Abstract:

Objective: The PIN1 gene plays a role in the growth and development of plant roots by regulating the polar transport of auxin. In this study, the full-length cloning and functional analysis of the PmPIN1 gene of Pinus massoniana was carried out in order to reveal the molecular mechanism of rooting for cutting propagation of P. massoniana. Method: The cDNA sequence of PmPIN1 gene of P. massoniana was successfully cloned by PCR and RACE. Bioinformatics software was used to analyze the nucleic acid sequence, amino acid sequence and construct a phylogenetic tree of PmPIN1 gene. The expression of PmPIN1 gene in young roots, annual branch, young leaf and flowers of 10-year-old P. massoniana was analyzed by real-time polymerase chain reaction. The PmPIN1 gene was overexpressed in tobacco(Nicotiana tabacum)by Agrobacterium infection method to obtain transgenic plants, and the phenotypic differences between transgenic plants and pCAMBIA-1302 blank vector were compared, and PmPIN1-GFP fluorescent protein was observed in transgenic tobacco roots by laser scanning confocal microscope. The expression of auxin in roots, rhizomes and leaves of transgenic tobacco and wild tobacco was determined by enzyme-linked immunosorbent assay (ELISA). Treatment of wild-type and transgenic tobacco with different concentrations of auxin polar transport inhibitor (1-N-naphthylphthalamic acid, NPA), the changes of auxin content in tobacco roots, rhizome junctions and leaves were analyzed. 16318-hGFP-PmPIN1 recombinant vector was transferred to Arabidopsis thaliana protoplasts by PEG-mediated method for subcellular localization analysis. Result: The PmPIN1 gene is 2 914 bp in length and contains a 2 085 bp ORF, which encodes a protein consisting of 695 amino acids, with a membrane transport protein at the N-terminus and C-terminus. The phylogenetic tree showed that P. massoniana and P. tabulaeformis are on the same branch of the development tree. The difference in expression of PmPIN1 in different tissues was found to be extremely significant by real-time polymerase chain reaction, high expression in annual branch and young roots, lowest in flowers; Transgenic tobacco obtained by Agrobacterium rhizogenes-mediated transformation had higher growth, increased rooting and longer root length compared with a blank vector. Laser confocal microscopy showed that there was obvious green fluorescence enrichment around the root of transgenic tobacco. ELISA was used to determine the content of auxin in the roots and rhizomes of transgenic tobacco, and the content was found higher than that in wild type, and less in leaves than in roots and rhizomes. After treatment with different concentrations of NPA, the auxin content of wild type tobacco roots changed extremely significantly, and the auxin content decreased with the increase of NPA treatment concentration. When the treatment concentration was 10 nmol·L^-1, the auxin content in leaves was the largest. When the concentration was 2 nmol·L^-1, the content of auxin in the rhizome junction increased more. After treatment with different concentrations of NPA, the changes of auxin content in different parts of transgenic tobacco did not reach extremely significant. Subcellular localization analysis indicated that PmPIN1 protein was mainly distributed on the cell membrane. Conclusion: PmPIN1 of P. massoniana and PIN1 of P. tabulaeformis had a higher affinity. PmPIN1 belongs to membrane protein. PmPIN1 transgenic tobacco increases the transport capacity of auxin from aboveground to underground. Transgenic tobacco reduces the effect of NPA on auxin content, indicating that PmPIN1 likely regulates the polar transport of auxin. PmPIN1-GFP green fluorescence was enriched in the middle of the root, PmPIN1 was highly expressed in young roots. Transgenic tobacco had more roots and longer roots than transgenic plasmid. The above findings suggest that the PmPIN1 gene plays an important role in root development.

Key words: Pinus massoniana, PmPIN1 gene, transgenic, functional analysis