EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings.
Methods Mol Biol
; 2505: 249-262, 2022.
Article
em En
| MEDLINE
| ID: mdl-35732950
ABSTRACT
Catharanthus roseus produces medicinal terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid biosynthesis in C. roseus . We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment.
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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Catharanthus
/
Alcaloides de Triptamina e Secologanina
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article