vvcyp76f14  (Addgene inc)

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Gene structure and amino acid sequence alignment analysis of VvCYP76F14 proteins. ( A ) Gene structure of VvCYP76F14 s derived from three mutant lines. ( B ) Acid sequence alignment of VvCYP76F14 proteins.

Article Snippet: The CDSs of the VvCYP76F14 s from ‘Yantai 2-2-08’, ‘Yantai 2-2-19’, ‘Yantai 2-3-37’, ‘Merlot’, and ‘L35’ were synthesized and verified by GenScript Co., Ltd. (Nanjing, China).

Techniques: Sequencing, Derivative Assay, Mutagenesis

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Subcellular localization of VvCYP76F14. The CDS of VvCYP76F14 derived from ‘Yantai 2-2-08’ and ‘Yantai 2-2-08’ and further cloned into the pBWA(V)HS-ccdb-GLosgfp vector. The GV3101 strain harboring the pBWA(V)HS-CYP76F14-Glosgfp or the empty vector was infiltrated into Arabidopsis mesophyll protoplasts. The GFP fluorescence and chloroplast autofluorescence were observed using the excitation/emission wavelengths 470/510 nm and 620/660 nm, respectively. Scale bar = 10 μm.

Article Snippet: The CDSs of the VvCYP76F14 s from ‘Yantai 2-2-08’, ‘Yantai 2-2-19’, ‘Yantai 2-3-37’, ‘Merlot’, and ‘L35’ were synthesized and verified by GenScript Co., Ltd. (Nanjing, China).

Techniques: Derivative Assay, Clone Assay, Plasmid Preparation, Fluorescence

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: In vitro activity analysis of VvCYP76F14 in Escherichia coli . The in vitro enzymatic activity of VvCYP76F14 was assayed by measuring the amount of remnant substrate. Data are shown as means ± SE ( n = 3). Letters indicate significant differences among five VvCYP76F14s at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test.

Article Snippet: The CDSs of the VvCYP76F14 s from ‘Yantai 2-2-08’, ‘Yantai 2-2-19’, ‘Yantai 2-3-37’, ‘Merlot’, and ‘L35’ were synthesized and verified by GenScript Co., Ltd. (Nanjing, China).

Techniques: In Vitro, Activity Assay

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Transient expression of VvCYP76F14 in berries of D299T mutant lines. The content levels of linalool, ( E )-8-hydroxylinalool, ( E )-8-oxolinalool, and ( E )-8-carboxylinalool in berries of ‘Yantai 2-2-08’ ( A ), ‘Yantai 2-2-19’ ( B ), and ‘Yantai 2-3-37’ ( C ) were assayed by UPLC-MS. Data are shown as means ± SE ( n = 3). Letters indicate significant differences among five VvCYP76F14s at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test.

Article Snippet: The CDSs of the VvCYP76F14 s from ‘Yantai 2-2-08’, ‘Yantai 2-2-19’, ‘Yantai 2-3-37’, ‘Merlot’, and ‘L35’ were synthesized and verified by GenScript Co., Ltd. (Nanjing, China).

Techniques: Expressing, Mutagenesis

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Determination of wine bouquet precursors from 5 wine grape varieties or offspring lines.

Article Snippet: The CDSs of the VvCYP76F14 s from ‘Yantai 2-2-08’, ‘Yantai 2-2-19’, ‘Yantai 2-3-37’, ‘Merlot’, and ‘L35’ were synthesized and verified by GenScript Co., Ltd. (Nanjing, China).

Techniques:

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Gene structure and amino acid sequence alignment analysis of VvCYP76F14 proteins. ( A ) Gene structure of VvCYP76F14 s derived from three mutant lines. ( B ) Acid sequence alignment of VvCYP76F14 proteins.

Article Snippet: Sequencing-verified VvCYP76F14 was cloned into the pMDC32-HPB (Addgene: 32078) to generate the recombinant plasmid pMDC32-HPB- CYP76F14 .

Techniques: Sequencing, Derivative Assay, Mutagenesis

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Subcellular localization of VvCYP76F14. The CDS of VvCYP76F14 derived from ‘Yantai 2-2-08’ and ‘Yantai 2-2-08’ and further cloned into the pBWA(V)HS-ccdb-GLosgfp vector. The GV3101 strain harboring the pBWA(V)HS-CYP76F14-Glosgfp or the empty vector was infiltrated into Arabidopsis mesophyll protoplasts. The GFP fluorescence and chloroplast autofluorescence were observed using the excitation/emission wavelengths 470/510 nm and 620/660 nm, respectively. Scale bar = 10 μm.

