WO2023087789A1 - 一种用于筛选抗氧化活性高的黄绿卷毛菇的dna条形码 - Google Patents

一种用于筛选抗氧化活性高的黄绿卷毛菇的dna条形码 Download PDF

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WO2023087789A1
WO2023087789A1 PCT/CN2022/109954 CN2022109954W WO2023087789A1 WO 2023087789 A1 WO2023087789 A1 WO 2023087789A1 CN 2022109954 W CN2022109954 W CN 2022109954W WO 2023087789 A1 WO2023087789 A1 WO 2023087789A1
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fragment containing
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repeat elements
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杨满军
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杨满军
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  • the invention relates to the technical field of edible fungus germplasm resource screening, in particular to a DNA barcode for screening yellow-green mushrooms with high antioxidant activity.
  • Yellow-green mushrooms are mainly distributed in the Qinghai-Tibet Plateau.
  • the main production areas are Damxung County in the Cambodia Autonomous Region, Qilian County in Qinghai City, and Shiqu County in Sichuan province. The quality of these three main production areas is also the best.
  • Yellow-green mushroom is a high-quality edible fungus with unique flavor, which cannot be artificially cultivated at present.
  • the main indicators for evaluating the nutritional value, flavor and biological activity of the yellow-green mushroom include: high content of total soluble protein, total soluble amino acid, total polyphenol, total polysaccharide and total fat, and strong antioxidant activity.
  • DNA barcode molecular identification technology is a molecular biology technique based on DNA barcode (conserved and stable genetic DNA sequence in the genome) to identify species and good quality. It is an effective supplement and expansion of traditional breeding methods, and it can accurately and effectively identify samples when their morphology is incomplete or lacks morphological structure (processed products such as powder, etc.).
  • ITS ribosomal RNA internal transcriptional spacer
  • RFLP restriction fragment length polymorphism
  • RAPD random amplified polymorphic DNA
  • the present invention provides a DNA barcode used for screening the yellow-green mushrooms with high antioxidant activity.
  • a DNA barcode for screening antioxidant activity indicators of Pleurotus pilosula includes:
  • SEQ ID NO: 17 SEQ ID NO: 18 and the combination of SEQ ID NO: 19 and SEQ ID NO: 20.
  • the present invention is based on the fluorescent PCR amplification of all simple sequence repeats (simple sequence repeat, SSR) in the whole genome of Pleurotus volvulus, and establishes a DNA barcode effectively corresponding to the antioxidant activity, and the amplified fragments are consistent with the DNA barcode of the present invention.
  • SSR simple sequence repeat
  • Another object of the present invention is to provide a primer set that can amplify the DNA barcode for the above-mentioned screening of the antioxidant activity index of Pleurotus chinensis, the nucleotide sequence of the primer set includes:
  • the nucleotide sequence of the primer set includes: such as SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 15 and SEQ ID NO: 16.
  • Different primer sets of the present invention can be used alone or in combination to screen the antioxidant activity of Pleurotus chinensis, and when all the primer sets are used together, the screening accuracy is the highest.
  • Another object of the present invention is to provide a method for screening yellow-green mushrooms with antioxidant activity indicators, including the following steps:
  • S2 Using S1 genomic DNA as a template, the above-mentioned one or more sets of primers are respectively subjected to fluorescent PCR amplification reactions to obtain amplification products;
  • the amplified products described in S3 and S2 are detected by capillary fluorescence electrophoresis, and determined by the number of fragments of the amplified product, the number of SSR sites, the SSR repeat elements and the number of repeats thereof.
  • the criteria for determining step S3 are:
  • the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 amplified to obtain a 256bp fragment containing 13 GAG repeat elements and a 274bp fragment containing 19 GAG repeat elements;
  • the primer set of SEQ ID NO: 5 and SEQ ID NO: 6 amplified to obtain a 257bp fragment containing 5 CAG repeat elements and a 266bp fragment containing 8 CAG repeat elements;
  • the primer set of SEQ ID NO: 10 and SEQ ID NO: 11 amplified to obtain a 254bp fragment containing 19 AT repeat elements and a 256bp fragment containing 20 AT repeat elements;
  • the reaction system of step S2 fluorescent PCR amplification reaction is:
  • the concentrations of the upstream primer, the downstream primer and the fluorescent M13 primer are all 10uM.
  • the fluorescent PCR amplification reaction procedure of step S2 is:
  • Another object of the present invention is to provide the application of the above-mentioned DNA barcode and/or the above-mentioned primer set in the preparation of products for screening Pleurotus volvulus with antioxidant activity indicators.
  • Another object of the present invention is to provide a product for screening high-quality yellow-green mushrooms with antioxidant activity indicators, which contains the above-mentioned one or more sets of primers, and meets the standards:
  • the primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 amplified to obtain a 256bp fragment containing 13 GAG repeat elements and a 274bp fragment containing 19 GAG repeat elements;
  • the primer set of SEQ ID NO: 5 and SEQ ID NO: 6 amplified to obtain a 257bp fragment containing 5 CAG repeat elements and a 266bp fragment containing 8 CAG repeat elements;
  • the primer set of SEQ ID NO: 10 and SEQ ID NO: 11 amplified to obtain a 254bp fragment containing 19 AT repeat elements and a 256bp fragment containing 20 AT repeat elements;
  • one or more of the 282bp fragment containing 10 GCT repeat elements and the 285bp fragment containing 11 GCT repeat elements are amplified by the primer set of SEQ ID NO: 15 and SEQ ID NO: 16.
  • the product is a kit.
