Cloning, Subcellular Localization and Expression Analysis of CnbHLH79 Transcription Factor from Camellia nitidissima

doi:10.13560/j.cnki.biotech.bull.1985.2022-1562

Abstract

Abstract:

In order to explore the regulation mechanism of CnbHLH79 in the flower color formation of Camellia nitidissima, the coding sequence of CnbHLH79 was cloned and analyzed by bioinformatics, and the subcellular localization of CnbHLH79 protein was performed. Subsequently the expression pattern of CnbHLH79 was analyzed by fluorescence quantitative PCR（RT-qPCR）in different tissues and petals of different flowering stages, and the correlation between the expression of CnbHLH79 and value of chromaticity b* and the content of flavonoid compounds was analyzed. It was found that the coding sequence of CnbHLH79, with bHLH_AtBPE_like conserved domain, was 855 bp encoding 284 amino acids. CnbHLH79 had the closest relationship with bHLH79 protein of Camellia sinensis. Subcellular localization analysis showed that CnbHLH79 mainly had functions in the nucleus. CnbHLH79 highly expressed in the roots and flowers of C. nitidissima and gradually decreased at flowering stages by RT-qPCR. In addition, correlation coefficient between the expression of CnbHLH79 and the value of chromaticity b* and content of Qu7G was -0.92 and -0.70 respectively, which had negative relationship. This study lays a foundation for elucidating the mechanism of CnbHLH79 transcription factor regulating the synthesis of flavonoids in C. nitidissima and the mechanism of regulating the yellow color of petals.

Key words: Camellia nitidissima, CnbHLH79 transcription factor, flower color formation, regulation of flavonoid synthesis, subcellular localization, quantitative analysis of expression, correlation analysis

LI Bo, LIU He-xia, CHEN Yu-ling, ZHOU Xing-wen, ZHU Yu-lin. Cloning, Subcellular Localization and Expression Analysis of CnbHLH79 Transcription Factor from Camellia nitidissima[J]. Biotechnology Bulletin, 2023, 39(8): 241-250.

Figures/Tables 10

Fig. 1 Different cultures of C. nitidissima for fluorescence quantitative analysis The cultures of C. nitidissima are lateral root, stem and leave from left to right. The different flowering stages are young bud（A）, early bud（B）, chromogenic stages of flowering（C）, the half-opening stage（D）and blooming stage（E）

Table 1 Primers used for cloning, subcellular localization and quantitative analysis of CnbHLH79

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Use
CnbHLH79-F	GATCCTCCGATAATCAATTTGACCTCTTTC	克隆Clone
CnbHLH79-R	TGCTGCTCTATCAAAACTGCTACCAAC
CnbHLH79-F	GGAACACCAACATTTGATGC	定量表达Quantitative expression
CnbHLH79-R	ATGAAGCCATTCGGTTTGT
CnbHLH79-F	ACTAGGGTCTCGCACCATGGATCCTCCGATAATCAATTTGACCTCTTTC	亚细胞定位Subcellular localization
CnbHLH79-R	ACTAGGGTCTCTCGCC TGCTGCTCTATCAAAACTGCTACCAAC
18S rRNA-F	CAACCATAAACGATGCCGA	内参基因Reference gene
18S rRNA-R	AGCCTTGCGACCATACTCC

Fig. 2 PCR amplification of CnbHLH79 gene cDNA in C. nitidissima

Fig. 3 Prediting functional domain of protein CnbHLH79 and amino acid multiple sequence aligning in C. nitidissima A: Protein functional domains prediction（bHLH_AtBPE_like: conserved domains; bHLH_SF: superfamily）. B: Homology comparison of putative amino acids sequence. The sequence underlined in red: conserved domains of bHLH_SF superfamily; conserved amino acids of bHLH_SF superfamily contained by bHLH79 protein; α1 and α2: α-helix; CnbHLH79: bHLH79 transcription factor of C. nitidissima; AtbHLH79: bHLH79 transcription factor of Arabidopsis thaliana（AT5G62610.1）; CsbHLH79_like: bHLH79 transcription factor of C. sinensis（XP_028119408.1）; ArBPEp: BPEp transcription factor of A. rufa（GFS32687.1）

Fig. 4 Tertiary structure prediction of CnbHLH79 protein in C. nitidissima

Fig. 5 Phylogenetic tree of protein CnbHLH79 in C. nitidissima

Fig. 6 Subcellular localization of the protein CnbHLH79（Bar = 50 μm）

Fig. 7 Relative expressions of CnbHLH79 in different culture ** indicates extremely significant difference in the expression of stem, leave and flower compared with root（P<0.01）. Reference gene: 18S rRNA; n=3

Fig. 8 Expressions of CnbHLH79 at different flowering stages A: Young bud; B: early bud; C: chromogenic stages of flowering; D: the half-opening stage; E: blooming stage. *** indicates the expressions of C, D, and E shows extremely significant difference compared with that of A（P<0.001）. ns means no significant difference. Reference gene: 18S rRNA; n=3

Fig. 9 Correlation analysis between expression of CnbH-LH79 and color index, content of flavonoid compounds

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