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Cloning and expression analysis of a pollen preferential rapid alkalinization factor gene, BoRALF1, from broccoli flowers

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Abstract

Rapid alkalinization factors (RALFs) are recently reported active peptide hormones and are considered to play important roles in plant development. We previously identified a differentially expressed cDNA fragment between cabbage flower buds of sterility lines and its maintainer line, which showed significant homology to Arabidopsis RALFL9. The novel RALF cDNA (BoRALF1) was isolated from broccoli flower buds by EST assembly. The open reading frame (ORF) comprises 240 bp, encoding a small putative preprotein of 79 amino acids (molecular weight of 8.72 kDa and a pI of 7.8), which contains the mature polypeptide at its C terminus. BoRALF1 shares 70.3% identity with Arabidopsis RALFL9, but has only moderate similarity with functionally characterized RALFs (ranging from 16.2% to 38.0%). BoRALF1 shows typical features of RALFs, including the 28-aa signal peptide, typical arrangement of four position conserved cysteines, the YIXY motif and a similar secondary structure. RT-PCR studies of different tissues and promoter-GUS fusions confirmed that BoRALF1 is expressed strictly in mature pollen grains and in the anther cells around the loculi. Based on in vivo transient assays, we found that BoRALF1 appears to be largely localized in the plasma membrane. Although the function of BoRALF1 remains to be determined, our experiments confirm the presence of RALF peptide in broccoli, and suggest it could have a role in anther or pollen development.

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Authors and Affiliations

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Beijing, 100081, People’s Republic of China

    Guo-yu Zhang, Jian Wu & Xiao-wu Wang

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  1. Guo-yu Zhang
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  2. Jian Wu
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  3. Xiao-wu Wang
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Correspondence to Xiao-wu Wang.

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Fig. 1

Structure and Phylogenetic analysis of BoRALF1. A, Nucleotide and deduced amino acid sequences of BoRALF1. The putative CAAT, TATA and TGAC boxes in the 5′-flanking region are underlined. The GTGA and AGAAA sequences, which are similar to the conserved sequence motifs of some other pollen-specific genes, are shown by open boxes. The instability motifs (ATTTA) in the 3′-UTR are shown as dotted lines. The mature peptide sequence of BoRALF1 is gray-shadowed. B, Sequences were aligned using the DNASTAR software package (version 7.1.0). The transmembrane regions are boxed; four conserved Cys residues constituting proposed disulfide bridges are marked with asterisks. The triangle indicates the predicted SP cleavage site and the black arrow shows the Arg-Arg motif. The dashed and dotted lines show the conserved motif (YIXY) and the C-terminal domain as designated by Ref. 14. C, Phylogenetic tree of RALFs. Amino acid sequences were aligned using ClustalW and the tree was inferred using MEGA-4.1 with the neighbor-joining method. Bootstrapped values are shown. RALFL21 was used as an outgroup as it was the most distantly related to other RALFs. Accession numbers are shown in parentheses, except for the following: LeRALF, ice plant, cotton, pea and wheat [9]. (JPG 3241 kb)

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Zhang, Gy., Wu, J. & Wang, Xw. Cloning and expression analysis of a pollen preferential rapid alkalinization factor gene, BoRALF1, from broccoli flowers. Mol Biol Rep 37, 3273–3281 (2010). https://doi.org/10.1007/s11033-009-9912-9

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  • DOI: https://doi.org/10.1007/s11033-009-9912-9

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