Cloning, Characterization and Expression Analysis of the Phosphate Starvation Response Gene, ClPHR1, from Chinese Fir

Wanting Chen,Mulualem Tigabu,Xiangqing Ma,Jiaoyang Li,Ying Zhang,Ming Li,Ranhong Chen

doi:10.3390/f11010104

Wanting Chen, Mulualem Tigabu + Show 5 more

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https://doi.org/10.3390/f11010104

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Abstract

The study on the function and sequence of PHR1 (Phosphate Starvation Response gene 1) gene, which plays a central role in plant phosphorus (Pi) signal regulatory network, is of great significance to further study response mechanisms to Pi deficiency. In this work, the previously selected Pi-efficient Chinese fir clone M32 was used as research material to obtain the full-length sequence of ClPHR1 transcription factors in Chinese fir by RACE (Rapid Amplification of cDNA Ends) full-length cloning technique, and the structure, function and subcellular localization of ClPHR1 gene encoding protein were analyzed. The temporal and spatial expression characteristics of ClPHR1 transcription factors in Chinese fir under low Pi stress were also analyzed, and the overexpression of ClPHR1 gene in transgenic Arabidopsis thaliana was obtained to verify the function of ClPHR1 gene under low Pi stress. The results showed that the length of the ClPHR1 gene obtained by rapid amplification of cDNA ends technique was 1954 bp, of which 1512 bp was an open reading frame. ClPHR1 was predicted to be an unstable hydrophilic protein with only one possible transmembrane domain. The ClPHR1 gene had a highly conserved MYB-CC domain, which is similar to the PHR1 gene of other plants. Phylogenetic tree analysis showed that the sequence had high homology with PHR1 genes in the Prunus species. The ClPHR1 was expressed in all organs of Chinese fir, with the highest expression in the roots, followed by the leaves with the lowest expression in stems. ClPHR1 expression in roots was reduced dramatically at the beginning of Pi stress treatment and followed by an increase at 7days; in leaves, it increased dramatically at the beginning of Pi starvation treatment and showed a decreasing trend after 3 days; in stems, the expression level of ClPHR1 increased after 7 days of Pi stress treatment. The transient expression vector was introduced into plant cells, and it was found that ClPHR1 was located in the nucleus and was a MYB-CC transcription factor expressed in the cell nucleus. The ClPHR1 overexpression vector was constructed, and then introduced into Arabidopsis thaliana by agrobacterium infection inflorescence method. The expressions of Pi transporter genes, AtPHT1;1, AtPHT1;2, AtPHT1;8 and AtPHT1;9, was significantly higher in the overexpressing strain than that in the wild type strain. The results suggest that the ClPHR1 transcription factor could regulate the regulation of downstream Pi transporter gene and increase Pi utilization efficiency of the Chinese fir under Pi stress.

Highlights

Plant-available phosphorus (Pi) is deficient worldwide, in extremely old and highly weathered soils, as Pi can be immobilized into insoluble phosphate by calcium, iron, aluminum and other metal ions or adsorbed by soil colloids as closed-form phosphorus [1,2,3]
To isolate the putative PHR1 gene from Cunninghamia lanceolate, we compared soybean, wheat, and European rape conserved sequences to design the degenerate primer, and a 314 bp intermediate conserved fragment was obtained by PCR amplification using one-year-old Chinese fir root complementary DNA (cDNA) as a template
The phosphate starvation response gene, ClPHR1, in Chinese fir was successfully obtained by rapid amplification of cDNA ends (RACE) technique

Summary

Introduction

Plant-available phosphorus (Pi) is deficient worldwide, in extremely old and highly weathered soils, as Pi can be immobilized into insoluble phosphate by calcium, iron, aluminum and other metal ions or adsorbed by soil colloids as closed-form phosphorus [1,2,3]. Screening Pi-efficient genotypes that are efficient in acquiring Pi from the soil and its subsequent utilization are the most sustainable method of Pi management [12,13,14]. A variety of adaptive strategies have been developed in the long-term adaptation of plants to low Pi environments [15,16]. Transcription factors, such as WRKY75 [17,18], ZAT6 [19,20], MYB62 [21], BHLH32 [22,23], AtPHR1 [24,25] and PHR1-LIKE1 are the major regulators of Pi homeostasis [26,27]

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