述的分离出的或纯化的木霉菌拮抗性基因作为检索序列与所述本地核苷酸数据库进行比对，若两种核苷酸序列的相似性大于90％，则待测木霉菌具有拮抗性。The application of an isolated or purified Trichoderma antagonistic characterization gene in evaluating the antagonism of Trichoderma is characterized in that it comprises: constructing the genome sequence of the Trichoderma strain to be detected into a local nucleotide database; The isolated or purified Trichoderma antagonistic gene described in claim 1 is used as a search sequence for comparison with the local nucleotide database. If the similarity between the two nucleotide sequences is greater than 90%, the Trichoderma to be tested It is antagonistic.
  7. 一种基于基因序列的木霉菌拮抗性评价扩增引物，其特征在于，包括根据权利要求2中所述的保守区段2设计的正向引物如SEQ ID No.6所示，反向引物如SEQ ID No.7所示。A gene sequence-based amplification primer for evaluating the antagonism of Trichoderma, characterized in that it includes a forward primer designed according to the conserved segment 2 described in claim 2 as shown in SEQ ID No. 6, and the reverse primer is as shown in SEQ ID No.7 is shown.
  8. 一种基于基因序列的木霉菌拮抗性评价扩增引物的应用，其特征在于，包括如下步骤：An application of amplification primers for evaluating the antagonism of Trichoderma based on gene sequence, characterized in that it comprises the following steps:
    S1：提取待测的木霉菌真菌样品的DNA，制得基因组DNA；S1: Extract the DNA of the Trichoderma fungus sample to be tested to obtain genomic DNA;
    S2：以步骤S1制得的基因组DNA为模板，利用权利要求7所述的正向引 物和反向引物进行PCR扩增，制得PCR扩增引物；S2: Using the genomic DNA prepared in step S1 as a template, the forward primer and the reverse primer according to claim 7 are used for PCR amplification to prepare PCR amplification primers;
    S3：对步骤S2制得的PCR扩增引物进行长度检测，若检测到扩增至长度为1000-2000bp的核苷酸片段，则待检测的木霉菌评价为拮抗性。S3: Perform length detection on the PCR amplification primer prepared in step S2. If a nucleotide fragment amplified to a length of 1000-2000 bp is detected, the Trichoderma to be detected is evaluated as antagonistic.
  9. 根据权利要求7所述的基于基因序列的木霉菌拮抗性评价扩增引物的应用，其特征在于，所述步骤S1具体为在超净台上，将待测的木霉菌株无菌接种到培养基中，在25-28℃条件在培养2-3天，收集木霉菌菌丝体，然后研磨木霉菌菌丝体，采用植物基因组提取试剂盒提取基因组DNA。The application of amplification primers for evaluating the antagonism of Trichoderma based on gene sequence according to claim 7, characterized in that, the step S1 is specifically inoculating the Trichoderma strain to be tested aseptically on a clean bench. In the base, culture for 2-3 days at 25-28°C, collect the Trichoderma mycelium, then grind the Trichoderma mycelium, and extract the genomic DNA using a plant genome extraction kit.
  10. 根据权利要求7所述的基于基因序列的木霉菌拮抗性评价扩增引物的应用，其特征在于，所述步骤S2中PCR反应体系如下：总体系为10μL，包括：Premix taq酶液5μl，DNA模板90-100ng，正向引物、反向引物的反应液终浓度均为0.2-1.0μmol/L，灭菌水补足10μL；The application of amplification primers for evaluating Trichoderma antagonism based on gene sequence according to claim 7, characterized in that the PCR reaction system in step S2 is as follows: the total system is 10 μL, including: Premix taq enzyme solution 5 μl, DNA The template is 90-100ng, the final concentration of the reaction solution of the forward primer and the reverse primer is 0.2-1.0μmol/L, and sterilized water makes up 10μL;
    PCR反应条件为：94℃预变性10min；94℃维持10s，55℃退火30s，72℃维持3min，30个循环；68℃延伸10min。The PCR reaction conditions were: 94°C pre-denaturation for 10 minutes; 94°C for 10 seconds, 55°C for 30 seconds, 72°C for 3 minutes, 30 cycles, and 68°C for 10 minutes.
  11. 权利要求6所述的基于基因序列的木霉菌拮抗性评价扩增引物在制备评价木霉菌拮抗性的产品中的应用。The use of the amplification primers for evaluating the antagonism of Trichoderma based on the gene sequence of claim 6 in the preparation of products for evaluating the antagonism of Trichoderma.
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