Identifying the interacting proteins of lignin biosynthetic P450 enzymes by affinity purification coupled with LC-MS/MS analysis

ABSTRACT: This project aims to identify the potential interacting proteins of lignin biosynthetic P450 enzymes by affinity purification and LC-MS/MS analysis. To this end, the genomic pieces of three lignin biosynthetic P450 genes C4H, C3’H and F5H were cloned into pMDCpC4H-HPB vector to generate HPB-fusion constructs driven by C4H promoter. The resulted constructs were then respectively transformed into ref3-3 (for C4H), Col-0 WT (for C3'H), and fah1-2 (for F5H) backgrounds and pMDCpC4H-HPB empty vector (as control) was transformed into Col-0 WT to generate transgenic plants. Arabidopsis T2 transgenic plants were grown for 5 weeks in soil and 15 grams of stem tissues were collected for protein extraction. Membrane protein complex was purified by affinity purification using streptavidin beads and subject to LC-MS/MS analysis. Two biological repeats were performed for each construct. For the first repeat (gel-base proteomics), each P450 construct has its control since each experiment was performed separately. For the second repeat (affinity purification,AP-MS), one control was shared for all the three P450 genes.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Shoot

SUBMITTER: Chang-Jun Liu  

LAB HEAD: Chang-Jun Liu

PROVIDER: PXD009033 | Pride | 2018-05-02

REPOSITORIES: Pride