Plants-Basel
·
2025
·Vol. 14
·期 17
·2664
·
IF 4.1
Dan Wang,Yin Liu,Yao Zhang,Xiumei Huang,Jiaxuan Wang,Yi Wang,Yue Liu,Chao Yan,Bingsheng Lv,Yue Jia
Welsh onion ( Allium fistulosum L.), a globally significant vegetable, flavoring agent, and phytomedicine resource, has remained unavailable with established transient expression platforms for functional genomic investigations. To address this critical methodological limitation, we present systematically optimized protocols for both Agrobacterium -mediated hairy root transformation and protoplast transient expression systems, achieving significant advances in transformation efficiency for this species. Through systematic optimization of key parameters, including Agrobacterium rhizogenes ( A. rhizogenes ) strain selection (with Ar.Qual demonstrating superior performance), explant type efficacy, bacterial suspension optical density (OD 600 = 0.3), and acetosyringone induction concentration (100 μM), we established a highly efficient stem disc infection methodology, achieving 88.75% hairy root induction efficiency. Subsequent optimization of protoplast isolation protocols identified the optimal enzymatic digestion conditions: 6-h dark digestion of young leaves using 1.0% ( w / v ) Cellulase R-10, 0.7% ( w / v ) Macerozyme R-10, and 0.4 M mannitol, yielding 3.3 × 10 6 viable protoplasts g −1 FW with 90% viability. System functionality validation through PEG-mediated transient transformation demonstrated successful green fluorescent protein (GFP) reporter gene expression, confirmed by fluorescence microscopy. As the first documented transient expression platforms for Welsh onion, these protocols enable essential molecular investigations, including in planta promoter activity profiling, subcellular protein localization, and CRISPR-based genome-editing validation. This methodological breakthrough overcomes previous technical constraints in Welsh onion molecular biology, providing critical tools for accelerated gene functional characterization in this agriculturally important species.
研究领域:
植物生物技术
·
分子生物学
·
遗传转化
·
功能基因组学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2017
·
IF 2.892
Yumin Fang,Zhuoni Hou,Xiaodan Zhang,Dongfeng Yang,Zongsuo Liang
Salvia miltiorrhiza Bunge ( S. miltiorrhiza ) and Salvia castanea Diels f. t omentosa Stib ( S. castanea ) are both important medicinal plants which have been widely used to treat cardiovascular diseases. Tanshinone and phenolic acid are active and specialized metabolites in the two species. However, the divergent mechanism of specialized metabolism in the two species is yet unknown. We found that S. castanea produced higher levels of cryptotanshinone, tanshinone IIA, caffeic acid, and rosmarinic acid, but lower levels of dihydrotanshinone I and salvianolic acid B than S. miltiorrhiza . Some key genes involved in specialized metabolism were potentially associated with the diverse metabolism between the two species. LH with 1 g l −1 contributed more to tanshinone accumulation in S. castanea than that in S. miltiorrhiza . Genes involved in tanshinone biosynthesis in S. castanea were also more sensitive to LH than those in S. miltiorrhiza . Knowledge about diverse specialized metabolism in the two species will help us understand mechanism of tanshinone and phenolic acid biosynthesis.
研究领域:
药理学
·
植物代谢
·
分子生物学
✓ 已验证
DOI
高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2021
·
IF 3.307
Yuanyuan Miao,Yanyun Hu,Shanyong Yi,Xuejia Zhang,Ninghua Tan
Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes -mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rol B and rol C genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g −1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g −1 DW for shoots and roots respectively) and calli (1.082 μg g −1 DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g −1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g −1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g −1 DW). Hairy root culture of R. yunnanensis , with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.
研究领域:
植物生物技术
·
生药学
·
分子生物学
·
药物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Yangzhen Liao,Min Wu,Jiahe Fan,Jingqiong Wan,Xin An,Xiaolan Li,Yuan Wei,Zhen Ouyang
1-Deoxynojirimycin (DNJ), a primary active component in mulberry leaves, has garnered significant attention due to its unique structure and notable pharmacological properties. Our previous investigations have elucidated the biosynthetic pathways of DNJ from lysine to 2-methylpiperidine. However, the hydroxylation process and its underlying mechanisms remain elusive. In this study, five CYP450s hydroxylase genes significantly correlated ( P < 0.05) with DNJ content in mulberry leaves at various time were screened through transcriptome profile. MaCYP71BG22 was first cloned and functionally characterized. This gene was shown to specifically catalyze the stereoselective hydroxylation of ( R )-2-methylpiperidine at the C4-position to produce (2 R , 4 R )-2-methylpiperidin-4-ol. In hairy roots of mulberry, overexpression of MaCYP71BG22 increased DNJ accumulation, while virus-induced gene silencing (VIGS) decreased its production. Furthermore, structural-function analysis pinpointed a critical residue, G460, in MaCYP71BG22, mutation of this residue to G460E enhanced the enzyme's catalytic efficiency. This study represents the first report of a CYP450 hydroxylase involved in the biosynthesis of piperidine alkaloids in mulberry leaves, and demonstrates that MaCYP71BG22 selectively catalyzes the C4-stereoselective hydroxylation of ( R )-2-methylpiperidine in DNJ biosynthesis. These findings further elucidate the DNJ biosynthetic pathway and provide new insights into the stereo- and regio-selective hydroxylation abilities of CYP450s hydroxylase in DNJ biosynthesis.
研究领域:
植物生物化学
·
分子生物学
·
药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）