滇山茶CrbHLH59基因克隆及其负调控花青素合成的功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2026-0514

摘要/Abstract

摘要：

目的明确滇山茶（Camellia reticulata）bHLH（basic helix-loop-helix）XII亚族成员CrbHLH59在花色形成中的功能及机制，为定向改良花色奠定理论基础。方法以滇山茶栽培品种‘童子面’（C. reticulata ‘Tongzimian’）为材料，利用RT-PCR技术克隆CrbHLH59基因，并对其进行生物信息学分析和表达模式分析。通过亚细胞定位实验确定CrbHLH59蛋白分布，同时构建烟草（Nicotiana tabacum）异源过表达株系分析其表型，结合靶向代谢组学分析过表达对烟草花冠中花青素及相关类黄酮代谢的影响，并通过RT-qPCR技术进一步检测花色合成途径关键基因的表达水平。结果 CrbHLH59基因序列全长为1 521 bp，编码506个氨基酸。系统发育分析结果表明，CrbHLH59与拟南芥（Arabidopsis thaliana）的AtbHLH48和AtbHLH60及柬埔寨龙血树（Dracaena cambodiana）的DcbHLH37共同聚类于bHLH转录因子家族的XII亚族。RT-qPCR结果显示，CrbHLH59在‘童子面’盛花期特异性高表达。亚细胞定位实验进一步证实CrbHLH59蛋白定位于细胞核。过表达CrbHLH59的转基因烟草花冠颜色显著变浅。与野生型烟草相比，OE-CrbHLH59烟草花冠中所有检测到的花青素组分含量均显著降低，同时多数黄酮类化合物和上游苯丙酸类物质的含量也显著减少。RT-qPCR结果进一步证实，在OE-CrbHLH59烟草花冠中NtPAL和Nt4CL表达水平显著下调。结论滇山茶CrbHLH59可能通过下调花色合成上游关键基因NtPAL和Nt4CL的表达，负向调控花青素及相关类黄酮代谢物的积累。

关键词: 滇山茶, CrbHLH59, 花色, 功能验证, XII亚族, 花青素, 负调控, 苯丙烷代谢

Abstract:

Objective The present study aims to elucidate the function and mechanism of CrbHLH59, a member of the bHLH subfamily XII, in the pigmentation of Camellia reticulata flowers. The findings provide a theoretical basis for targeted improvements in flower color in this species. Method The C. reticulata ‘Tongzimian’ cultivar was used as experimental material. The CrbHLH59 gene was cloned by RT-PCR, and bioinformatics analysis as well as expression profiling were performed. Subcellular localization experiments were conducted to determine the distribution of the CrbHLH59 protein. Meanwhile, heterologous overexpression lines were generated in Nicotiana tabacum, and their phenotypes were analyzed. Targeted metabolomics was combined to assess the impact of overexpression on anthocyanin and related flavonoid metabolism in tobacco corollas, and RT-qPCR was further used to detect the expression levels of key genes in the flower color synthesis pathway. Result The full-length CrbHLH59 gene was 1 521 bp and encoded 506 amino acids. Phylogenetic analysis showed that CrbHLH59 clustered with AtbHLH48 and AtbHLH60 from Arabidopsis thaliana and DcbHLH37 from Dracaena cambodiana in subfamily XII of the bHLH family. RT-qPCR revealed that CrbHLH59 was highly expressed during peak flowering in ‘Tongzimian’. Subcellular localization further confirmed that CrbHLH59 was localized in the nucleus. Overexpression experiments in transgenic tobacco showed that the corolla color was significantly lighter in CrbHLH59-overexpressing plants. Compared with wild-type tobacco, the contents of all detected anthocyanin components in OE-overexpressing CrbHLH59 tobacco corollas were significantly reduced, while the contents of most flavonoids and upstream phenylpropanoids also decreased significantly. RT-qPCR further confirmed that the expression levels of NtPAL and Nt4CL were significantly downregulated in OE-CrbHLH59 tobacco corollas. Conclusion CrbHLH59 in C. reticulata may negatively regulate anthocyanin and related flavonoid accumulation by downregulating the expression of the key upstream genes NtPAL and Nt4CL of the flower color synthesis pathway.

Key words: Camellia reticulata, CrbHLH59, flower color, functional validation, subfamily XII, anthocyanins, negative regulation, phenylpropanoid metabolism

周麟, 苏文坤, 段浦东, 程方怡, 刘子嫣, 屈燕. 滇山茶CrbHLH59基因克隆及其负调控花青素合成的功能验证[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0514.

