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Linkage mapping, molecular cloning and functional analysis of soybean gene Fg2 encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase

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Abstract

There are substantial genotypic differences in the levels of flavonol glycosides (FGs) in soybean leaves. The first objective of this study was to identify and locate genes responsible for FG biosynthesis in the soybean genome. The second objective was to clone and verify the function of these candidate genes. Recombinant inbred lines (RILs) were developed by crossing the Kitakomachi and Koganejiro cultivars. The FGs were separated by high performance liquid chromatography (HPLC) and identified. The FGs of Koganejiro had rhamnose at the 6″-position of the glucose or galactose bound to the 3-position of kaempferol, whereas FGs of Kitakomachi were devoid of rhamnose. Among the 94 RILs, 53 RILs had HPLC peaks classified as Koganejiro type, and 41 RILs had peaks classified as Kitakomachi type. The segregation fitted a 1:1 ratio, suggesting that a single gene controls FG composition. SSR analysis, linkage mapping and genome database survey revealed a candidate gene in the molecular linkage group O (chromosome 10). The coding region of the gene from Koganejiro, designated as GmF3G6″Rt-a, is 1,392 bp long and encodes 464 amino acids, whereas the gene of Kitakomachi, GmF3G6″Rt-b, has a two-base deletion resulting in a truncated polypeptide consisting of 314 amino acids. The recombinant GmF3G6″Rt-a protein converted kaempferol 3-O-glucoside to kaempferol 3-O-rutinoside and utilized 3-O-glucosylated/galactosylated flavonols and UDP-rhamnose as substrates. GmF3G6″Rt-b protein had no activity. These results indicate that GmF3G6″Rt encodes a flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase and it probably corresponds to the Fg2 gene. GmF3G6″Rt was designated as UGT79A6 by the UGT Nomenclature Committee.

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Acknowledgments

The authors are grateful to Dr. Joseph G. Dubouzet (Scion Research, New Zealand) for critical reading of the manuscript, and to Mr. Tetsuya Mori (RIKEN CSRS, Japan) for his technical support. This study was partially supported by the Japanese Government (MEXT) Scholarship (to F.R.R. and T.O.R) and the JSPS KAKENHI Grant Number 25440148 (to K.Y.-S.).

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Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan

    Felipe Rojas Rodas, Tito O. Rodriguez & Ryoji Takahashi

  2. Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, 305-0005, Japan

    Yoshinori Murai & Tsukasa Iwashina

  3. RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan

    Satoko Sugawara, Makoto Suzuki, Ryo Nakabayashi, Keiko Yonekura-Sakakibara & Kazuki Saito

  4. Showa Pharmaceutical University, Machida, Tokyo, 194-8543, Japan

    Junichi Kitajima

  5. National Institute of Crop Science, Tsukuba, Ibaraki, 305-8518, Japan

    Kyoko Toda & Ryoji Takahashi

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  1. Felipe Rojas Rodas
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Correspondence to Ryoji Takahashi.

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11103_2013_133_MOESM1_ESM.eps

Multiple alignment of flavonoid glycoside glycosyltransferases (GGTs). The PSPG motif is within the box. Amino acid residues conserved in flavonoid GGTs are highlighted in black. Amino acid residues conserved in flavonoid G6″GTs and G2″GTs are highlighted in red and blue, respectively. (EPS 1059 kb)

Chemical data for identification of flavonol glycosides. (DOC 51 kb)

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Rojas Rodas, F., Rodriguez, T.O., Murai, Y. et al. Linkage mapping, molecular cloning and functional analysis of soybean gene Fg2 encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase. Plant Mol Biol 84, 287–300 (2014). https://doi.org/10.1007/s11103-013-0133-1

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