WO2021047032A1 - 芬娜金针菇新菌株的分子标记的引物和分子标记方法 - Google Patents

芬娜金针菇新菌株的分子标记的引物和分子标记方法 Download PDF

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WO2021047032A1
WO2021047032A1 PCT/CN2019/119579 CN2019119579W WO2021047032A1 WO 2021047032 A1 WO2021047032 A1 WO 2021047032A1 CN 2019119579 W CN2019119579 W CN 2019119579W WO 2021047032 A1 WO2021047032 A1 WO 2021047032A1
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flammulina
fennae
new strain
molecular labeling
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French (fr)
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吴清平
胡惠萍
刘远超
谢意珍
张智
吴晓贤
王傲
卓丽君
史钏
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广东省微生物研究所（广东省微生物分析检测中心）
广东粤微生物科技有限公司
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  • the invention relates to a molecular labeling primer and a molecular labeling method of a new strain, in particular to a molecular labeling primer and a molecular labeling method of a new strain of Fenneria Flammulina.
  • Flammulina velutipes is one of the edible fungi cultivated widely at present, and it is also a kind of edible and medicinal fungus with high economic value. Flammulina velutipes contains a variety of nutrients. Among them, the 8 amino acids required for human growth are relatively high. The content of lysine and arginine is particularly rich, which can promote the healthy growth and intellectual development of children, so it is often called "enhancement”. Zhi Mushroom”. Flammulina velutipes contains simple mushrooms, which has significant anti-cancer function. Regular consumption of Flammulina velutipes can also prevent high blood pressure and treat diseases such as hepatitis and gastrointestinal ulcers.
  • the present invention provides a molecular labeling primer and a molecular labeling method for the new strain of Fenner Flammulina velutipes.
  • the present invention provides a molecular marker primer for a new strain of Fenner Flammulina velutipes.
  • the new strain is isolated from the dead wood of broad-leaved trees in Laotudingzi Mountain, Huanren County, Benxi City, Liaoning province, and the original strain is obtained by tissue separation and purification of the fruiting bodies. It was identified as a new strain of Flammulina fennae by morphology and molecular biology, and the strain was named Flammulina fennae HMGIM-A151357, which was deposited to the China Center for Type Culture Collection (CCTCC for short) on June 20, 2019 , Wuhan, China), the deposit number is: CCTCC NO: M 2019478.
  • the hyphae of the strain isolated by the inventors are vigorous, and the fruit bodies have been obtained through artificial domestication and cultivation, and the commercial traits are excellent. And completed the sequencing and assembly of the whole genome of this strain, and through comparison with the genome of edible fungi, it belongs to the species of Flammulina fennae Bas, specifically related to Agaricomycetes from the Fungi phylum Basidiomycota (Agaricomycetes) Agaricales (Agaricales) Physalacriaceae (Physalacriaceae) Flammulina (Flammulina).
  • the Fenner Flammulina velutipes fruit body of the present invention is yellow, the cap is light yellow, in a hemispherical shape, large, not easy to open the umbrella, and has a thin gelatinous skin on the surface; the stalk is slender, light yellow to brown, and the base of the stalk is larger than the stalk.
  • the top color should be dark; the fungus flesh is white and thin; the gills are nearly cream or light yellow, concave or extended.
  • the growth cycle is 60 days to 70 days, and the fruiting management time is 10 days to 13 days.
  • the optimum temperature for mycelium is 22-24°C, primordium formation is 15-16°C, and the optimum temperature for fruit body growth is 16-18°C.
  • the growth rate of mycelium is moderate on ordinary PDA medium, and the hyphae are slightly dense, white to off-white in the early stage, and light yellow in the later stage, and powder spores are produced.
  • the Fenna Flammulina velutipes of the present invention has a richer calcium content, a higher fruiting tolerance temperature, is conducive to energy saving, and the cap is not easy to open the umbrella, has excellent commercial properties, and has low cellulose content. It is not easy to stuff teeth and has a better taste.
  • the molecularly labeled primers of the new strain of Fenna Flammulina fennae HMGIM-A151357 including FAA-F-3 and FAA-R-4, or FAA-F-5 and FAA-R-7;
  • FAA-R-7 5’---CGTCCTATGTCGTAGAGTAGCG---3’.
  • the two pairs of specific nucleotide primer sequences of the present invention combined with conventional PCR and electrophoresis methods can quickly and accurately identify the Fenner Flammulina velutipes species, and are used to distinguish common Flammulina velutipes species on the market.
