Identification of the bHLH Gene Family and Selection of Genes Related to Color Formation in Camellia reticulata

doi:10.13560/j.cnki.biotech.bull.1985.2024-0257

Abstract

Abstract:

【Objective】The preliminary screening of bHLH genes related to C. reticulata flower color by bioinformatics method provides support for further study of C. reticulata flower color.【Method】Based on the full-length transcriptome data of C. reticulata, the members of the bHLH gene family were identified by bioinformatics method, and their physical and chemical properties, secondary structure, phylogenetic relationships and domains were analyzed. The second-generation transcriptome and metabolome data were used to mine the bHLH genes related to the flower color of C. reticulata. Real-time quantitative polymerase chain reaction（RT-qPCR）was used to verify the expression of bHLH gene during flowering of C. reticulata among different varieties.【Result】Total 71 CrbHLHs genes were identified in the full-length transcriptome of C. reticulata. Their molecular weight ranged from 22 994.58 to 102 996.96 Da, and their equipotential ranged from 4.98 to 9.51. All of them were hydrophilic proteins. The cluster analysis with Arabidopsis thaliana showed that the bHLH gene family members of C. reticulata could be distributed into 14 subfamilies. Multiple omics analysis showed that CrbHLH8, CrbHLH61 and CrbHLH63 were positively correlated with cyanidin, while CrbHLH59 was negatively correlated with cyanidin. CrbHLH13 and CrbHLH71 were positively correlated with proanthocyanidins, and CrbHLH8 and CrbHLH61 were members of the IIIf subfamily.【Conclusion】The 71 CrbHLHs family members were identified from the full-length transcriptome of C. reticulata, of which 6 CrbHLHs were related to C. reticulata flower color.

Key words: Camellia reticulata, bHLH gene family, flower color, phylogenetic tree, full-length transcriptome, bioinformatics

ZHOU Lin, HUANG Shun-man, SU Wen-kun, YAO Xiang, QU Yan. Identification of the bHLH Gene Family and Selection of Genes Related to Color Formation in Camellia reticulata[J]. Biotechnology Bulletin, 2024, 40(8): 142-151.

Figures/Tables 9

Table 1 Primers used for RT-qPCR

基因Gene	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
CrbHLH8	GCAGTTGTGCCCTCAACAAG	CACTCACCTCCATTGGCTGT
CrbHLH13	CGGCTGAATGCTCGTTGATG	CTGAAATTGGCGGTGTTGGG
CrbHLH59	ACCCCATTGAATTCCCCACC	TACAAATCCAGGATCCGCGG
CrbHLH61	GCAGTTGTGCCCTCAACAAG	CACTCACCTCCATTGGCTGT
CrbHLH63	TGTCCATTGGATGCTCACCC	CAGCACCATCTTGAGTCCGT
CrbHLH71	AGCCACGTACTGCTGAACAA	TCTGCGCTATTGCCAGAACA
CrActin	GTCTGTTCCCTCTCCTCCCT	GAAGGGATCGTTGACAGCGA

Fig. 1 Phylogenetic tree of bHLH gene family of C. reticulata and A. thaliana

Fig. 2 Phylogenetic tree of IIIf subgroup members of bHLH family in C. reticulata and other species

Fig. 3 Analysis of conserved motif and conserved domain of the bHLH gene family of C. reticulate A: Cluster analysis of bHLH gene family members of C. reticulata. B: Conserved motif analysis of bHLH gene members of C. reticulata. C: Conserved domain of the bHLH gene family members of C. reticulata

Fig. 4 Differential bHLH analysis of C. reticulate A: Veen map of C. reticulata varieties. B: Veen map of C. reticulata at different developmental stages. C: Differential bHLH gene combination heat map. S: C. reticulata ‘Shizitou’. T: C. reticulata ‘Tongzimian’. 1: Bud stage. 2: Full flowering stage. -1, -2, and -3 indicate three repetitions. The same below

Fig. 5 Floral phenotype（A）, and key pigment content（B）in C. reticulata ‘Shizitou’ and C. reticulata ‘Tongzimian’ Different lower letters indicate significant differences at P<0.05 level. The same below

Fig. 6 Network regulation of C. reticulata's bHLH with cyanidin glycosides and proanthocyanidins

Fig. 7 Analysis of gene expression pattern in the bHLH family of C. reticulata

Fig. 8 bHLH regulates the synthesis pathway of color pathways in C. reticulata

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