  • the present invention discloses a DNA barcode for screening yellow-green mushrooms with high antioxidant activity. Compared with the prior art, the present invention can accurately and quickly
  • the DNA barcoding technology for identifying the strains of Pleurotus pilosula and realizing high-quality breeding at the same time has the characteristics of low cost, high efficiency, easy operation, stable and reliable results, and good repeatability.
  • the present invention Compared with the traditional breeding method and other existing DNA barcode technologies, the present invention has the advantages of saving time, effort, money, accuracy and high efficiency, and plays an active role in the identification of the origin of high-quality yellow-green mushrooms and genetic breeding. It also provides an effective method for the identification and protection of germplasm resources.
  • Figure 1 is a diagram showing the comparative results of the antioxidant activity content of Examples of the present invention, Comparative Examples 1 and 2, wherein from left to right are Comparative Example 1, Comparative Example 2 and Examples.
  • FIG. 2 is a diagram showing the comparative results of PCR amplification of Comparative Examples 1 and 2 and Examples using primer 1 according to the present invention.
  • Figure 3 is a diagram showing the comparison results of Comparative Examples 1 and 2 and Examples using primer 2 PCR amplification in the present invention.
  • Fig. 4 is a diagram showing the comparative results of Comparative Examples 1 and 2 and Examples using primer 3 PCR amplification in the present invention.
  • Fig. 5 is a diagram showing comparison results of comparative examples 1 and 2 and examples of PCR amplification using primer 4 according to the present invention.
  • the embodiment of the present invention discloses a DNA barcode for screening the yellow-green mushroom with high antioxidant activity.
  • Genome sequencing was carried out on the samples of Dangxiong County in Cambodia Autonomous Region, Qilian County in Qinghai City, and Shiqu County in Sichuan province.
  • the SSR loci in the genome sequences were analyzed using the MISA program.
  • the samples from the above three origins were respectively amplified using effective primers and detected by capillary electrophoresis.
  • the simple sequence repeat (SSR) site corresponding to the antioxidant activity was established through analysis.
  • 4 pairs of primers were obtained (see Table 1), and the fragment polymorphism obtained by using these 4 pairs of primers to amplify the sample genome can assist in the screening of strains with good antioxidant activity.
  • Comparative Example 1 Samples from Qilian County, Qinghai City (the processing method is the same as above).
  • Comparative Example 2 Samples from Shiqu County, Sichuan City (the processing method is the same as above).
  • the antioxidant activity of the extract was determined by 1,1-diphenyl-2-trinitrophenylhydrazine ethanol solution.
  • 1,1-diphenyl-2-trinitrophenylhydrazine (1,1-diphenyl-2-picrylhydrazyl, DPPH) is often used to determine the total antioxidant activity of food, expressed as a clearance percentage.
  • the determination method is such as Tian Pingping (Chinese Agricultural Sciences, 2016, 49(3): 543-553), expressed as the percentage of scavenging DPPH free radicals.
  • the DPPH free radical scavenging rate of samples from Qilian County, Qinghai province was 81.3% ( ⁇ 0.59%)
  • the DPPH free radical scavenging rate of samples from Shiqu County, Sichuan province was 71.53% ( ⁇ 0.95%)
  • the DPPH free radical scavenging rate of samples from Damxung County, Cambodia Autonomous Region was 81.3% ( ⁇ 0.59%).
  • the rate was 91.18% ( ⁇ 0.39%).
  • the sample from Qilian County, Qinghai province was determined as Comparative Example 1
  • the sample from Shiqu County, Sichuan province was determined as Comparative Example 2
  • the sample from Damxung County, Cambodia Autonomous Region was determined as a test example (see accompanying drawing 1).
  • Fluorescent PCR amplification reaction system (10 ⁇ L): 2 ⁇ Taq PCR MasterMix 5 ⁇ L, template (genomic DNA) 1 ⁇ L, upstream primer 0.1 ⁇ L, downstream primer 0.4 ⁇ L (concentration of both upstream and downstream primers is 10 uM), fluorescent M13 primer (concentration 10uM) 0.4 ⁇ L, dilute to 10 ⁇ L with sterile deionized water;
  • Reaction conditions pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 s, drop PCR annealing at 62 to 55°C for 30 s, extension at 72°C for 30 s, a total of 10 cycles; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s, a total of 25 cycles 72°C final extension for 20min; 4°C incubation for 6h for fluorescence capillary electrophoresis detection.
  • the internal standard is LIZ-500 Molecular weight internal standard (also known as molecular weight internal control, internal lane standards) is composed of 16 double-stranded DNA fragments labeled with LIZ fluorescein (orange), and the molecular weights are: 35, 50, 75, 100 , 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500bp.
  • the size of the fragment in the amplification result electrophoresis is equal to the actual bp number of the amplified fragment plus the M13 fluorescent primer (about 18bp), the error range is 1-2bp, the peak number of the amplified capillary electrophoresis combined with the sequencing result indicates that the gene heterozygote is amplified number of fragments.
  • the amplification result of primer 1 is shown in Figure 2.
  • primer 1 was used for fluorescent PCR amplification
  • 2 fragments (2 peaks) were amplified, containing 2 SSR sites, and the SSR repeating element was GAG.
  • the characteristic information of the amplified fragment obtained in the test example is that the amplified fragment is a 256bp fragment containing 13 repeated GAGs and a 274bp fragment containing 19 repeated GAGs.
  • Primer 1 amplified fragment (The statistical fragment length of the electropherogram includes the M13 fluorescent primer. The specific sequence display removes the M13 fluorescent primer sequence (19bp), the error is 1bp, and the underlined part is the SSR repeat element.)