  • the identification method is simple and has high specificity.
  • Figure 1 shows the ITS sequencing results of the new strain of the present invention in Example 1.
  • Figure 2 shows a different site of Example 2.
  • FIG. 3 is another difference site of Example 2.
  • FIG. 3 is another difference site of Example 2.
  • FIG. 4 is a PCR amplification electrophoresis pattern of the primer pair of Example 3.
  • FIG. 4 is a PCR amplification electrophoresis pattern of the primer pair of Example 3.
  • FIG. 5 is a PCR amplification electrophoresis pattern of another primer pair of Example 3.
  • FIG. 5 is a PCR amplification electrophoresis pattern of another primer pair of Example 3.
  • the present invention provides a new Fenna Flammulina velutipes strain obtained through field collection and isolation, which is identified as a new strain through molecular biological identification and traits, and its whole genome data is determined and compared with the genes of Flammulina velutipes widely used in the market. Design specific primers to complete their molecular markers, realize the protection of their own strains, and provide property rights protection for commercial applications.
  • the original strain is obtained by tissue separation and purification of the fruiting bodies. It is identified as Fenne Flammulina velutipes by morphology and molecular biology
  • the new strain of Flammulina fennae named Flammulina fennae HMGIM-A151357, has been deposited to the China Center for Type Culture Collection (CCTCC, Wuhan, China) on June 20, 2019, and the deposit number is: CCTCC NO: M 2019478.
  • the similarity reached more than 98%, indicating that the two pairs of nucleic acid probe primers can specifically amplify the Fenner Flammulina velutipes strain to distinguish the Flammulina velutipes strains circulating in the market.
  • the molecularly labeled primers of the new strain of Fenna Flammulina fennae HMGIM-A151357 including FAA-F-3 and FAA-R-4, or FAA-F-5 and FAA-R-7;
  • the molecular labeling method of the new strain of Fenna Flammulina (Flammulina fennae HMGIM-A151357), using primer pairs FAA-F-3 and FAA-R-4, or FAA-F-5 and FAA-R-7 for PCR amplification A specific amplified band was obtained, and it was identified as a new strain of Flammulina fennae (Flammulina fennae HMGIM-A151357).
  • the molecular labeling method further includes using primer pairs FAA-F-3 and FAA-R-4, or FAA-F-5 and FAA-R-7 to perform PCR amplification of common Flammulina velutipes, showing no amplified bands, indicating Identification specificity of a new strain of Fenna Flammulina (Flammulina fennae HMGIM-A151357).
  • the molecular labeling method further includes using ITS1/ITS4 as a primer pair to perform control amplification, and the amplified bands indicate the effectiveness of the PCR amplification system.
  • the blast program was used on the NCBI website to compare and analyze this sequence, and the identification result was Flammulina fennae.
  • the purified and separated Fenner Flammulina velutipes strains were inoculated into a 13cm ⁇ 25cm polypropylene bag containing the original seed culture material.
  • the original seed culture material consisted of 98% sorghum grains, 2% calcium carbonate, and cultivated at 24-25°C in the dark.
  • the original seed is obtained after the mycelium is overgrown with the bag. Inoculate the original seed into a polypropylene fruiting bag of the cultivar culture material.
  • the composition of the cultivar culture material is 50% cotton seed hull, 38% sawdust, 10% bran, 2% calcium carbonate, and placed at 24-25°C in air.
  • the above primer pair and PCT system were used for identification.
  • the PCR reaction system was 50 ⁇ L, DNA polymerase mixture (Premix Taq TM , RR003A) 25 ⁇ L, Fenner Flammulina velutipes genomic DNA template 5 ⁇ L, each primer was 5 ⁇ L, ddH 2 O 10 ⁇ L, the control was ordinary Flammulina velutipes genomic DNA is used as a template, and the reaction procedure is shown in Table 1.
  • both Fenna Flammulina and Common Flammulina can amplify bands when using ITS1/ITS4 as primers. Therefore, the amplified products of this primer pair cannot directly distinguish the two types of Flammulina, but they are specific
  • the primers FAA-F-3/FAA-R-4, FAA-F-5/FAA-R-7 can specifically amplify the sequence of Fenner Flammulina velutipes by PCR, and the electropherogram of the PCR product can quickly distinguish between the two Enoki mushroom.
  • the amplified products of these two pairs of specific primers are 620-630bp and 560-570bp, respectively.
  • the combination of the two pairs of specific nucleotide primer sequences of the present invention with conventional PCR and electrophoresis methods can quickly and accurately identify the Fenner Flammulina velutipes species, so as to distinguish common Flammulina velutipes species on the market.