Article Snippet: Sequencing-verified VvCYP76F14 was cloned into the pMDC32-HPB (Addgene: 32078) to generate the recombinant plasmid pMDC32-HPB- CYP76F14 .

Techniques: Derivative Assay, Clone Assay, Plasmid Preparation, Fluorescence

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: In vitro activity analysis of VvCYP76F14 in Escherichia coli . The in vitro enzymatic activity of VvCYP76F14 was assayed by measuring the amount of remnant substrate. Data are shown as means ± SE ( n = 3). Letters indicate significant differences among five VvCYP76F14s at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test.

Article Snippet: Sequencing-verified VvCYP76F14 was cloned into the pMDC32-HPB (Addgene: 32078) to generate the recombinant plasmid pMDC32-HPB- CYP76F14 .

Techniques: In Vitro, Activity Assay

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Transient expression of VvCYP76F14 in berries of D299T mutant lines. The content levels of linalool, ( E )-8-hydroxylinalool, ( E )-8-oxolinalool, and ( E )-8-carboxylinalool in berries of ‘Yantai 2-2-08’ ( A ), ‘Yantai 2-2-19’ ( B ), and ‘Yantai 2-3-37’ ( C ) were assayed by UPLC-MS. Data are shown as means ± SE ( n = 3). Letters indicate significant differences among five VvCYP76F14s at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test.

Article Snippet: Sequencing-verified VvCYP76F14 was cloned into the pMDC32-HPB (Addgene: 32078) to generate the recombinant plasmid pMDC32-HPB- CYP76F14 .

Techniques: Expressing, Mutagenesis

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Gene structure and amino acid sequence alignment analysis of VvCYP76F14 proteins. ( A ) Gene structure of VvCYP76F14 s derived from three mutant lines. ( B ) Acid sequence alignment of VvCYP76F14 proteins.

Article Snippet: To evaluate the enzymatic activity of VvCYP76F14 in berries, the CDS of VvCYP76F14 was isolated from ‘L35’, ‘Merlot’, or D299T mutant lines, respectively, and synthesized by GenScript Co., Ltd. (Nanjing, China).

Techniques: Sequencing, Derivative Assay, Mutagenesis

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Subcellular localization of VvCYP76F14. The CDS of VvCYP76F14 derived from ‘Yantai 2-2-08’ and ‘Yantai 2-2-08’ and further cloned into the pBWA(V)HS-ccdb-GLosgfp vector. The GV3101 strain harboring the pBWA(V)HS-CYP76F14-Glosgfp or the empty vector was infiltrated into Arabidopsis mesophyll protoplasts. The GFP fluorescence and chloroplast autofluorescence were observed using the excitation/emission wavelengths 470/510 nm and 620/660 nm, respectively. Scale bar = 10 μm.

Article Snippet: To evaluate the enzymatic activity of VvCYP76F14 in berries, the CDS of VvCYP76F14 was isolated from ‘L35’, ‘Merlot’, or D299T mutant lines, respectively, and synthesized by GenScript Co., Ltd. (Nanjing, China).

Techniques: Derivative Assay, Clone Assay, Plasmid Preparation, Fluorescence

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: In vitro activity analysis of VvCYP76F14 in Escherichia coli . The in vitro enzymatic activity of VvCYP76F14 was assayed by measuring the amount of remnant substrate. Data are shown as means ± SE ( n = 3). Letters indicate significant differences among five VvCYP76F14s at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test.

Article Snippet: To evaluate the enzymatic activity of VvCYP76F14 in berries, the CDS of VvCYP76F14 was isolated from ‘L35’, ‘Merlot’, or D299T mutant lines, respectively, and synthesized by GenScript Co., Ltd. (Nanjing, China).

Techniques: In Vitro, Activity Assay

Journal: Genes

Article Title: D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape

doi: 10.3390/genes15111478

Figure Lengend Snippet: Transient expression of VvCYP76F14 in berries of D299T mutant lines. The content levels of linalool, ( E )-8-hydroxylinalool, ( E )-8-oxolinalool, and ( E )-8-carboxylinalool in berries of ‘Yantai 2-2-08’ ( A ), ‘Yantai 2-2-19’ ( B ), and ‘Yantai 2-3-37’ ( C ) were assayed by UPLC-MS. Data are shown as means ± SE ( n = 3). Letters indicate significant differences among five VvCYP76F14s at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test.

Article Snippet: To evaluate the enzymatic activity of VvCYP76F14 in berries, the CDS of VvCYP76F14 was isolated from ‘L35’, ‘Merlot’, or D299T mutant lines, respectively, and synthesized by GenScript Co., Ltd. (Nanjing, China).