  • the amplification result of primer 2 is shown in Figure 3.
  • 3 fragments (3 peaks) were amplified, containing 3 SSR sites, and the SSR repeating element was CAG.
  • the characteristic information of the amplified fragments obtained in the test example are 257bp and 266bp fragments of 5 and 8 repetitions of CAG respectively.
  • Primer 2 amplified fragment (wherein the statistical fragment length of the electropherogram includes the M13 fluorescent primer, the specific sequence display removes the M13 fluorescent primer sequence (18bp), the 263bp amplified fragment electropherogram statistical fragment includes the M13 fluorescent primer sequence (17bp), The error is 1bp, the underlined part is the SSR repeat element.)
  • the amplification result of primer 3 is shown in Figure 4.
  • 3 fragments (3 peaks) were amplified, containing 3 SSR sites, and the SSR repeating element was AT.
  • the characteristic information of the amplified fragment obtained in the test example is 254bp and 256bp fragments of 19 and 20 repeated ATs respectively.
  • Primer 3 amplified fragment (The statistical fragment length of the electropherogram includes the M13 fluorescent primer, the specific sequence shows that the M13 fluorescent primer sequence (18bp) is removed, and the underlined part is the SSR repeat element.)
  • the amplification result of primer 4 is shown in Figure 5.
  • 4 fragments (4 peaks) were amplified, containing 4 SSR sites, and the SSR repeating element was ATG.
  • the characteristic information of the amplified fragment obtained in the test example is a 282bp fragment and a 285bp fragment containing 10 and 11 repeated GCTs respectively.
  • Primer 4 amplified fragment (The statistical fragment length of the electropherogram includes the M13 fluorescent primer, the specific sequence display removes the M13 fluorescent primer sequence (18bp), and the underlined part is the SSR repeat element.)
  • Primer 1 amplifies a 256bp fragment containing 13 GAG repeat elements (as shown in SEQ ID NO: 3) and a 274bp fragment containing 19 GAG repeat elements (as shown in SEQ ID NO: 4), and primer 2 amplifies A 257bp fragment containing 5 CAG repeat elements (as shown in SEQ ID NO: 7) and a 266bp fragment containing 8 CAG repeat elements (as shown in SEQ ID NO: 9); Primer 3 amplifies 19 AT repeats The 254bp fragment (as shown in SEQ ID NO: 13) of element) and the 256bp fragment (as shown in SEQ ID NO: 14) containing 20 AT repeat elements; Primer 4 amplifies the 282bp fragment containing 10 GCT repeat elements (as shown in SEQ ID NO: 19) and a 285bp fragment (as shown in SEQ ID NO: 20) containing 11 GCT repeat elements.
  • primers 1, 2, 3, and 4 were used for comprehensive detection and judgment
  • the second step test using primers (SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 15 and SEQ ID NO: 16) was amplified and subjected to capillary electrophoresis.
