金色毛壳菌XBAa1对烟草的促生作用及其在烟草根内的定殖

doi:10.13560/j.cnki.biotech.bull.1985.2025-0732

摘要/Abstract

摘要：

目的明确金色毛壳菌（Arcopilus aureus）XBAa1对烟草的促生作用及其在根内的定殖动态规律。方法通过重建烟草-XBAa1共生体，验证菌株的内生性并测定其促生作用；采用聚乙二醇（PEG）介导制备XBAa1原生质体，将携带绿色荧光蛋白（GFP）基因的P3641质粒整合至菌株基因组，获得稳定表达GFP的转化子；通过转化子侵染烟草根系，结合激光共聚焦显微镜和绝对荧光定量PCR法检测XBAa1菌株侵染部位及其在烟草根内的定殖量变化。结果 XBAa1可内生定殖于烟草根内，显著促进烟草生长；地上部及根系生物量明显增加，叶片叶绿素a+b含量较对照组提升63.17%；调控烟草内源激素水平，细胞分裂素和脱落酸含量分别较对照组增加143.65%和降低46.56%；同时降低烟草氧化损伤程度，丙二醛含量较对照组下降38.02%。XBAa1原生质体制备细胞壁最佳裂解条件为12.5 mg/mL Megalyase酶解液酶解3 h；获得GFP标记的XBAa1-Y3转化子稳定定殖于烟草根组织细胞间隙。温室无菌基质条件下，接种30 d内，XBAa1菌株在烟草根内的定殖量呈“先升后降”趋势，接种15 d时达到峰值（1.23×10⁷ copies/mL）；30 d时，定殖量降至8.12×10⁶ copies/mL。结论金色毛壳菌XBAa1能在烟草根内稳定定殖，显著促进烟草生长，在农业上具有一定的应用潜力；获得的GFP遗传转化子表明原生质体可作为后续遗传转化材料，为深入理解XBAa1在烟草体内迁移及其生防功能奠定研究基础。

关键词: 金色毛壳菌, 内生真菌, 原生质体, 内源激素, 定殖

Abstract:

Objective This work is to investigate the growth-promoting effects of Arcopilus aureus strain XBAa1 on tobacco and the colonization dynamics in roots. Method By reconstruction of the tobacco-XBAa1 fungal symbiosis, its endophytic performance and plant growth promotion was evaluated. Based on the PEG-mediated protoplast transformation method, GFP gene carried by the P3641 plasmid was integrated into A. aureus XBAa1 genome, the transformants stably expressing GFP were obtained. Transformants were used to infect tobacco roots. The infection sites and colonization levels of strain XBAa1 within tobacco roots were detected using laser confocal microscopy (LCM) and quantitative absolute real-time PCR assays. Result A. aureus XBAa1 endophytically colonized in tobacco roots, which significantly promoted tobacco growth. The biomasses of the shoots and roots increased remarkably, with the chlorophyll a+b contents in leaves enhanced by 63.17% compared with CK. It regulated the changes in hormones of tobacco, with cytokinin content increased by 143.65% and abscisic acid content decreased by 46.56% relative to CK, respectively. Meanwhile, it reduced the degree of oxidative damage in tobacco, and the malondialdehyde content decreased by 38.02% compared with CK. The optimal cell wall lysis conditions for protoplast preparation of XBAa1 were enzymatic hydrolysis with 12.5 mg/mL megalyase solution treated for 3 h; for the obtained XBAa1-Y3 transformant, laser confocal microscopy observations revealed that it colonized stably in the intercellular spaces of tobacco root tissue cells. Under the condition of sterile substrate in the greenhouse, the colonization biomasses of XBAa1-GFP strain in tobacco roots showed a “first increasing and then decreasing” trend within 30 d post-inoculation (dpi), reaching a peak of 1.23×10⁷ copies/mL at 15 dpi; at 30 dpi, the colonization density decreased to 8.12×10⁶ copies/mL. Conclusion A. aureus XBAa1 had stable colonization in tobacco roots and remarkable growth-promoting effects on host plants, indicating considerable application potential in agriculture. The successfully obtained GFP-tagged XBAa1 transformant verified that protoplasts are feasible materials for subsequent genetic transformation, establishing a critical basis for an in-depth clarifying the migration traits of XBAa1 in tobacco plants and its biocontrol functions.

Key words: Arcopilus aureus, endophytic fungi, protoplast, endogenous hormones, colonization

耿铭言, 冯慧, 何雷, 张国政, 王静. 金色毛壳菌XBAa1对烟草的促生作用及其在烟草根内的定殖[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0732.

GENG Ming-yan, FENG Hui, HE Lei, ZHANG Guo-zheng, WANG Jing. The Growth-promoting Effect of Arcopilus aureus XBAa1 on Tobacco and Its Colonization in Tobacco Root[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0732.

图/表 9

图1 烟苗根内XBAa1菌株再分离A，C：等量无菌水对照；B，D：菌株XBAa1

Fig. 1 Re-isolation of strain XBAa1 in tobacco rootA, C: Sterile water control. B, D: Strain XBAa1

图2 组培和无菌基质条件下XBAa1对烟草早期生长的促生作用A，B：组培共生体烟苗；C，D，E，F：盆栽共生体烟苗地上部和根；G：根鲜重；H：地上部鲜重（*P<0.05，**P<0.01，***P<0.001，下同）

Fig. 2 Promoting effects of XBAa1 on early growth of tobacco under tissue culture and sterile substrate conditionsA, B: Tobacco-XBAa1 symbiosis in tissue culture. C, D, E, F: Above ground and root of the tobacco-XBAa1 symbiosis in the greenhouse. G: Root weight. H: Shoot weight (* P<0.05, **P<0.01, ***P<0.001, the same below)

图3 菌株XBAa1对烟草叶绿素、细胞分裂素、脱落酸及丙二醛含量的影响A：叶绿素； B：细胞分裂素； C：脱落酸； D：丙二醛

Fig. 3 Effects on chlorophyll, cytokinin, ABA and MDA contents of tobacco by strain XBAa1A: Chlorophyll. B: Cytokinin. C: Abscisic acid. D: MDA

图4 野生型金色毛壳菌XBAa1对Neo的敏感性

Fig. 4 Sensitivity of wild type Arcopilus aureus XBAa1 to Neo

图5 XBAa1原生质体的释放A：顶端释放；B：侧位释放；C：原位释放

Fig. 5 Release of strain XBAa1 protoplastsA: Top release. B: Lateral release. C: In-situ release

图6 XBAa1-GFP标记菌株PCR检测及其在烟草根内定殖的共聚焦显微镜观察（40×）

Fig. 6 PCR detection of strain XBAa1-GFP and observation in tobacco root colonization by confocal microscope (40×)

图7 XBAa1-GFP荧光菌株与XBAa1野生菌株的菌落性状A：XBAa1野生菌株；B：XBAa1-GFP标记菌株；C：菌株生长速度

Fig. 7 Colony characteristics of tagged strain XBAa1-GFP and wild type strain XBAa1A: Wild type XBAa1. B: XBAa1-GFP strain. C: Growth rates of strains

图8 菌株XBAa1荧光转化子在烟草根内定殖的共聚焦显微镜观察

Fig. 8 Observation of colonization of XBAa1 in tobacco root by confocal microscopy

图9 菌株XBAa1转化子在烟草根内的定殖动态

Fig. 9 Colonization dynamics of strain XBAa1 in tobacco roots

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