bar基因筛选标记在金针菇遗传转化中的应用

doi:10.19586/j.2095-2341.2026.0048

生物技术进展 ›› 2026, Vol. 16 ›› Issue (4): 952-959.DOI: 10.19586/j.2095-2341.2026.0048

bar基因筛选标记在金针菇遗传转化中的应用

毛阿秀¹^,²(), 尚也然¹^,², 尚晓冬², 唐桂容²(), 张中林¹()

^1.上海师范大学生命科学学院，上海 201418
^2.上海市农业科学院食用菌研究所，上海 201403

收稿日期:2026-03-02 接受日期:2026-03-31 出版日期:2026-07-25 发布日期:2026-09-11
通信作者: 唐桂容,张中林
作者简介:毛阿秀E-mail: 18369555248@163.com
基金资助:
上海市农业科学院卓越团队建设计划项目(沪农科卓[2022]001)

Application of bar Gene Screening Markers in Genetic Transformation of Flammulina filiformis

Axiu MAO¹^,²(), Yeran SHANG¹^,², Xiaodong SHANG², Guirong TANG²(), Zhonglin ZHANG¹()

^1.College of Life Sciences，Shanghai Normal University，Shanghai 201418，China
^2.Institute of Edible Fungi，Shanghai Academy of Agricultural Sciences，Shanghai 201403，China

Received:2026-03-02 Accepted:2026-03-31 Online:2026-07-25 Published:2026-09-11
Contact: Guirong TANG,Zhonglin ZHANG

摘要/Abstract

摘要：

金针菇（Flammulina filiformis）是一种重要的食药用真菌。然而，其遗传转化长期依赖潮霉素抗性作为唯一的筛选标记，单一标记的限制制约了多基因协同调控机制的研究。使用以bar基因为筛选标记的pAgbar与pAgbarG 两种载体，成功建立了以草铵膦为筛选剂的根癌农杆菌（Agrobacterium tumefaciens）介导的金针菇遗传转化体系。其中，pAgbar携带由双孢蘑菇（Agaricus bisporus）组成型启动子gpd调控的bar基因，pAgbarG则包含bar-egfp融合表达框。将该体系应用于单核菌株Dan3，获得了遗传稳定的草铵膦抗性转化子。筛选效率比较结果表明，M-100培养基的筛选效果显著优于PDA培养基。PCR及Southern blot杂交分析证实，外源bar基因已成功整合至金针菇基因组中。同时，bar-egfp融合表达系统实现了荧光信号的有效表达，可作为阳性转化子快速、直观的可视化指示标记。研究构建了以bar为抗性筛选标记的金针菇遗传操作平台，为功能基因解析、分子辅助育种及次生代谢通路调控等研究提供了稳定且可靠的技术支撑。

关键词: 金针菇, bar, 筛选标记, 遗传转化

Abstract:

Flammulina filiformis is an economically important edible and medicinal mushroom. However， its genetic transformation has historically relied solely on hygromycin resistance as a selectable marker and this limitation has impeded research into the coordinated regulation of multiple genes. In this study， we established an Agrobacterium tumefaciens-mediated genetic transformation system for F. filiformis using the herbicide resistance gene bar and glufosinate-ammonium as the selectable marker and agent， respectively. Two vectors were used， pAgbar， in which the bar gene is driven by the constitutive gpd promoter from Agaricus bisporus； and pAgbarG， which harbors a bar-egfp translational fusion expression cassette. Both constructs were delivered into the monokaryotic strain Dan3， yielding stable， glufosinate-ammonium-resistant transformants. Transformation efficiency was markedly higher on M-100 medium than on PDA. PCR and Southern blot hybridization confirmed the integration of the exogenous bar gene into the F. filiformis genome. Moreover， the bar-egfp fusion system enabled effective fluorescence signal expression， providing a rapid and visual means of identifying positive transformants. This study establishes a genetic manipulation platform for F. filiformis using bar as a resistance selectable marker， offering reliable technical support for functional gene analysis， molecular-assisted breeding， and mechanistic studies of secondary metabolic pathway regulation.

Key words: Flammulina filiformis, bar, selection marker, genetic transformation

中图分类号:

Q786

毛阿秀, 尚也然, 尚晓冬, 唐桂容, 张中林. bar基因筛选标记在金针菇遗传转化中的应用[J]. 生物技术进展, 2026, 16(4): 952-959.