  • the primer set can be amplified using one or more pairs of combinations to distinguish the blind test sample from the DNA barcode characteristics of the test example;
  • the third step was to unblind, and the results are shown in Table 3.
  • the results of unblinding of 48 samples were all correct based on the barcode characteristics of antioxidant activity to distinguish the antioxidant activity. This shows that the DNA barcode of antioxidant activity is suitable for the screening of antioxidant activity traits.

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Abstract

本发明公开了一种用于筛选抗氧化活性高的黄绿卷毛菇的DNA条形码，属于食用菌种质资源筛选技术领域。本发明具有省时、省力、省钱、准确、高效的优点，在优质黄绿卷毛菇的遗传育种上发挥积极作用，同时也为种质资源的鉴定及保护提供了一种有效方法。

Description

一种用于筛选抗氧化活性高的黄绿卷毛菇的DNA条形码 技术领域
本发明涉及食用菌种质资源筛选技术领域，更具体的说是涉及一种用于筛选抗氧化活性高的黄绿卷毛菇的DNA条形码。
背景技术
黄绿卷毛菇，色泽呈金黄色，又称为黄蘑菇、金蘑菇。黄绿卷毛菇主要分布于青藏高原，主产区为西藏自治区当雄县、青海省祁连县以及四川省石渠县等，也以这三个主产区的品质最佳。黄绿卷毛菇是一种风味独特的优质食用菌，当前不能人工栽培。评价黄绿卷毛菇营养价值风味及生物学活性的主要指标包括：总可溶性蛋白、总可溶性氨基酸、总多酚、总多糖和总脂肪含量高，抗氧化活性强。按传统方法，要筛选出优质菌株十分困难，此外，由于其所分布的主产区海拔较高，样本采集也十分困难。为了实现黄绿卷毛菇的开发利用，利用DNA条形码分子鉴定技术辅助筛选优质黄绿卷毛菇菌株显得尤为重要和迫切。黄绿卷毛菇不同产地具有不同营养价值，不同风味，不同生物学活性，不同市场价格。以往对黄绿卷毛菇的选育主要利用形态学方法结合上述有益指标测定进行。
但是受到特殊的青藏高气候原环境的影响不同地区所产的黄绿卷毛菇常常出现同名异物和同物异名的现象，因此形态学鉴别法难以有效区分。更为困难的是，不能通过形态学方法来筛选出总可溶性蛋白、总可溶性氨基酸、总多酚、总多糖和总脂肪含量高，抗氧化活性强的优质菌株。DNA条形码分子鉴定技术是基于DNA条形码(基因组中保守且稳定遗传DNA序列)来进行物种和优良品质识别鉴定的分子生物学技术。它是传统育种方法的有效补充和拓展，能够在样品形态不完整或缺乏形态结构(加工制品如粉末等)时对样品进行精准和有效地鉴定。
现有的DNA条形码技术中，ITS(核糖体RNA内转录间隔区)和线粒体体中的非编码区或保守基因序列主要用于物种物鉴定；限制性片段长度多态性(restriction fragment length polymorphism，RFLP)操作十分繁复，结果的可靠性和可重复性较差，随机扩增多态性DNA(random amplified polymorphic DNA，RAPD)易受干扰，对操作者技术水平要求较高，在辅助育种工作中难以推广；单核苷酸多态性(single nucleotide polymorphism，SNP)对设备要求高，成本也高。
因此针对传统育种方法选育黄绿卷毛菇菌株不够准确费时费力的缺点，如何提供一种可以准确、快捷鉴别黄绿卷毛菇的所属菌株，同时实现优质品质选育的DNA条形码，具有成本低，效率高，操作简便，结果稳定可靠性重复性好的特点是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种用于筛选抗氧化活性高的黄绿卷毛菇的DNA条形码。
为了实现上述目的，本发明采用如下技术方案：
一种筛选黄绿卷毛菇抗氧化活性指标的DNA条形码，DNA条形码的核苷酸序列包括：
如SEQ ID NO：3；
和/或SEQ ID NO：4；
和/或SEQ ID NO：3和SEQ ID NO：4组合；
和/或SEQ ID NO：9；
和/或SEQ ID NO：7和SEQ ID NO：8组合；
和/或SEQ ID NO：7和SEQ ID NO：9组合；
和/或SEQ ID NO：12；
和/或SEQ ID NO：13；
和/或SEQ ID NO：14；
和/或SEQ ID NO：13和SEQ ID NO：14组合；
和/或SEQ ID NO：17；
和/或SEQ ID NO：18；
和/或SEQ ID NO：17和SEQ ID NO：18组合；
和/或SEQ ID NO：19和SEQ ID NO：20组合；
和/或SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19和SEQ ID NO：20组合中的一种或多种。
本发明基于黄绿卷毛菇全基因组中所有简单重复序列(simple sequence repeat，SSR)进行荧光PCR扩增，确立了与抗氧化活性有效对应的DNA条形码，扩增所得片段与本发明的DNA条形码进行比对，可以快速、准确地筛选出黄绿卷毛菇抗氧化活性高的菌株，为黄绿卷毛菇的育种提供有利辅助。
本发明的又一目的是，提供可扩增上述筛选黄绿卷毛菇抗氧化活性指标的DNA条形码的引物组，引物组的核苷酸序列包括：
如SEQ ID NO：1和SEQ ID NO：2，
和/或SEQ ID NO：5和SEQ ID NO：6，
和/或SEQ ID NO：10和SEQ ID NO：11，
和/或SEQ ID NO：15和SEQ ID NO：16中的一组或多组。
作为本发明优选的技术方案，引物组的核苷酸序列包括：如SEQ ID NO：1和SEQ ID NO：2、SEQ ID NO：5和SEQ ID NO：6、SEQ ID NO：10和SEQ ID NO：11、SEQ ID NO：15和SEQ ID NO：16。