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Abstract

一种芬娜金针菇新菌株的分子标记的引物和分子标记方法。芬娜金针菇Flammulina fennae HMGIM-A151357的分子标记的引物，包括FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7；FAA-F-3：GCATTGGCAAGAGTTTCA，FAA-R-4：AGGCAAGTCATCCGTTTT；FAA-F-5：GCATTTGGACTATTGCCTCA，FAA-R-7：CGTCCTATGTCGTAGAGTAGCG。两对特异性核苷酸引物序列结合常规PCR、电泳方法可以快速准确鉴定此芬娜金针菇菌种，区分市场上的普通金针菇菌种，鉴别方法简单且特异性高。

Description

芬娜金针菇新菌株的分子标记的引物和分子标记方法 技术领域
本发明涉及一种新菌株的分子标记引物和分子标记方法，尤其涉及一种芬娜金针菇新菌株的分子标记的引物和分子标记方法。
背景技术
目前，食用菌的产业发展迅猛，我国食用菌每年的产量达到3000万吨以上，占全球75％以上，从业人员超过2000万人，食用菌产业在种植业中排在除了粮、菜果、油之后的第五位，超过了棉、茶叶和蚕桑。
在食用菌产业蓬勃发展的今天，越来越多的珍稀食用菌品种逐渐进入人们的视野，许多原有的珍稀品种逐渐被驯化，如竹荪、茶薪菇、离褶伞等。但是也有大批的野生食药用菌由于未能被人类认识，没有得到研究。在目前有的15万种大型真菌中，仅仅有80种左右的野生食药用菌被人类驯化，而且规模化栽培的品种更只有20多种。随着人们生活水平的逐步上升，对于生活品质的要求更高，而大型真菌由于其富含具有营养及功能作用的各种成分，包括真菌多糖、三萜类、甾醇等，对于人体健康具有非常良好的作用，日益受到人们的重视。
金针菇(Flammulina velutipes)是目前人工广泛栽培的食用菌之一，也是一种经济价值很高的食药用真菌。金针菇含有多种营养成分，其中人体生长所需的8种氨基酸含量较高，尤以赖氨酸和精氨酸含量特别丰富，能促进儿童的健康成长和智力发育，因此常被称为“增智菇”。金针菇含有朴菇素，具有显著的抗癌功能，经常食用金针菇还可以预防高血压，治疗肝炎及肠胃溃疡等疾病。
基于不同的金针菇菌种，需要进行鉴别区分。然而，操作简单并且鉴别特异性高的鉴别方法是迫切需要的。
发明内容
针对以上不足，本发明提供一种芬娜金针菇新菌株的分子标记的引物和分子标记方法。
本发明提供一种芬娜金针菇新菌株的分子标记的引物，该新菌种分离自辽宁省本溪市桓仁县老秃顶子山阔叶树枯木上，通过对子实体进行组织分离并纯化得到原始菌株，经形态学及分子生物学鉴定为芬娜金针菇Flammulina fennae新菌株，将该菌株命名为芬娜金针菇Flammulina fennae HMGIM-A151357，已于2019年6月20日保藏至中国典型培养物保藏中心(简称CCTCC，中国武汉)，保藏编号为：CCTCC NO：M 2019478。
发明人分离得到的该菌株菌丝活力旺盛，当前已经通过人工驯化栽培获得子实体，商品 性状优良。并且对该菌株完成其全基因组的测序及组装，通过与食用菌基因组比对，属于芬娜金针菇(Flammulina fennae Bas)菌种，具体涉及来自真菌界(Fungi)担子菌门(Basidiomycota)伞菌纲(Agaricomycetes)伞菌目(Agaricales)膨瑚菌科(Physalacriaceae)金针菇属(Flammulina)。
本发明的芬娜金针菇子实体为黄色，菌盖浅黄色，呈半球形，较大，不易开伞，表面有胶质的薄皮；菌柄细长，浅黄色至褐色，菌柄基部比菌柄顶部颜色要深；菌肉白色，较薄；菌褶近奶油色或浅黄色，凹生或延生。生长周期为60天-70天，出菇管理时间为10天-13天。菌丝体最适温度22-24℃，原基形成15-16℃，子实体生长最适温度为16-18℃。菌丝体在普通PDA培养基上生长速度适中，菌丝稍浓密，初期为白色至灰白色，后期呈现出淡黄色，并产生粉孢子。
与普通金针菇相比，本发明的芬娜金针菇具有更为丰富的钙含量，出菇耐受温度更高，有利于节能，并且菌盖不易开伞，商品性状优，纤维素含量低，食用时不易塞牙，口感更优。
芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)的分子标记的引物，包括FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7；
其中，
FAA-F-3：5’---GCATTGGCAAGAGTTTCA---3’
FAA-R-4：5’---AGGCAAGTCATCCGTTTT---3’
FAA-F-5：5’---GCATTTGGACTATTGCCTCA---3’
