Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.)
Abstract
:1. Introduction
2. Results
2.1. The Identified Anthocyanin Biosynthesis-Related Blueberry bHLH Genes
2.2. Physiochemical Properties Analysis of Blueberry bHLH Proteins
2.3. Gene Structrues of VcbHLH Genes and Conserved Conmains and Motifs in Their Encoded Proteins
2.4. Phylogenetic Analysis of Blueberry Anthocyanin Biosynthesis-Related bHLHs
2.5. Chromosomal Location Analysis of Blueberry Anthocyanin Biosynthesis-Related bHLH Genes
2.6. cis-Acting Elements and TFBSs Distribution in Promoters of Blueberry Anthocyanin Biosynthesis-Related bHLH Genes
2.7. Protein-Protein Interaction Analysis of Blueberry bHLH Proteins
2.8. Cloning and Expression Analysis of Blueberry Anthocyanin Biosynthesis-Related bHLH Genes
3. Discussion
3.1. The Identified Blueberry Anthocyanin Biosynthesis-Related bHLHs Contained Conserved bHLH Domains and Key Amino Acids Required for Their DNA Binding Activity and Functions
3.2. Blueberry Anthocyanin Biosynthesis-Related bHLHs Could Be Classified into Two Groups, and the Functions of Each Group Differed from Each Other
3.3. There Are Many Factors Influencing the Expression and Functions of VcbHLH Genes
4. Materials and Methods
4.1. Plant Materials
4.2. Identification of Anthocyanin Biosynthesis-Related Blueberry bHLH Genes
4.3. Bioinformatic Analysis of bHLH Genes and Their Encoded Proteins
4.4. Identification and Analysis of cis-Acting Elements and Transcription Factor Binding Sites in Promoter of Blueberry bHLH Genes
4.5. Gene Cloning and Sequencing of Blueberry bHLH Genes
4.6. Quantitative Real Time PCR Analysis of Blueberry bHLH Genes
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene Name | Gene ID | Homologous Gene Name | Homologous Gene ID | Identity/% | Reference |
---|---|---|---|---|---|
VcAN1 | VaccDscaff11-processed-gene-379.7 | SmelAN1 | SMEL_009g326640.1.01 | 77.12 | [33] |
AcbHLH42 | MH643775 | 74.42 | [34] | ||
MdbHLH3 | ADL36597.1 | 65.08 | [14,35] | ||
Noemi | Cs5g31400 | 63.08 | [15,16] | ||
SlAN1 | KR076778 | 52.03 | [37] | ||
IbbHLH2 | itf14g18730.t2 | 46.45 | [11] | ||
VcbHLH42-1 | VaccDscaff24-augustus-gene-24.28 | AcbHLH42 | MH643775 | 79.10 | [34] |
AtTT8 | At4G09820 | 73.21 | [13] | ||
MdbHLH3 | ADL36597.1 | 69.03 | [14,35] | ||
Noemi | Cs5g31400 | 67.25 | [15,16] | ||
BjTT8 | BjuB004115 | 58.48 | [38] | ||
SlAN1 | KR076778 | 54.77 | [37] | ||
SmelAN1 | SMEL_009g326640.1.01 | 50.14 | [33] | ||
VcbHLH42-2 | VaccDscaff15-augustus-gene-371.25 | AcbHLH42 | MH643775 | 79.37 | [34] |
MdbHLH3 | ADL36597.1 | 69.30 | [14,35] | ||
Noemi | Cs5g31400 | 67.11 | [15,16] | ||
SlAN1 | KR076778 | 54.77 | [37] | ||
SmelAN1 | SMEL_009g326640.1.01 | 49.72 | [33] | ||
VcbHLH42-3 | VaccDscaff19-augustus-gene-381.30 | AcbHLH42 | MH643775 | 79.23 | [34] |
MdbHLH3 | ADL36597.1 | 69.30 | [14,35] | ||
Noemi | Cs5g31400 | 67.11 | [15,16] | ||
SlAN1 | KR076778 | 54.77 | [36] | ||
SmelAN1 | SMEL_009g326640.1.01 | 49.72 | [33] | ||
