A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean

Nishizawa, Keito; Kita, Yoshio; Kitayama, Masahiko; Ishimoto, Masao

doi:10.1007/s00299-006-0210-x

Cited by 57 publications

(38 citation statements)

References 16 publications

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“…First, an expression cassette of a red fluorescent protein (DsRed2) was retrieved from pUR (Nishizawa et al, 2006) by XbaI and KpnI digestion and then blunt-ended with T4 DNA polymerase. To generate a binary vector expressing DsRed2 (pPZP:DsRed), the DsRed2 expression cassette was inserted into pPZP2028 (Kuroda et al, 2010) that is a T-DNA binary vector based on pPZP202 (Hajdukiewicz et al, 1994), which had been digested with BamHI and HindIII and blunt-ended.…”

Section: Complementation Analysis Of Sg-1 In Transgenic Soybeansmentioning

confidence: 99%

The Sg-1 Glycosyltransferase Locus Regulates Structural Diversity of Triterpenoid Saponins of Soybean

et al. 2012

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Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar-dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-ag, the Sg-1 a allele encodes the xylosyltransferase UGT73F4, whereas Sg-1 b encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1 a and Gly-138 in Sg-1 b proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-1 0 is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.

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Section: Complementation Analysis Of Sg-1 In Transgenic Soybeansmentioning

confidence: 99%

The Sg-1 Glycosyltransferase Locus Regulates Structural Diversity of Triterpenoid Saponins of Soybean

et al. 2012

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“…A transgenic line transformed with the empty vector, pUHR (Nishizawa et al, 2006a(Nishizawa et al, , 2006b, was used as a control.…”

Section: Western-blot Analysismentioning

confidence: 99%

Accumulation of β-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between α′- and α-Subunits

et al. 2012

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b-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean b-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the a#-and a-subunits of b-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the b-conglycinin complexes containing the disulfide-linked a#/a-subunits were complexes of more than 720 kD. The a#-and a-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between a#/a-subunits residing in different b-conglycinin hexamers, but the binding of P34 to a#-and a-subunits reduces the linkage between b-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when b-conglycinin was expressed under nonreducing conditions. The large majority of seed storage proteins from the leguminous species are globulins with sedimentation coefficients of 7 to 8 S (vicilin) and 11 to 12 S (legumin). The corresponding proteins from soybean (Glycine max) are called b-conglycinin and glycinin, respectively (Casey, 1999). The b-conglycinin from soybean seeds is isolated as a trimer with sedimentation coefficients between 7 and 8 S (Thanh and Shibasaki, 1976;Nielsen and Nam, 1999). The size of b-conglycinin complexes depends on the ionic strength of the solution. b-Conglycinin is a trimer at higher than 0.2 M ionic strength, but at neutral pH, it associates into hexamers at decreasing ionic strength (Thanh and Shibasaki, 1979). The b-conglycinin trimers are mixtures of isoforms consisting of different combinations of the three types of subunits, designated a#, a with M r of 57,000, and b with M r of 42,000 (Thanh and Shibasaki, 1978;Davies et al., 1985;Nielsen and Nam, 1999). The a#-, a-, and b-subunits are synthesized in the endoplasmic reticulum (ER) of the cotyledon cell as polypeptides with a signal peptide and a propeptide (prepro a#-and prepro a-subunits) or as polypeptides with only a signal peptide (pre-b-subunit; Sengupta et al

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“…Despite the discovery of a multiplicity of fluorescent proteins in the red spectral range in recent years [6], so far almost exclusively different forms of DsRed have been used for studies in molecular cell biology in plants [8-12]. These proteins are applied in dual-labeling experiments together with GFP or alone to report on promoter activity or as a marker in transgenic plants.…”

Section: Introductionmentioning

confidence: 99%

The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants

Forner

Binder

2007

BMC Plant Biol

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Background: Intrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual-or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.

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A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean

Cited by 57 publications

References 16 publications

The Sg-1 Glycosyltransferase Locus Regulates Structural Diversity of Triterpenoid Saponins of Soybean

The Sg-1 Glycosyltransferase Locus Regulates Structural Diversity of Triterpenoid Saponins of Soybean

Accumulation of β-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between α′- and α-Subunits

The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants

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