FAA-R-7：5’---CGTCCTATGTCGTAGAGTAGCG---3’。
本发明两对特异性核苷酸引物序列结合常规PCR、电泳方法可以快速准确鉴定此芬娜金针菇菌种，用以区分市场上的普通金针菇菌种，鉴别方法简单并且特异性高。
附图说明
图1为实施例1的本发明的新菌株的ITS测序结果。
图2为实施例2的一个差异性位点。
图3为实施例2的另一差异性位点。
图4为实施例3的引物对的PCR扩增电泳图谱。
图5为实施例3的另一引物对的PCR扩增电泳图谱。
具体实施方式
本发明提供了一株通过野外采集分离得到的芬娜金针菇新菌株，通过分子生物学鉴定以及性状鉴别为新菌株，测定其全基因组数据并与市场上广泛使用的金针菇菌种基因进行比较 分析，设计特异性引物完成其分子标记，实现自有菌株的保护，为商业化应用提供产权保障。
一种芬娜金针菇新菌株，分离自辽宁省本溪市桓仁县老秃顶子山阔叶树枯木上，通过对子实体进行组织分离并纯化得到原始菌株，经形态学及分子生物学鉴定为芬娜金针菇Flammulina fennae新菌株，将该菌株命名为芬娜金针菇Flammulina fennae HMGIM-A151357，已于2019年6月20日保藏至中国典型培养物保藏中心(简称CCTCC，中国武汉)，保藏编号为：CCTCC NO：M 2019478。
对该菌株完成其全基因组的测序及组装，通过与市场上常见金针菇基因进行比对分析，筛选获得差异基因集，设计特异性引物及PCR扩增，将扩增产物电泳检测并测序。结果表明，本发明的两对核酸探针引物针对该芬娜金针菇菌株可以扩增出特异性条带，而对于普通金针菇则无条带，并且成功扩增出来的条带经送检测序后，与芬娜金针菇基因组比对，相似性达到98％以上，表明这两对核酸探针引物可以特异性扩增芬娜金针菇菌株，用以区别市场上流通的金针菇菌株。
芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)的分子标记的引物，包括FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7；
其中，
FAA-F-3：5’---GCATTGGCAAGAGTTTCA---3’
FAA-R-4：5’---AGGCAAGTCATCCGTTTT---3’
FAA-F-5：5’---GCATTTGGACTATTGCCTCA---3’
FAA-R-7：5’---CGTCCTATGTCGTAGAGTAGCG---3’
芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)的分子标记方法，采用引物对FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7进行PCR扩增，得到特异性扩增条带，鉴定为芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)。
所述分子标记方法进一步包括采用引物对FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7进行普通金针菇的PCR扩增，显示无扩增条带，表明芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)的鉴定特异性。
所述分子标记方法进一步包括采用ITS1/ITS4为引物对进行对照扩增，扩增出条带表明PCR扩增体系的有效性。
以下结合具体实施例对本发明进行进一步说明。
实施例1分子生物学鉴定
分离自辽宁省本溪市桓仁县老秃顶子山阔叶树枯木上的芬娜金针菇新菌株芬娜金针菇 Flammulina fennae HMGIM-A151357，采用Magen(美基)生物HiPure Fungal DNA Mini Kit II真菌基因组DNA抽提试剂盒提取菌丝体DNA，以ITS1(TCCGTAGGTGAACCTGCGG)、ITS4(TCCTCCGCTTATTGATATGC)为引物进行基因组ITS区域PCR试验，将扩增产物送至上海美吉生物测序，得到碱基数为760bp的序列，序列信息如图1所示。
在NCBI网站使用blast程序将此序列比对分析，鉴定结果为Flammulina fennae。
实施例2
人工栽培方法
将纯化分离的芬娜金针菇菌种接种至装有原种培养料的13cm×25cm聚丙烯袋中，原种培养料成分为98％高粱粒，2％碳酸钙，24-25℃避光培养至菌丝长满菌袋后得到原种。将原种接种至栽培种培养料的聚丙烯出菇袋中，栽培种培养料成分为50％棉籽壳，38％木屑，10％麸皮，2％碳酸钙，置于24-25℃、空气相对湿度70％左右的培养室中避光培养至菌丝长满菌袋。然后开袋催蕾，并增加湿度至95％左右，待培养料表面出现水滴后，每天换风2次培养4天左右。然后降温至6℃、空气相对湿度85％左右、光照2h(每天)进行冷刺激，处理时间为4天左右。然后将温度升至18℃，空气相对湿度为85％左右，无光照，并控制二氧化碳浓度小于5％，等待幼菇长高至10cm以上、菌盖开始平展时即可采收。
