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Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.

Di S, Yan F, Rodas FR, Rodriguez TO, Murai Y, Iwashina T, Sugawara S, Mori T, Nakabayashi R, Yonekura-Sakakibara K, Saito K, Takahashi R - BMC Plant Biol. (2015)

Bottom Line: GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee.Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. dishaokang@affrc.go.jp.

ABSTRACT

Background: Flavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.

Results: HPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].

Conclusion: GmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

No MeSH data available.


Related in: MedlinePlus

Outline and results of dCAPS analysis of GmF3G2″Gt in soybean. (A) Schematic presentation of dCAPS analysis. Partial nucleotide sequences around the region polymorphic between Harosoy and Nezumisaya are exhibited. A mismatched base in the forward primer is highlighted in gray. Polymorphic nucleotides are highlighted in black. (B) Results of dCAPS analysis of the parents and the recombinant inbred lines derived from a cross between Nezumisaya and Harosoy. PCR products amplified with dCAPS primers were digested by KpnI and the digests were separated on an 8% polyacrylamide gel. ϕ, molecular marker ϕx174/HaeIII; N, Nezumisaya; H, Harosoy. FG pattern of the recombinant inbred lines is exhibited below the gel. H, Harosoy-type; N, Nezumisaya-type, 3, type 3; 4, type 4. The migration of size markers (bp) is shown to the left of the gel.
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Fig5: Outline and results of dCAPS analysis of GmF3G2″Gt in soybean. (A) Schematic presentation of dCAPS analysis. Partial nucleotide sequences around the region polymorphic between Harosoy and Nezumisaya are exhibited. A mismatched base in the forward primer is highlighted in gray. Polymorphic nucleotides are highlighted in black. (B) Results of dCAPS analysis of the parents and the recombinant inbred lines derived from a cross between Nezumisaya and Harosoy. PCR products amplified with dCAPS primers were digested by KpnI and the digests were separated on an 8% polyacrylamide gel. ϕ, molecular marker ϕx174/HaeIII; N, Nezumisaya; H, Harosoy. FG pattern of the recombinant inbred lines is exhibited below the gel. H, Harosoy-type; N, Nezumisaya-type, 3, type 3; 4, type 4. The migration of size markers (bp) is shown to the left of the gel.

Mentions: PCR products with molecular size of 230 bp were amplified with the dCAPS primers in Nezumisaya and Harosoy (Figure 5). KpnI digestion generated a band of 213 bp in Harosoy, whereas the PCR amplicon of Nezumisaya was unaffected. Banding patterns co-segregated with FG patterns; RILs with FGs of the Harosoy type and ‘type 3’ had bands of Harosoy type, whereas RILs with FGs of the Nezumisaya type and ‘type 4’ had bands of Nezumisaya type (Figure 5).Figure 5


Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.

Di S, Yan F, Rodas FR, Rodriguez TO, Murai Y, Iwashina T, Sugawara S, Mori T, Nakabayashi R, Yonekura-Sakakibara K, Saito K, Takahashi R - BMC Plant Biol. (2015)

Outline and results of dCAPS analysis of GmF3G2″Gt in soybean. (A) Schematic presentation of dCAPS analysis. Partial nucleotide sequences around the region polymorphic between Harosoy and Nezumisaya are exhibited. A mismatched base in the forward primer is highlighted in gray. Polymorphic nucleotides are highlighted in black. (B) Results of dCAPS analysis of the parents and the recombinant inbred lines derived from a cross between Nezumisaya and Harosoy. PCR products amplified with dCAPS primers were digested by KpnI and the digests were separated on an 8% polyacrylamide gel. ϕ, molecular marker ϕx174/HaeIII; N, Nezumisaya; H, Harosoy. FG pattern of the recombinant inbred lines is exhibited below the gel. H, Harosoy-type; N, Nezumisaya-type, 3, type 3; 4, type 4. The migration of size markers (bp) is shown to the left of the gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494776&req=5

Fig5: Outline and results of dCAPS analysis of GmF3G2″Gt in soybean. (A) Schematic presentation of dCAPS analysis. Partial nucleotide sequences around the region polymorphic between Harosoy and Nezumisaya are exhibited. A mismatched base in the forward primer is highlighted in gray. Polymorphic nucleotides are highlighted in black. (B) Results of dCAPS analysis of the parents and the recombinant inbred lines derived from a cross between Nezumisaya and Harosoy. PCR products amplified with dCAPS primers were digested by KpnI and the digests were separated on an 8% polyacrylamide gel. ϕ, molecular marker ϕx174/HaeIII; N, Nezumisaya; H, Harosoy. FG pattern of the recombinant inbred lines is exhibited below the gel. H, Harosoy-type; N, Nezumisaya-type, 3, type 3; 4, type 4. The migration of size markers (bp) is shown to the left of the gel.
Mentions: PCR products with molecular size of 230 bp were amplified with the dCAPS primers in Nezumisaya and Harosoy (Figure 5). KpnI digestion generated a band of 213 bp in Harosoy, whereas the PCR amplicon of Nezumisaya was unaffected. Banding patterns co-segregated with FG patterns; RILs with FGs of the Harosoy type and ‘type 3’ had bands of Harosoy type, whereas RILs with FGs of the Nezumisaya type and ‘type 4’ had bands of Nezumisaya type (Figure 5).Figure 5

Bottom Line: GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee.Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. dishaokang@affrc.go.jp.

ABSTRACT

Background: Flavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.

Results: HPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].

Conclusion: GmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

No MeSH data available.


Related in: MedlinePlus