Optimization of Agrobacterium-Mediated Genetic Transformation System of Armillaria gallica
10.13422/j.cnki.syfjx.20220314
- VernacularTitle:农杆菌介导的高卢蜜环菌遗传转化体系的优化
- Author:
Xi-ying TENG
1
;
Peng-jie HAN
2
;
Xue-yan ZHANG
2
;
Jun-hui ZHOU
2
;
Yu-yang ZHAO
2
;
Lu-qi HUANG
2
;
Yuan YUAN
2
Author Information
1. School of Pharmacy，Jiangsu University，Zhenjiang 212013，China
2. National Resource Center for Chinese Materia Medica，China Academy of Chinese Medical Sciences，Beijing 100700，China
- Publication Type:Journal Article
- Keywords:
genetic transformation;
Armillaria gallica;
system optimization
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2022;28(6):124-130
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo optimize the existing genetic transformation system of Armillaria gallica to improve the transformation efficiency and lay a foundation for the follow-up research on Armillaria molecular marker-assisted breeding and gene function. MethodThe genetically transformed plasmid pH101-PAgGPD-GFP-TrpC was constructed，transformed into Escherichia coli，amplified， and cultured，and the plasmid was extracted. The extracted plasmid was transformed into four different agrobacteria LBA4404，EHA105，GV3101，and AGL-1，respectively. The transformed agrobacteria were used for impregnating A. gallica，and the agrobacteria with the highest conversion rate were screened out. Then the agrobacterium-mediated genetic transformation system of A. gallica was optimized from the type and concentration of antibiotics，co-culture time，concentration of bacterial solution， and impregnation method. The phenotype profiles of A. gallica under different conditions were observed using Synbiosis ProtoCol 3. ResultThe optimized genetic transformation conditions of A. gallica were as follows： the Agrobacterium strain of EHA105 at absorbance A600 nm=0.6， the co-culture time of 2 d， the infection mode of negative pressure impregnation for 10 min， the primary screening medium of PDA medium containing 400 mg·L-1 cefotaxime sodium and 10 mg·L-1 hygromycin，and the secondary screening medium of PDA medium containing 12 mg·L-1 hygromycin. ConclusionIn this study，the existing genetic transformation system of A. gallica was optimized，and there was a significant difference in the transformation rate before and after optimization （P<0.05）. After optimization，the transformation efficiency of A. gallica was about 4.33%，which was about eight times higher than that before optimization.
