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Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean
Published on: December 23, 2017
12.8K
A plant CitPITP1 protein-coding exon sequence serves as a promoter in bacteria
Qingjiang Wu1, Jialing Fu1, Juan Sun1
1Key Laboratory of Horticultural Plant Biology of Ministry of Education, Huazhong Agricultural University, Wuhan, 430000, China.
Journal of Biotechnology
|July 23, 2021
Summary
A plant gene sequence (key64) with a CRAL_TRIO domain functions as a bacterial promoter. This discovery reveals cross-kingdom gene element neofunction and aids in designing novel promoters.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The functional roles of DNA sequences are not fully understood, despite extensive genetic manipulation in prokaryotes.
- Plant genes are increasingly utilized in molecular biology, but their inherent functionalities require deeper investigation.
Purpose of the Study:
- To identify and characterize novel prokaryotic promoter sequences within plant genes.
- To investigate the cross-kingdom functionality of gene elements, specifically exploring plant gene sequences for promoter activity in bacteria.
- To understand the structural requirements for promoter activity and its potential for modular promoter design.
Main Methods:
- Characterization of the plant protein-coding gene CitPITP1 from Citrus, focusing on its CRAL_TRIO domain.
- Identification and isolation of a critical 64-bp sequence (key64) responsible for prokaryotic promoter activity.
- In vitro binding assays using bacterial RNA polymerase subunit RpoD and the key64 sequence.
- Bioinformatic analysis to identify key64-like sequences in fungi, plants, and animals.
- Site-directed mutagenesis to determine crucial nucleotides for transcription activity.
- Expression analysis of recombinant CitPITP1 in E. coli and CitPITP1-GFP fusion protein in plant cells.
Main Results:
- A 64-bp sequence (key64) within the plant gene CitPITP1 exhibits significant prokaryotic promoter activity.
- Bacterial RNA polymerase subunit RpoD specifically binds to the key64 sequence.
- Key64-like sequences were identified in diverse species including Amborella, Rice, Arabidopsis, and Citrus, functioning as prokaryotic promoters.
- Two conserved motifs were identified in these sequences, with specific nucleotides (positions 7, 29, 30) being critical for activity.
- While exon 4 of CitPITP1 contained key64, it did not show promoter activity in plants, indicating context-dependent functionality.
Conclusions:
- The study identifies a novel basal promoter derived from a plant gene sequence, demonstrating cross-kingdom neofunction of genetic elements.
- The findings provide valuable insights into the modular design of synthetic promoters by understanding the essential components and their functions.
- This research expands the toolkit for genetic engineering by revealing a plant-derived sequence capable of driving gene expression in prokaryotes.
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