实施例3特异性引物设计
通过将此芬娜金针菇菌株的编码蛋白质序列(Coding sequence,简称CDs序列，下同)与普通金针菇CDs序列进行比对，确定了差异基因，通过筛选，确定该菌株的gene02699基因与普通金针菇基因相似性仅为75％，具体差异位置如图2所示。
通过筛选，确定该菌株的gene11357基因与普通金针菇基因相似性仅为76％，具体差异位置图如图3所示。
根据图2和图3的差异位点，设计了2对特异性引物对，分别为引物对FAA-F-3和FAA-R-4，引物对FAA-F-5和FAA-R-7，具体的序列信息如下：
FAA-F-3：5’---GCATTGGCAAGAGTTTCA---3’
FAA-R-4：5’---AGGCAAGTCATCCGTTTT---3’
FAA-F-5：5’---GCATTTGGACTATTGCCTCA---3’
FAA-R-7：5’---CGTCCTATGTCGTAGAGTAGCG---3’
实施例4特异性引物验证
采用上述引物对及PCT体系进行鉴定，PCR反应体系为50μL，DNA聚合酶混合液(Premix Taq TM，RR003A)25μL，芬娜金针菇基因组DNA模板5μL，引物各5μL，ddH 2O10 μL，对照以普通金针菇基因组DNA为模板，反应程序表1所示。
表1：反应程序
Figure PCTCN2019119579-appb-000001
PCR反应结束后，将产物及DNA Marker分别上样置于2％琼脂糖凝胶中，120V，电泳25min，然后取出凝胶，荧光下观察、拍照，如图4和图5所示。
从图4和图5可以看出，芬娜金针菇和普通金针菇在以ITS1/ITS4为引物时，均可以扩增出条带，因此该引物对扩增的产物不能直接区分两种金针菇，但是特异性引物FAA-F-3/FAA-R-4、FAA-F-5/FAA-R-7能够通过PCR专一性地扩增出芬娜金针菇序列，且PCR产物电泳图可以快速区分两种金针菇。这两对特异性引物扩增产物大小分别为620-630bp、560-570bp。
因此，本发明两对特异性核苷酸引物序列结合常规PCR、电泳方法可以快速准确鉴定此芬娜金针菇菌种，用以区分市场上的普通金针菇菌种。
以上所述，仅为本发明的较佳的具体实施例，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其构思加以等同替换或改变，都应涵盖在本发明的保护范围内。

Claims (4)

  1. 芬娜金针菇新菌株的分子标记的引物，所述芬娜金针菇新菌株为芬娜金针菇Flammulina fennae HMGIM-A151357，保藏编号为：CCTCC NO：M 2019478，其特征在于，所述芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)的分子标记的引物，包括FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7；
    其中，
    FAA-F-3：5’---GCATTGGCAAGAGTTTCA---3’
    FAA-R-4：5’---AGGCAAGTCATCCGTTTT---3’
    FAA-F-5：5’---GCATTTGGACTATTGCCTCA---3’
    FAA-R-7：5’---CGTCCTATGTCGTAGAGTAGCG---3’。
  2. 芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)的分子标记方法，其特征在于，采用引物对FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7进行PCR扩增，得到特异性扩增条带，鉴定为芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)。
  3. 根据权利要求2所述的分子标记方法，其特征在于，所述分子标记方法进一步包括采用引物对FAA-F-3和FAA-R-4，或FAA-F-5和FAA-R-7进行普通金针菇的PCR扩增，显示无扩增条带，表明芬娜金针菇新菌株(芬娜金针菇Flammulina fennae HMGIM-A151357)的鉴定特异性。
  4. 根据权利要求2或3所述的分子标记方法，其特征在于，所述分子标记方法进一步包括采用ITS1/ITS4为引物对进行对照扩增，扩增出条带表明PCR扩增体系的有效性。
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