本发明不同的引物组可以单独或组合使用筛选黄绿卷毛菇的抗氧化活性，当所有引物组共同使用时，筛选的准确率最高。
本发明的再一目的是，提供一种以抗氧化活性指标筛选黄绿卷毛菇的方法，包括如下步骤：
S1、提取待测样品基因组DNA；
S2、以S1基因组DNA为模板，上述的一组或多组引物分别进行荧光PCR扩增反应，得扩增产物；
S3、S2所述扩增产物经毛细管荧光电泳检测，通过扩增产物的片段数、SSR位点数、SSR重复元件及其重复次数进行判定。
作为本发明优选的技术方案，所述步骤S3的判定标准为：
SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含13次GAG重复元件的256bp片段和含19次GAG重复元件的274bp片段；
和/或SEQ ID NO：5和SEQ ID NO：6引物组扩增得到含5次CAG重复元件的257bp片段和含8次CAG重复元件的266bp片段；
和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含19次AT重复元件的254bp片段和含20次AT重复元件的256bp片段；
和/或SEQ ID NO：15和SEQ ID NO：16引物组扩增得到含10次GCT重复元件的282bp片段和含11次GCT重复元件的285bp片段时，判定该黄绿卷毛菇为抗氧化活性高的黄绿卷毛菇。
作为本发明优选的技术方案，步骤S2荧光PCR扩增反应的反应体系为：
2×Taq PCRMasterMix 5μL，基因组DNA 1μL，上游引物0.1μL，下游引物0.4μL，带荧光的M13引物0.4μL，用无菌去离子水定容至10μL。
更优选的，上游引物、下游引物和带荧光的M13引物浓度均为10uM。
作为本发明优选的技术方案，步骤S2荧光PCR扩增反应程序为：
95℃预变性3min；95℃变性30s，62至55℃降落PCR退火30s，72℃延伸30s，共10个循环；95℃变性30s，52℃退火30s，72℃延伸30s，共25个循环；72℃终延伸20min；4℃保温6h后用于荧光毛细管电泳检测。
本发明的再一目的是，提供上述DNA条形码和/或上述引物组在制备以抗氧化活性指标筛选黄绿卷毛菇的产品中的应用。
本发明的再一目的是，提供一种以抗氧化活性指标筛选优质黄绿卷毛菇的产品，含有上述的一组或多组引物组，且符合标准：
SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含13次GAG重复元件的256bp片段和含19次GAG重复元件的274bp片段；
和/或SEQ ID NO：5和SEQ ID NO：6引物组扩增得到含5次CAG重复元件的257bp片段和含8次CAG重复元件的266bp片段；
和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含19次AT重复元件的254bp片段和含20次AT重复元件的256bp片段；
和/或SEQ ID NO：15和SEQ ID NO：16引物组扩增得到含10次GCT重复元件的282bp片段和含11次GCT重复元件的285bp片段中的一个或多个。
作为本发明优选的技术方案，产品为试剂盒。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种用于筛选抗氧化活性高的黄绿卷毛菇的DNA条形码，相较于现有技术本发明可以准确、快捷鉴别黄绿卷毛菇菌株，同时实现优质品质选育的DNA条形码技术，具有成本低，效率高，操作简便，结果稳定可靠性重复性好的特点。
本发明与传统育种方法及其他现有DNA条形码技术相比较，它具有省时、省力、省钱、准确、高效的优点，在优质黄绿卷毛菇原产地鉴别和遗传育种上发挥积极作用，同时也为种质资源的鉴定及保护提供了一种有效方法。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明实施例、对比例1和2的抗氧化活性含量对比结果图，其中由左至右依次为对比例1、对比例2和实施例。
图2附图为本发明利用引物1PCR扩增对比例1、2和实施例对比结果图。
图3附图为本发明利用引物2PCR扩增对比例1、2和实施例对比结果图。
图4附图为本发明利用引物3PCR扩增对比例1、2和实施例对比结果图。
图5附图为本发明利用引物4PCR扩增对比例1、2和实施例对比结果图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例公开了一种用于筛选抗氧化活性高的黄绿卷毛菇的DNA条形码。
实施例1
黄绿卷毛菇DNA条形码的构建
采集西藏自治区当雄县、青海省祁连县、四川省石渠县的黄绿卷毛菇样品进行基因组测序，使用MISA程序对基因组序列中的SSR位点进行分析。
设计引物对这些SSR位点进行PCR扩增，保留能扩增出对应片段的引物，舍弃无效引物。
选取西藏自治区当雄县、青海省祁连县、四川省石渠县的黄绿卷毛菇样品测定抗氧化活性。
利用有效引物对上述三个产地样品分别扩增并通过毛细管电泳检测。经分析建立抗氧化活性对应的简单重复序列(simple sequence repeat，SSR)位点。最终获得4对引物(见表1)，利用这4对引物对样品基因组进行扩增所得片段多态性可辅助筛选抗氧化活性好的黄绿卷毛菇菌株。
表1 黄绿卷毛菇抗氧化活性好的菌株筛选特异引物
Figure PCTCN2022109954-appb-000001
实施例2
黄绿卷毛菇高抗氧化活性菌株SSR特异引物扩增
(1)抗氧化活性测定
以西藏自治区当雄县样品为本发明试验例，将子实体样品冻干粉碎过50目筛，以1克干粉加20mL双蒸水，用300W超声波辅助提取30min，然后5000转每分钟离心30min后取上清液制备成水提取液。
对比例1：青海省祁连县样品(处理方法同上)。
对比例2：四川省石渠县样品(处理方法同上)。
提取物抗氧化活性测定用1,1-二苯基-2-三硝基苯肼乙醇溶液测定。1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl，DPPH)常用于测定食品的总抗氧化活性，用清除百分比表示。测定方法如田平平等(中国农业科学,2016,49(3):543-553)，以清除DPPH自由基百分率表示。
其中青海省祁连县样品DPPH自由基清除率为81.3％(±0.59％)，四川省石渠县样品DPPH自由基清除率为71.53％(±0.95％)，西藏自治区当雄县样品DPPH自由基清除率为91.18％(±0.39％)，青海省祁连县样品确定为对比例1，四川省石渠县样品确定为对比例2，西藏自治区当雄县样品确定为试验例(参见附图1)。
