scholarly journals Subcellular Localization and Functional Analysis of the Arabidopsis GTPase RabE

2009 ◽  
Vol 149 (4) ◽  
pp. 1824-1837 ◽  
Author(s):  
Elena Bray Speth ◽  
Lori Imboden ◽  
Paula Hauck ◽  
Sheng Yang He
Keyword(s):  
Functional Analysis ◽  
Subcellular Localization

scholarly journals Functional Analysis of HIV-1 Vpr: Identification of Determinants Essential for Subcellular Localization

Virology ◽  
1995 ◽  
Vol 212 (2) ◽  
pp. 331-339 ◽  
Author(s):  
S. Mahalingam ◽  
Ronald G. Collman ◽  
Mamata Patel ◽  
Claude E. Monken ◽  
A. Srinivasan
Keyword(s):  
Functional Analysis ◽  
Subcellular Localization ◽  
Hiv 1

scholarly journals Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuanyuan Cheng ◽  
Xiaoli Wang ◽  
Li Cao ◽  
Jing Ji ◽  
Tengfei Liu ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Subcellular Localization ◽  
Root System ◽  
Hairy Root ◽  
Protein Interactions ◽  
Gene Editing ◽  
Transformation Frequency ◽  
Highly Efficient ◽  
Root Transformation ◽  
Gene Functional Analysis

Abstract Background Agrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function. Results In this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean. Conclusion A rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

Cloning, functional analysis, and subcellular localization of two isoforms of NADH:cytochrome b5 reductase from developing seeds of tung (Vernicia fordii)

Plant Science ◽  
2005 ◽  
Vol 169 (2) ◽  
pp. 375-385 ◽  
Author(s):  
Jay M. Shockey ◽  
Preetinder K. Dhanoa ◽  
Tammy Dupuy ◽  
Dorselyn C. Chapital ◽  
Robert T. Mullen ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Subcellular Localization ◽  
Vernicia Fordii ◽  
Developing Seeds

scholarly journals Microinjection Method for Analyzing Zebrafish Early Stage Oocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Manami Kobayashi ◽  
Allison Jamieson-Lucy ◽  
Mary C. Mullins
Keyword(s):  
Functional Analysis ◽  
Embryonic Development ◽  
Subcellular Localization ◽  
Germ Cell ◽  
Analytical Methods ◽  
Early Stage ◽  
Cell Formation ◽  
Maternal Factors ◽  
Early Stages ◽  
Germ Cell Formation

Maternal factors which accumulate and establish oocyte polarity during the early stages of oogenesis play key roles in embryonic development, as well as germ cell formation. However, vertebrate oogenesis, especially early stages of oogenesis, is not well understood due to the difficulty of accessing these oocytes and the lack of analytical methods. Here, we report on a microinjection method for analyzing zebrafish early-stage oocytes and some artifacts to be aware of when performing oocyte injections or analyzing oocytes. Using this method, we successfully injected mRNAs encoding fluorescent-tagged proteins into early-stage oocytes and observed subcellular localization in the live oocytes. This method is expected to advance the functional analysis of genes involved in oogenesis.

Investigating the functional link between TMEM165 and SPCA1

2019 ◽  
Vol 476 (21) ◽  
pp. 3281-3293 ◽  
Author(s):  
Elodie Lebredonchel ◽  
Marine Houdou ◽  
Hans-Heinrich Hoffmann ◽  
Kateryna Kondratska ◽  
Marie-Ange Krzewinski ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Complete Loss ◽  
Protein Family ◽  
Uncharacterized Protein ◽  
Functional Link ◽  
P Type

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

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