Abstract
The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3′-end and 5′-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%–86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.
Highlights
M. pinnata (Pongamia Pinnata) belongs to the semi-mangrove plant which is an intervenient species between halophytes and glycophytes
It was observed that the Millettia pinnata chalcone isomerase (MpCHI) transcription level was significantly enhanced (p < 0.01, n = 4) in leaves at 4 h (19 fold), 8 h (9 fold) and 12 h (6 fold) after salt-treatment, with the highest MpCHI mRNA amount at 4 h, verifying that the MpCHI cloned in this study is associated with salt-tolerance in M. pinnata
Slightly but significantly (p < 0.01, n = 4) incremental transcription quantity was observed at 12 h (2 fold) after salt-treatment, indicating the expression of the MpCHI mainly occur in leaves
Summary
M. pinnata (Pongamia Pinnata) belongs to the semi-mangrove plant which is an intervenient species between halophytes and glycophytes. It has been reported that the PAL enzyme is related to plants’ disease resistance [3,4]; CHS and CHI are the two key enzymes in plant flavonoid biosynthesis and were confirmed to be associated with UV protection [5,6,7]; while F3H and DFR, locating in the later steps next to CHS and CHI in phenylpropanoid biosynthesis pathway (Figure 1), are mainly responsible for the biosynthesis of anthocyanins which possess of photoprotection function [8] The variations in their mRNA expression imply that CHS and CHI enzymes or their catalyzed flavonoids are closely associated with M. pinnata salt tolerance since both were improved in mRNA expression by salt stress. S. cerevisiae deletion mutant strains: Δnha1 [12] and Δnhx1 [13] increased in tolerance to NaCl (1.2 M) in contrast to the pYES2-vector, without enzyme substrate fed into the culture medium, indicating one possibility that the MpCHI could regulate the response of M. pinnata to salt stress directly through changing its mRNA level or protein level
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