Production of recombinant miraculin protein in carrot callus via Agrobacterium-mediated transformation

Park, Yun-Ji; Han, Jong-Eun; Lee, Hyoshin; Lee, Ji-Young; Ho, Thanh-Tam; Park, Soyoung

doi:10.1007/s11240-021-02032-3

Cited by 5 publications

(1 citation statement)

References 31 publications

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“…The Agrobacterium tumefaciens -mediated transformation system (ATMTs) is widely used for exogenous gene function analysis in plant. For example, ATMTs has been used to introduce a miraculin gene into callus of carrot ( Daucus carota L.), and during cell division, the miraculin gene was highly expressed in transgenic callus lines [ 17 ]. In addition, ATMTs was also used to transfect the sections of young cotyledon in tomato ( Solanum lycopersicum L.) to examine overexpression of target genes [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

An efficient overexpression method for studying genes in Ricinus that transport vectorized agrochemicals

Xiao

Zhang

et al. 2022

Plant Methods

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Background Plant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals. Therefore, transporters associated with phloem loading of vectorized agrochemicals have drawn increasing attention. As a model system, castor bean (Ricinus communis L.) has been widely used to detect the phloem mobility of agrochemicals. However, there is still a lack of an efficient protocol for the Ricinus seedling model system that can be directly used to investigate the recognition and phloem loading functions of plasmalemma transporters toward vectorized agrochemicals. Results Here, using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Strong fluorescence signals were observed in leaves, demonstrating that exogenous genes can be successfully overexpressed in seedlings. Subsequently, gene expression time and vacuum infiltration parameters were optimized. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Parameter optimization showed Agrobacterium concentration at OD600 = 1.2, and infiltration for 20 min (0.09 MPa), return to atmospheric pressure, and then infiltration for another 20 min, were the suitable transformation conditions. To test the application of vacuum agroinfiltration in directly examining the loading functions of plasma membrane transporters to vectorized agrochemicals in seedlings, two LHT (lysine/histidine transporter) genes, RcLHT1 and RcLHT7, were overexpressed. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. Furthermore, the concentrations of l-Val-PCA (l-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of l-Val-PCA. Conclusions We successfully constructed a transient expression system in Ricinus seedlings and laid the foundation for researchers to directly investigate the loading functions of plasma membrane transporters to vectorized agrochemicals in the Ricinus system.

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Section: Introductionmentioning

confidence: 99%