Construction of ATMT System for Clonostachys rosea and Its Conditional Optimization of Colonization into Glycyrrhiza uralensis Seeds
10.13422/j.cnki.syfjx.20220748
- VernacularTitle:粉红粘帚菌的ATMT体系构建及定殖甘草条件优化
- Author:
Qijin SHI
1
;
Xiaohan WANG
1
;
Guangxi REN
1
;
Dan JIANG
1
;
Chunsheng LIU
1
Author Information
1. School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China
- Publication Type:Journal Article
- Keywords:
Clonostachys rosea;
Agrobacterium tumefaciens-mediated transformation (ATMT);
green fluorescent protein;
β-glucuronidase (GUS) staining;
Glycyrrhiza uralensis;
transformant;
orthogonal test
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2023;29(4):126-133
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveAgrobacterium tumefaciens-mediated transformation (ATMT) of Clonostachys rosea, an endophytic fungus of Glycyrrhiza uralensis seeds, was established and optimized, and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds, so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. MethodThe conditions of ATMT were optimized from three aspects, including the concentration of acetosyringone, co-culture time and the concentration of conidia of recipient fungi. Then, high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature, co-culture time and spore concentration as factors and colonization rate as index. ResultWhen spore concentration was 1×107 cfu·mL-1, acetosyringone concentration was 150 μmol·L-1 and the co-culture time was 60 h, the transformation efficiency of C. rosea was the highest, which was 135 transformants per 1×107 recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein (GFP) and β-glucuronidase (GUS) staining. When co-culture temperature was 25 ℃, co-culture time was 36 h and the spore concentration was 1×106 cfu·mL-1, the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest, which was 71.11%. ConclusionThis study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea, but also of colonization of the transformants into G. uralensis seeds, which can lay a foundation for the development of biofertilizer of G. uralensis.