(2)利用生工生物工程(上海)有限公司Ezup柱式真菌基因组DNA抽提试剂盒(货号B518259)提取黄绿卷毛菇样品基因组，稀释至20ng/μL用于荧光PCR扩增。
(3)利用表1中引物进行荧光PCR扩增SSR DNA条形码。
荧光PCR扩增反应体系(10μL)：2×Taq PCR MasterMix 5μL，模板(基因组DNA)1μL，上游引物0.1μL，下游引物0.4μL(上下游引物浓度均为10uM)，带荧光的M13引物(浓度10uM)0.4μL，用无菌的去离子水定容至10μL；
反应条件：95℃预变性3min；95℃变性30s，62至55℃降落PCR退火30s，72℃延伸30s，共10个循环；95℃变性30s，52℃退火30s，72℃延伸30s，共25个循环；72℃终延伸20min；4℃保温6h后用于荧光毛细管电泳检测。
(4)将PCR产物进行定量稀释后，取1μL PCR稀释产物加9μL甲酰胺(含1％内标)变性后上DNA测序仪ABI 3730xl进行毛细管荧光电泳检测。内标为LIZ-500分子量内标(又称分子量内对照，internal lane standards)由16条带有LIZ荧光素(橙色)标记的双链DNA片段组成，分子量分别是：35、50、75、100、139、150、160、200、250、300、340、350、400、450、490和500bp。扩增结果电泳图中片段大小等于所扩增片段实际bp数加上M13荧光引物(约18bp)，误差范围1-2bp，扩增毛细管电泳峰结合测序结果，峰数量表示该基因杂合子扩增片段数量。
(5)采用以上方法对试验例、对比例1和对比例2的黄绿卷毛菇进行鉴定。
引物1扩增结果如附图2所示，当使用引物1进行荧光PCR扩增时，扩增得到2个片段(2个峰)，含有2个SSR位点，SSR重复元件为GAG。其中试验例所得扩增片段的特征信息为扩增片段为含13次重复GAG的256bp片段和含19次重复GAG的274片段。
引物1扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(19bp)，误差为1bp，下划线部分为SSR重复元件。)
256bp扩增片段序列：
TGTCGCTGAAGTGAAAGGCTCTGTGAGTAGATGTGAGCCGACAGAGAGATATACCGCATACTTTAGTTGTGTATGAGTGGAACCAAATAGCTGCCTCTGATGAAGTGTTTTGAGTCGTTTAGATGGTATGGGTGAGGGTGATGATGAA GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGATGAGACGGATGACGAAGAATCAGAATCAGAGTCCGAAGTGTTAGACTCGTCCGAAGTGTCAGGTGAA，如SEQ ID No.3；
274bp扩增片段序列：
TGTCGCTGAAGTGAAAGGCTCTGTGAGTAGATGTGAGCCGACAGAGAGATATACCGCATACTTTAGTTGTGTATGAGTGGAACCAAATAGCTGCCTCTGATGAAGTGTTTTGAGTCGTTTAGATGGTATGGGTGAGGGTGATGATGAA GAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAGGATGAGACGGATGACGAAGAATCAGAATCAGAGTCCGAAGTGTTAGACTCGTCCGAAGTGTCAGGTGAA，如SEQ ID No.4；
引物2扩增结果如附图3所示，当使用引物2进行荧光PCR扩增时，扩增得到3个片段(3个峰)，含有3个SSR位点，SSR重复元件为CAG。其中试验例所得扩增片段的特征信息为分别5和8次重复CAG的257bp和266bp片段。
引物2扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(18bp)，263bp的扩增片段电泳图统计片段包括M13荧光引物序列(17bp)，误差为1bp，下划线部分为SSR重复元件。)
257bp扩增片段序列：
AGCGATGCAACAACAACGTCAACATGAGCAGCAACAGCAACAACAGCAACAACAGCAACAGCAACAGGCACAACAAGGTCAAATGCATCGTACAGTAGGTCCTAGTGGTATTGCAATTGGTAATGCACAGTTAGCGGCTATGCAACAACATCAGCAGCAGCAACAGCAGCAACATCAACACCAAC AGC AGCAGCAGCAGCAACACCAACAGCATCTCTCGCAGCAACAAGGAATGGGCGGAATGGGAATGGGTGGAA，如SEQ ID No.7；
263bp扩增片段序列：
AGCGATGCAACAACAACGTCAACATGAGCAGCAACAGCAACAACAGCAACAACAGCAACAGCAACAGGCACAACAAGGTCAAATGCATCGTACAGTAGGTCCTAGTGGTATTGCAATTGGTAATGCACAGTTAGCGGCTATGCAACAACATCAGCAGCAGCAACAGCAGCAACATCAACACCAAC AGC AGCAGCAGCAGCAGCAGCAACACCAACAGCATCTCTCGCAGCAACAAGGAATGGGCGGAATGGGAATGGGTGGAA，如SEQ ID No.8；
266bp扩增片段序列：
AGCGATGCAACAACAACGTCAACATGAGCAGCAACAGCAACAACAGCAACAACAGCAACAGCAACAGGCACAACAAGGTCAAATGCATCGTACAGTAGGTCCTAGTGGTATTGCAATTGGTAATGCACAGTTAGCGGCTATGCAACAACATCAGCAGCAGCAACAGCAGCAACATCAACACCAAC AGC AGCAGCAGCAGCAGCAGCAGCAACACCAACAGCATCTCTCGCAGCAACAAGGAATGGGCGGAATGGGAATGGGTGGAA，如SEQ ID No.9；
引物3扩增结果如附图4所示，当使用引物3进行荧光PCR扩增时，扩增得到3个片段(3个峰)，含有3个SSR位点，SSR重复元件为AT。其中 试验例所得扩增片段的特征信息为分别19和20次重复AT的254bp和256bp片段。
引物3扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(18bp)，下划线部分为SSR重复元件。)
