Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes

Hu, Zhoubo and Ghosh, Ajit and Stolze, Sara C and Horváth, Mihály and Bing, Bai and Schaefe, Sabine and Zündorf, Simone and Liu, Shanda and Harzen, Anne and Hajheidai, Mohsen and Sarnowski, Tomasz J. and Nakagami, Hirofumi and Koncz, Zsuzsa and Koncz, Csaba (2019) Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes. PLANT JOURNAL, AiP. ISSN 0960-7412


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To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

Item Type: Article
Uncontrolled Keywords: affinity purification; recombineering; technical advance; fluorescent reporters; DNA replication-dependent HISTONE H3.1; TFIIH protein kinases; site-directed gene modification;
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia
Q Science / természettudomány > QK Botany / növénytan
Depositing User: MTMT SWORD
Date Deposited: 21 Nov 2019 19:06
Last Modified: 21 Nov 2019 19:06

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