Techniques: Expressing, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: Cytochrome P450 VvCYP76F14 dominates the production of wine bouquet precursors in wine grapes

doi: 10.3389/fpls.2024.1450251

Figure Lengend Snippet: SDS-PAGE of MBP fusion analysis of VvCYP76F14s. SDS-PAGE analysis of the recombinant VvCYP76F14 from the Full-bodied variety of ‘Yanniang No.2’ in E. coli . The pMAL-c6T-VvCYP76F14s (Biomarker Co., Ltd, Beijing, China) constructs were expressed in the E. coli BL21(DE3) strain. The protein standard marker (M) was loaded on the gel. Lane 1 represents purified MBP-VvCYP76F14 eluted from amylose column with maltose. Lane 2 represents purified VvCYP76F14 after TEV protease cleavage. Lane 3 represents isolated and partially purified VvCYP76F14.

Article Snippet: To enhance production and facilitate the folding of recombinant VvCYP76F14 proteins, the pMAL-c6T vector (New England Biolabs, Beijing, China) containing a maltose-binding protein (MBP) tag was used for heterologous expression ( ).

Techniques: SDS Page, Recombinant, Biomarker Assay, Construct, Marker, Purification, Isolation

Journal: Frontiers in Plant Science

Article Title: Cytochrome P450 VvCYP76F14 dominates the production of wine bouquet precursors in wine grapes

doi: 10.3389/fpls.2024.1450251

Figure Lengend Snippet: Enzyme kinetics of VvCYP76F14 and its site-directed mutant proteins (VvCYP76F14-SMs) using linalool, ( E )-8-hydroxylinalool and ( E )-8-oxolinalool as substrate, respectively. VvCYP76F14 was isolated from the Full-Bodied variety ‘Yanniang No.2’. The reaction product was collected and analyzed using HPLC-HRMS (Waters, Milford, MA, USA). Boiled protein (nonfunctional) served as a control. For the determination of kinetic parameters, substrate reduction was both qualitatively and quantitatively determined by HPLC-HRMS (Waters, Milford, MA, USA). The turnover number ( k cat ) and affinity ( k m ) were individually calculated. Data were presented as the means ± SE ( n = 3). Letters represent significant differences among VvCYP76F14 and VvCYP76F14-SMs at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test.

Article Snippet: To enhance production and facilitate the folding of recombinant VvCYP76F14 proteins, the pMAL-c6T vector (New England Biolabs, Beijing, China) containing a maltose-binding protein (MBP) tag was used for heterologous expression ( ).

Techniques: Mutagenesis, Isolation, Control

Journal: Frontiers in Plant Science

Article Title: Cytochrome P450 VvCYP76F14 dominates the production of wine bouquet precursors in wine grapes

doi: 10.3389/fpls.2024.1450251

Figure Lengend Snippet: Transient expression of Full-Bodied type VvCYP76F14 in three wine bouquet type varieties. The CDS of VvCYP76F14 was isolated from the Full-Bodied variety ‘Yanniang No.2’ and further cloned into the pMDC32-HPB (Addgene: 32078) to generate the recombinant plasmid pMDC32-HPB- CYP76F14 . The pMDC32-HPB- VvCYP76F14 overexpression vector and empty vector were transferred into Agrobacterium GV3101 (WEIDI, Shanghai, China) strain, respectively. The Agrobacterium GV3101 suspension with an OD 600 of 0.8 was injected into the berries of ‘Yanniang No.2’ at 110 DAFB (A) , ‘Marselan’ at 100 DAFB (B) , or ‘Italian Riesling’ at 90 DAFB (C) , respectively. After injection for 3 days, the contents of linalool, ( E )-8-hydroxylinalool, ( E )-8-oxolinalool and ( E )-8-carboxylinalool in the grape berries were determined using HPLC-HRMS. Data are presented as means ± SEs ( n = 3). Letters represent significant differences among wild type berries, berries transformed with empty vector, and berries transformed with VvCYP76F14 at a significance level of p ≤ 0.05, as determined using ANOVA followed by Fisher’s LSD test. FW means fresh weight.

Article Snippet: To enhance production and facilitate the folding of recombinant VvCYP76F14 proteins, the pMAL-c6T vector (New England Biolabs, Beijing, China) containing a maltose-binding protein (MBP) tag was used for heterologous expression ( ).

Techniques: Expressing, Isolation, Clone Assay, Recombinant, Plasmid Preparation, Over Expression, Suspension, Injection, Transformation Assay