金花茶CnbHLH79转录因子的克隆、亚细胞定位及表达分析

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

摘要/Abstract

摘要：

为探究CnbHLH79转录因子在金花茶花色形成中的作用机理，克隆了金花茶CnbHLH79转录因子的编码序列，对其进行生物信息学和亚细胞定位分析，利用荧光定量PCR技术分析了CnbHLH79转录因子在金花茶不同组织，以及不同发育阶段的花瓣中的表达模式，并分析CnbHLH79转录因子的表达量与色相b*、类黄酮物质含量的相关关系。研究结果表明，CnbHLH79转录因子的开放阅读框长度为855 bp，共编码284个氨基酸，具有bHLH_AtBPE_like保守结构域。系统进化分析发现，金花茶CnbHLH79蛋白与茶树bHLH79蛋白的亲缘关系最近。亚细胞定位分析显示，CnbHLH79转录因子主要在细胞核中发挥功能。对CnbHLH79转录因子进行实时荧光定量PCR分析发现，该转录因子在金花茶的根、花等组织中表达量较高；还发现在花朵开放过程中，CnbHLH79转录因子的表达量总体呈先高后低，逐步下降的趋势；此外，CnbHLH79的表达量与色相b*值、槲皮素-7-O-葡糖苷的相关性系数分别为-0.92、-0.7，它们之间的负相关性较高。本研究将为阐明CnbHLH79转录因子在金花茶中调控类黄酮的合成机制，以及调控花瓣显黄色的作用机理奠定基础。

关键词: 金花茶, CnbHLH79转录因子, 花色形成, 类黄酮合成调控, 亚细胞定位, 表达定量, 相关分析

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

李博, 刘合霞, 陈宇玲, 周兴文, 朱宇林. 金花茶CnbHLH79转录因子的克隆、亚细胞定位及表达分析[J]. 生物技术通报, 2023, 39(8): 241-250.

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.

图/表 10

图1 用于荧光定量表达的金花茶不同组织从左到右分别是侧根、茎、叶；不同的开花时期分别为幼蕾期（A）、初蕾期（B）、显色期（C）、半开期（D）、盛开期（E）

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）

表1 CnbHLH79转录因子克隆、亚细胞定位以及表达定量所用引物

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

图2 金花茶CnbHLH79基因cDNA 的PCR扩增

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

图3 金花茶CnbHLH79蛋白功能结构域预测及氨基酸多重序列比对 A：蛋白功能结构域预测（bHLH_AtBPE_like：保守结构域；bHLH_SF：超基因家族）；B：氨基酸序列同源性比较；红色划线区域：bHLH_SF基因家族保守结构域，bHLH79蛋白在该区域的氨基酸序列高度保守；α1和α2：α-螺旋元件；CnbHLH79：金花茶bHLH79转录因子；AtbHLH79：拟南芥bHLH79转录因子（AT5G62610.1）；CsbHLH79_like：茶树bHLH79转录因子（XP_028119408.1）；ArBPEp：褐枝猕猴桃BPEp转录因子（GFS32687.1）

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）

图4 金花茶CnbHLH79蛋白的三维结构预测

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

图5 金花茶CnbHLH79蛋白系统发生树

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

图6 CnbHLH79蛋白的亚细胞定位

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

图7 CnbHLH79在不同组织中的基因表达量 **代表茎、叶、花的表达量相对于根，差异极显著（P<0.01）；内参基因：18S rRNA；n=3

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

图8 CnbHLH79转录因子在不同开花时期的表达量 A：幼蕾期；B：初蕾期；C：显色期；D：半开期；E：盛开期；***代表C、D、E的表达量相对于A，差异极显著（P<0.001）；ns代表没有显著区别；内参基因：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

图9 CnbHLH79的表达量与花色指标、类黄酮化合物的相关性分析

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

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