Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain
Abstract
:1. Introduction
2. Results
2.1. Sequence Analysis of CYP75B3s and CYP75B4s
2.2. Yeast Expression of CYP75Bs and CYP75B4s and Enzyme Assays
2.3. Gene Expression Analysis
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Isolation of CYP75B3 and CYP75B4 cDNAs from Non-Pigmented and Pigmented Rice
4.3. Yeast Expression of CYP75B3s and CYP75B4s and Substrate Feeding Assay
4.4. Microsomal Protein Preparation
4.5. Standard Enzyme Assay
4.6. HPLC Analysis
4.7. Immunoblot Analysis
4.8. Gene Expression Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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OsF3′Hs | Substrates | ||||
---|---|---|---|---|---|
NAR | API | DHK | KAM | ||
CYP75B3-IM (CYP75B3-JJJ) | Km (µM) | 0.286 ± 0.015 | 0.072 ± 0.011 | 2.494 ± 0.350 | 0.110 ± 0.008 |
Vmax (μM∙min−1·mg−1) | 0.214 ± 0.015 | 0.131 ± 0.011 | 0.430 ± 0.05 | 0.240 ± 0.014 | |
Vmax/Km (1∙min−1·mg−1) | 0.746 ± 0.067 | 1.809 ± 0.168 | 0.173 ± 0.018 | 2.178 ± 0.282 | |
CYP75B3-HN | Km (μM) | 0.108 ± 0.009 | 0.085 ± 0.015 | 1.034 ± 0.107 | 0.098 ± 0.013 |
Vmax (μM∙min−1·mg−1) | 0.193 ± 0.029 | 0.161 ± 0.008 | 0.281 ± 0.023 | 0.270 ± 0.021 | |
Vmax/Km (1∙min−1·mg−1) | 1.796 ± 0.240 | 1.887 ± 0.156 | 0.271 ± 0.034 | 2.755 ± 0.323 | |
CYP75B4-IM | Km (μM) | ND | 0.240 ± 0.042 | * | 2.948 ± 0.330 |
Vmax (μM∙min−1·mg−1) | ND | 0.008 ± 0.001 | * | 0.007 ± 0.001 | |
Vmax/Km (1∙min−1·mg−1) | ND | 0.034 ± 0.003 | * | 0.002 ± 0.001 |
Target | Locus ID | Forward (5′ to 3′) | Reverse (5′ to 3′) |
---|---|---|---|
qPCR | |||
CYP75B3 | Os10g0320100 | ACGGATTCATCAACGAAAGG | AGCAGCACGCTTAGAAGGTC |
CYP75B4 | Os10g0317900 | TCTCCCATCCGCTTACAATA | ACCAATCTACCAACATACAACAA |
CHS | Os11g0530600 | GGGCTCATCTCGAAGAACAT * | CCTCATCCTCTCCTTGTCCA * |
CHI | Os03g0819600 | AATCGAGCTGCGAATTAACC * | CGCGATTTCTCCTTTCCTTT * |
FNSII | Os04g0101400 | GATTGGCAGTGCATGGACA | TTGTATTACGGTGCGTTACAGG |
F3H | Os04g0662600 | AGCACAGAAGCCCAAGTCTC * | CTTCGATTTTCGACGGAAGA * |
DFR | Os01g0633500 | GCGAGAAGGAACCGATACTG * | TCCAAATCTCGCATTGTGAA * |
ANS | Os01g0372500 | GCATCGAACGGAATGAGAAC * | TTCGCTTCCGTTGAACATTA * |
UBQ5 | Os01g0328400 | GAAGTAAGGAAGGAGGAGGA * | AAGGTGTTCAGTTCCAAGG * |
Cloning | |||
CYP75B3 | CACCATGGACGTTGTGCCTCTCCCGC | TACTCCATAAGCCGATGGAAGCAGC | |
CYP75B4 | CACCATGGAGGTCGCCGCCATGG | TGCAATATTGTAAGCGGATGGGAGAAG |
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Park, S.; Choi, M.J.; Lee, J.Y.; Kim, J.K.; Ha, S.-H.; Lim, S.-H. Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain. Int. J. Mol. Sci. 2016, 17, 1549. https://doi.org/10.3390/ijms17091549
Park S, Choi MJ, Lee JY, Kim JK, Ha S-H, Lim S-H. Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain. International Journal of Molecular Sciences. 2016; 17(9):1549. https://doi.org/10.3390/ijms17091549
Chicago/Turabian StylePark, Sangkyu, Min Ji Choi, Jong Yeol Lee, Jae Kwang Kim, Sun-Hwa Ha, and Sun-Hyung Lim. 2016. "Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain" International Journal of Molecular Sciences 17, no. 9: 1549. https://doi.org/10.3390/ijms17091549