VcbHLH1-1 | VaccDscaff28-augustus-gene-45.27 | PalbHLH1 | PAYT030711.1 | 57.12 | [38] |
PPLS1 | Seita.7G195400 | 53.85 | [17] | ||
SmelJAF13 | SMEL_008g319200.1.01 | 49.69 | [33] | ||
AtEGL3 | At1G63650 | 48.23 | [39] | ||
AtGL3 | At5G41315 | 47.77 | [39] | ||
ThMYC4E | KX914905 | 37.54 | [18] | ||
VcbHLH1-2 | VaccDscaff44-augustus-gene-0.19 | PalbHLH1 | PAYT030711.1 | 55.61 | [38] |
SmelJAF13 | SMEL_008g319200.1.01 | 48.55 | [33] | ||
AtEGL3 | At1G63650 | 46.95 | [39] | ||
AtGL3 | At5G41315 | 46.42 | [39] |
Protein Name | Gene ID | CDS Length/bp | Protein Size/aa | Molecular Weight/Da | PI | Instability Index | GRAVY | Subcellular Localization |
---|---|---|---|---|---|---|---|---|
VcAN1 | VaccDscaff11-processed-gene-379.7 | 2328 | 775 | 85,474.08 | 5.55 | 67.34 | −0.595 | Nuclear |
VcbHLH42-1 | VaccDscaff24-augustus-gene-24.28 | 2190 | 729 | 80,213.15 | 5.46 | 69.43 | −0.618 | Nuclear |
VcbHLH42-2 | VaccDscaff15-augustus-gene-371.25 | 2190 | 729 | 80,320.22 | 5.45 | 70.96 | −0.64 | Nuclear |
VcbHLH42-3 | VaccDscaff19-augustus-gene-381.30 | 2190 | 729 | 80,280.2 | 5.49 | 69.96 | −0.629 | Nuclear |
VcbHLH1-1 | VaccDscaff28-augustus-gene-45.27 | 1803 | 600 | 67,581.48 | 6.25 | 55.99 | −0.388 | Nuclear |
VcbHLH1-2 | VaccDscaff44-augustus-gene-0.19 | 1860 | 619 | 69,831.17 | 6.16 | 56.26 | −0.358 | Nuclear |
Target Gene | Primer Name | Primer Sequence | Target Length (bp) | Tm (°C) | Application |
---|---|---|---|---|---|
VcAN1 | VcAN1-F | GCAACCCTCTCTCTTTCACT | 2885 | 60.5 | Gene cloning |
VcAN1-R | CCATTTCATTACCGCAAGC | ||||
VcbHLH1-1 | VcbHLH1-1-F | GCACAGAATCAATGGCTTC | 2043 | 60 | |
VcbHLH1-1-R | CGTGTTTGTTGGTTTGGC | ||||
VcbHLH1-2 | VcbHLH1-2-F | TTCCCTTACCCATCTTCCT | 2415 | 60 | |
VcbHLH1-2-R | CTGTGTTTGTTGGTTTGGC | ||||
VcAN1 | VcAN1-qF | CACCCTCCACAGCCTCCGAA | 242 | 59 | qRT-PCR |
VcAN1-qR | CTATTCCGCCCCGTGTCAGC | ||||
VcbHLH1-1 | VcbHLH1-1-qF | CTTGGAATGGGGTGATGGGT | 210 | 59 | |
VcbHLH1-1-qR | CACTCGGTATCGGTCAGGTC | ||||
VcbHLH1-2 | VcbHLH1-2-qF | AGCTGGAGAGAAGGGTCGAA | 155 | 59 | |
VcbHLH1-2-qR | GTCCCGTGCTTTCCTCTTGT | ||||
GAPDH | GAPDH-qF | ACTACCATCCACTCTATCACCG | 116 | 59 | Reference gene |
GAPDH-qR | AACACCTTACCAACAGCCTTG |
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Zhang, Y.; Liu, F.; Wang, B.; Wu, H.; Wu, J.; Liu, J.; Sun, Y.; Cheng, C.; Qiu, D. Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.). Int. J. Mol. Sci. 2021, 22, 13274. https://doi.org/10.3390/ijms222413274
Zhang Y, Liu F, Wang B, Wu H, Wu J, Liu J, Sun Y, Cheng C, Qiu D. Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.). International Journal of Molecular Sciences. 2021; 22(24):13274. https://doi.org/10.3390/ijms222413274
Chicago/Turabian StyleZhang, Yongyan, Fan Liu, Bin Wang, Huan Wu, Junwei Wu, Jiapeng Liu, Yueting Sun, Chunzhen Cheng, and Dongliang Qiu. 2021. "Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.)" International Journal of Molecular Sciences 22, no. 24: 13274. https://doi.org/10.3390/ijms222413274