Axiu MAO, Yeran SHANG, Xiaodong SHANG, Guirong TANG, Zhonglin ZHANG. Application of bar Gene Screening Markers in Genetic Transformation of Flammulina filiformis[J]. Current Biotechnology, 2026, 16(4): 952-959.

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链接本文: https://swjsjz.magtechjournal.com/CN/10.19586/j.2095-2341.2026.0048

https://swjsjz.magtechjournal.com/CN/Y2026/V16/I4/952

图/表 9

表1 引物名称和序列

Table 1 Primer names and sequences

引物名称	引物序列（5'→3'）
AbbarF	5'-CTGCAGCCCGGGGGATCCGAAGAAGCAT TAAGAGGTCCG-3'
baregfpR	5'-CTCCTCGCCCTTGCTCACGATCTCGGTGA CGGGCA-3'
BaregfpF	5'-TGCCCGTCACCGAGATCGTGAGCAAGGG CGAGGAG-3'
BaregfpR	5'-CGGCCGCTCTAGAACTAGTCTATTACTTGTACAGCTCGTCCAT-3'
M13F	5'-CAGGGTTTTCCCAGTCACG-3'
M13R	5'-GAGCGGATAACAATTTCACAC-3'
YZbarF	5'-AGCCCAGAACGACGCCCGGC-3'
YZbarR	5'-GATCTCGGTGACGGGCAGGAC-3'
YZegfpF	5'-GGCGAGGAGCTGTTCACCG-3'
YZegfpR	5'-GCTCGTCCATGCCGAGAG-3'
ActF	5'-GATCGTATGCAGAAGGAGTTGACAC-3'
ActR	5'-CCACTCTCGTCGTACTCTTGCTTG-3'

图1 Dan3菌株在不同培养基中对GA的敏感性分析

Fig. 1 Sensitivity analysis of Dan3 strain to GA in different culture medium concentrations

图2 含pAgbar质粒的农杆菌菌株的PCR鉴定注：M—DNA marker；1~8—农杆菌AGL1工程菌株；pc—以质粒pAgbar为模板作阳性对照；nc—以水为模板作空白对照。

Fig. 2 PCR identification of Agrobacteriumtumefaciens strains containing the pAgbar plasmid

表2 金针菇GA抗性转化子的筛选结果

Table 2 Screening of GA resistant transformants in F. filiformis

复筛培养基	总转化子数	抗性转化子数	转化效率/%
M-100	162	9	5.56
PDA	158	6	3.80

图3 金针菇GA抗性转化子的筛选A：转化子在含GA的M-100培养基上的初筛观察；B：转化子在含GA的M-100复筛培养基上培养7 d的情况；C：转化子在含GA的PDA复筛培养基上培养15 d的情况

Fig. 3 Screening of GA-resistant transformants of F. filiformis

图4 PCR和Southern印迹杂交分析金针菇GA抗性转化子A：金针菇转化子bar和actin基因的PCR扩增；1～5为传代前GA抗性转化子，6～10为传代后GA抗性转化子，wt为金针菇Dan3，pc为pAgbar质粒，nc为以水作模板的阴性对照，M为DNA分子量标准；B：Southern blot验证结果；1为GA抗性转化，wt为金针菇Dan3，pc为pAgbar质粒，M为DIG标记的DNA分子量标准

Fig. 4 PCR and Southern blot hybridization analysis of GA-resistant transformants in F. filiformis

图5 GA抗性菌株的菌落形态与生长速率A：转化子在PDA培养基中生长情况；B：转化子在含GA的PDA培养基中生长情况；C：PDA培养基中菌丝生长速率；D：含GA的PDA培养基中菌丝生长速率；*、***分别表示差异在P<0.05、P<0.001水平上具有统计学意义。

Fig. 5 Colony morphology and growth rate of GA-resistant strains

图6 pAgbarG质粒构建示意图

Fig. 6 Construction of pAgbarG plasmid

图7 显微镜观察含egfp的GA抗性转化子A：在PDA平板上观察转化子egfp的表达情况；B：转化子菌丝显微观察照片；C：pAgbarG载体转化菌株PCR验证电泳图

Fig. 7 Microscopic observation of GA-resistant transformants expressing egfp

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bar基因筛选标记在金针菇遗传转化中的应用

Application of bar Gene Screening Markers in Genetic Transformation of Flammulina filiformis

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