236bp扩增片段序列：
AACCGTTTGTCCTTGCCGTACTTCCGAGTCTACTTCGTGCAAATGCCTCGAATGACTTTCATTTA ATATATATATATATATATATCCCGAGAAAATATAAAACTGCAAGCATTGGCTTGCATCCAGTCGGCTGTTCATGGTACATACAAATTGATTTATATAGATTGGCCAGTCAATGTGTCTAATATTGAAAACCCGGAAAAATTCCACAATGTAAAGAAACGATCCAGGCGTCC，如SEQ ID No.12；
254bp扩增片段序列：
AACCGTTTGTCCTTGCCGTACTTCCGAGTCTACTTCGTGCAAATGCCTCGAATGACTTTCATTTA ATATATATATATATATATATATATATATATA TATATATCCCGAGAAAATATAAAACTGCAAGCATTGGCTTGCATCCAGTCGGCTGTTCATGGTACATACAAATTGATTTATATAGATTGGCCAGTCAATGTGTCTAATATTGAAAACCCGGAAAAATTCCACAATGTAAAGAAACGATCCAGGCGTCC，如SEQ ID No.13；
256bp扩增片段序列：
AACCGTTTGTCCTTGCCGTACTTCCGAGTCTACTTCGTGCAAATGCCTCGAATGACTTTCATTTA ATATATATATATATATATATATATATATATA TATATATATCCCGAGAAAATATAAAACTGCAAGCATTGGCTTGCATCCAGTCGGCTGTTCATGGTACATACAAATTGATTTATATAGATTGGCCAGTCAATGTGTCTAATATTGAAAACCCGGAAAAATTCCACAATGTAAAGAAACGATCCAGGCGTCC，如SEQ ID No.14；
引物4扩增结果如附图5所示，当使用引物4进行荧光PCR扩增时，扩增得到4个片段(4个峰)，含有4个SSR位点，SSR重复元件为ATG。其中试验例所得扩增片段的特征信息为分别含10和11次重复GCT的282bp片段和285bp片段。
引物4扩增片段：(其中电泳图统计片段长度包括M13荧光引物，具体序列展示去掉了该M13荧光引物序列(18bp),下划线部分为SSR重复元件。)
276bp扩增片段序列：
GTCTGCAGACTTCCGGAACAGTTGGAGGGCTTCAAGTTCATCCTTGCTGAGATAGTCCTTTGTGTTGAGCTGGACAGCATACTGGAGGATAGAGTCTGGGAGAAGAGAGTTGTCTGGAGGAGGGTTTGGCTGAGAGAATTTGTTGAGCAGGC ATGATGATGATGATGATGATGATGAGGAAAGGATGGGGACAGAGAGAGATTTTATATATTGGAATAAAACATATTATTATATCAAA GATCTAGATTCTAGACTTGGCTAGACCTTTCGTGCGAT，如SEQ ID No.17；
279bp扩增片段序列：
GTCTGCAGACTTCCGGAACAGTTGGAGGGCTTCAAGTTCATCCTTGCTGAGATAGTCCTTTGTGTTGAGCTGGACAGCATACTGGAGGATAGAGTCTGGGAGAAGAGAGTTGTCTGGAGGAGGGTTTGGCTGAGAGAATTTGTTGAGCAGGC ATGATGATGATGATGATGATGATGATGAGGAAAGGATGGGGACAGAGAGAGATTTTATATATTGGAATAAAACATATTATTATATCAAGAATCTAGATTCTAGACTTGGCTAGACCTTTCGTGCGAT，如SEQ ID No.18；
282bp扩增片段序列：
GTCTGCAGACTTCCGGAACAGTTGGAGGGCTTCAAGTTCATCCTTGCTGAGATAGTCCTTTGTGTTGAGCTGGACAGCATACTGGAGGATAGAGTCTGGGAGAAGAGAGTTGTCTGGAGGAGGGTTTGGCTGAGAGAATTTGTTGAGCAGGC ATGATGATGATGATGATGATGATGATGATGAGGAAAGGATGGGGACAGAGAGAGATTTTATATATTGGAATAAAACATATTATTATAATCAAGATCTAGATTCTAGACTTGGCTAGACCTTTCGTGCGAT，如SEQ ID No.19；
285bp扩增片段序列：
GTCTGCAGACTTCCGGAACAGTTGGAGGGCTTCAAGTTCATCCTTGCTGAGATAGTCCTTTGTGTTGAGCTGGACAGCATACTGGAGGATAGAGTCTGGGAGAAGAGAGTTGTCTGGAGGAGGGTTTGGCTGAGAGAATTTGTTGAGCAGGC ATGATGATGATGATGATGATGATGATGATGATGAGGAAAGGATGGGGACAGAGAGAGATTTTATATATTTGGAATAAAACATATTATTATATCAAGATCTAGATTCTAGACTTGGCTAGACCTTTCGTGCGAT，如SEQ ID No.20；
通过对试验例、对比例1和对比例2图谱和测序结果综合分析，获得抗氧化活性好的黄绿卷毛菇的DNA条形码特征信息如表2。
表2 抗氧化活性高的黄绿卷毛菇的DNA条形码特征
Figure PCTCN2022109954-appb-000002
Figure PCTCN2022109954-appb-000003
引物1扩增出含13次GAG重复元件的256bp片段(如SEQ ID NO：3所示)和含19次GAG重复元件的274bp片段(如SEQ ID NO：4所示)，引物2扩增出含5次CAG重复元件的257bp片段(如SEQ ID NO：7所示)和含8次CAG重复元件的266bp片段(如SEQ ID NO：9所示)；引物3扩增出含19次AT重复元件的254bp片段(如SEQ ID NO：13所示))和含20次AT重复元件的256bp片段(如SEQ ID NO：14所示)；引物4扩增出含10次GCT重复元件的282bp片段(如SEQ ID NO：19所示)和含11次GCT重复元件的285bp片段(如SEQ ID NO：20所示)。当同时分别使用上述引物1、2、3、4进行综合检测判断时，黄绿卷毛菇抗氧化活性指标筛选的准确性最好。
实施例3
黄绿卷毛菇抗氧化活性指标筛选验证
通过盲试试验验证黄绿卷毛菇抗氧化活性的DNA条形码。
第一步盲试，以抗氧化活性DPPH自由基清除率高于或等于91.1％的西藏自治区当雄县样品为试验例，以低于91.1％(显著性p<0.05)的青海省祁连县和四川省石渠县样品为对比1组和对比2组，各取16份样品进行盲试；
第二步测试，利用引物(SEQ ID NO：1和SEQ ID NO：2，SEQ ID NO：5和SEQ ID NO：6，SEQ ID NO：10和SEQ ID NO：11，SEQ ID NO：15和SEQ ID NO：16)扩增并进行毛细管电泳。引物组可使用一对或多对组合扩增以试验例DNA条形码特征区分盲试样品；
第三步揭盲，结果如表3所示，以抗氧化活性条形码特征区分抗氧化活性共48份样品揭盲结果全部正确。由此说明抗氧化活性的DNA条形码适用于抗氧化活性性状的筛选。
表3 抗氧化活性DNA条形码特征揭盲鉴定结果
Figure PCTCN2022109954-appb-000004
Figure PCTCN2022109954-appb-000005
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种筛选黄绿卷毛菇抗氧化活性指标的DNA条形码，其特征在于，所述DNA条形码的核苷酸序列包括：
    如SEQ ID NO：3；
    和/或SEQ ID NO：4；
    和/或SEQ ID NO：3和SEQ ID NO：4组合；
    和/或SEQ ID NO：9；
    和/或SEQ ID NO：7和SEQ ID NO：8组合；
    和/或SEQ ID NO：7和SEQ ID NO：9组合；
    和/或SEQ ID NO：12；
    和/或SEQ ID NO：13；
    和/或SEQ ID NO：14；
    和/或SEQ ID NO：13和SEQ ID NO：14组合；
    和/或SEQ ID NO：17；
    和/或SEQ ID NO：18；
    和/或SEQ ID NO：17和SEQ ID NO：18组合；
    和/或SEQ ID NO：19和SEQ ID NO：20组合；
    和/或SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19和SEQ ID NO：20组合中的一种或多种。
  2. 一种扩增如权利要求1所述筛选黄绿卷毛菇抗氧化活性指标的DNA条形码的引物组，其特征在于，所述引物组的核苷酸序列包括：
    如SEQ ID NO：1和SEQ ID NO：2；
    和/或SEQ ID NO：5和SEQ ID NO：6；
    和/或SEQ ID NO：10和SEQ ID NO：11；
    和/或SEQ ID NO：15和SEQ ID NO：16中的一组或多组。
  3. 根据权利要求2所述的引物组，其特征在于，所述引物组的核苷酸序列包括：如SEQ ID NO：1和SEQ ID NO：2、SEQ ID NO：5和SEQ ID NO：6、SEQ ID NO：10和SEQ ID NO：11、SEQ ID NO：15和SEQ ID NO：16。
  4. 一种以抗氧化活性指标筛选黄绿卷毛菇的方法，其特征在于，包括如下步骤：
    S1、提取待测样品基因组DNA；
    S2、以S1基因组DNA为模板，选择权利要求2所述的一组或多组引物分别进行荧光PCR扩增反应，得扩增产物；
    S3、S2所述扩增产物经毛细管荧光电泳检测，通过扩增产物的片段数、SSR位点数、SSR重复元件及其重复次数进行判定。
  5. 根据权利要求4所述的以抗氧化活性指标筛选黄绿卷毛菇的方法，其特征在于，所述步骤S3的判定标准为：
    SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含13次GAG重复元件的256bp片段和含19次GAG重复元件的274bp片段；
    和/或SEQ ID NO：5和SEQ ID NO：6引物组扩增得到含5次CAG重复元件的257bp片段和含8次CAG重复元件的266bp片段；
    和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含19次AT重复元件的254bp片段和含20次AT重复元件的256bp片段；
    和/或SEQ ID NO：15和SEQ ID NO：16引物组扩增得到含10次GCT重复元件的282bp片段和含11次GCT重复元件的285bp片段时，判定该黄绿卷毛菇为抗氧化活性高的黄绿卷毛菇。
  6. 根据权利要求4所述的以抗氧化活性指标筛选黄绿卷毛菇的方法，其特征在于，步骤S2所述荧光PCR扩增反应的反应体系为：
    2×Taq PCR Master Mix 5μL，基因组DNA 1μL，上游引物0.1μL，下游引物0.4μL，带荧光的M13引物0.4μL，用无菌去离子水定容至10μL。
  7. 根据权利要求6所述的以抗氧化活性指标筛选黄绿卷毛菇的方法，其特征在于，所述上游引物、下游引物和带荧光的M13引物浓度均为10uM。
  8. 根据权利要求4所述的以抗氧化活性指标筛选黄绿卷毛菇的方法，其特征在于，步骤S2所述荧光PCR扩增反应程序为：
    95℃预变性3min；95℃变性30s，62至55℃降落PCR退火30s，72℃延伸30s，共10个循环；95℃变性30s，52℃退火30s，72℃延伸30s，共25个循环；72℃终延伸20min；4℃保温6h后用于荧光毛细管电泳检测。
  9. 权利要求1所述DNA条形码和/或权利要求2所述引物组在制备以抗氧化活性指标筛选黄绿卷毛菇的产品中的应用。
  10. 一种以抗氧化活性指标筛选优质黄绿卷毛菇的产品，其特征在于，含有权利要求2所述的一组或多组引物组，且符合标准：
    SEQ ID NO：1和SEQ ID NO：2引物组扩增得到含13次GAG重复元件的256bp片段和含19次GAG重复元件的274bp片段；
    和/或SEQ ID NO：5和SEQ ID NO：6引物组扩增得到含5次CAG重复元件的257bp片段和含8次CAG重复元件的266bp片段；
    和/或SEQ ID NO：10和SEQ ID NO：11引物组扩增得到含19次AT重复元件的254bp片段和含20次AT重复元件的256bp片段；
    和/或SEQ ID NO：15和SEQ ID NO：16引物组扩增得到含10次GCT重复元件的282bp片段和含11次GCT重复元件的285bp片段中的一个或多个。
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