ZHOU Lin, SU Wen-kun, DUAN Pu-dong, CHENG Fang-yi, LIU Zi-yan, QU Yan. Cloning of the CrbHLH59 Gene in Camellia reticulata and Validation of Its Role in Downregulating Anthocyanin Synthesis[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0514.

图/表 5

图1 滇山茶CrbHLH59与其他物种bHLH蛋白的系统发育树及多序列比对分析A：滇山茶CrbHLH59与其他物种同源蛋白的系统发育树；B：CrbHLH59与其他物种bHLH蛋白序列比对分析

Fig. 1 Phylogenetic tree and multiple sequence alignment analysis of CrbHLH59 from C. reticulata and bHLH proteins from other speciesA: Phylogenetic tree of CrbHLH59from C. reticulata and its homologs from other species. B: Sequence alignment of CrbHLH59with bHLH proteins from other species

图2 滇山茶‘狮子头’与‘童子面’不同发育阶段的表型及生理分子特征A：滇山茶‘狮子头’和‘童子面’不同发育阶段表型；B：各发育时期花瓣颜色参数；不同小写字母表示组间差异显著（P<0.05）；C：各发育时期花瓣花青素含量；D：CrbHLH59在‘狮子头’和‘童子面’不同发育阶段的表达模式分析；E：表型参数、CrbHLH59表达量与花青素含量的相关性分析；F：CrbHLH59编码蛋白的亚细胞定位；S1：‘狮子头’花蕾期；S2：‘狮子头’盛花期；T1：‘童子面’花蕾期；T2：‘童子面’盛花期；下同

Fig. 2 Phenotypic, physiological, and molecular characteristics of C. reticulata 'Shizitou' and 'Tongzimian' at different developmental stagesA: Phenotypes of the C. reticulata varieties ‘Shizitou’ and ‘Tongzimian’ at different developmental stages. B: Petal color parameters at each developmental stage. Different lowercase letters indicate significant differences between groups (P<0.05). C: Anthocyanin content in petals at each developmental stage. D: Analysis of the expression patterns of CrbHLH59 in ‘Shizitou’ and ‘Tongzimian’ at different developmental stages. E: Correlation analysis of phenotypic parameters, CrbHLH59 expression level and anthocyanin content. F: Subcellular localization of protein encoded by CrbHLH59. S1: ‘Shizitou’ bud stage; S2: ‘Shizitou’ full bloom stage; T1: ‘Tongzimian’ bud stage; T2: ‘Tongzimian’ full bloom stage. The same below

图3 CrbHLH59转基因烟草的鉴定及表型分析A：CrbHLH59阳性转基因烟草植株的PCR鉴定结果（M：Marker；0：水；-：阴性对照；+：阳性对照；1‒20：PCR产物）；B：野生型和过表达CrbHLH59烟草花冠表型；C：野生型和过表达CrbHLH59烟草花冠颜色参数；D：半定量检测野生型和过表达CrbHLH59烟草花冠CrbHLH59表达情况；E：RT-qPCR检测烟草花冠CrbHLH59表达情况；WT：野生型；OE-CrbHLH59：过表达CrbHLH59植株；* P<0.05，** P<0.01

Fig. 3 Identification and phenotypic analysis of CrbHLH59 transgenic tobaccoA: PCR identification results of CrbHLH59-positive transgenic tobacco plants (M: Marker; 0: H₂O; -: negative control; +: positive control; 1-20: PCR products). B: Phenotypes of wild-type and CrbHLH59-overexpressing tobacco corollas. C: Color parameters of wild-type and CrbHLH59-overexpressing tobacco corollas. D: Semi-quantitative analysis of CrbHLH59 expression in wild-type and CrbHLH59-overexpressing tobacco corollas. E: RT-qPCR analysis of CrbHLH59 expression in tobacco corollas. WT: Wild-type; OE-CrbHLH59: overexpressing CrbHLH59 plants. * P<0.05, ** P<0.01

图4 野生型和过表达CrbHLH59烟草花冠靶向代谢组学分析A：花青素靶向代谢组二级分类物质含量图；B：黄酮靶向代谢组二级分类物质含量图。*表示存在显著差异；t检验，*P<0.05，**P<0.01，***P<0.001，****P<0.000 1。下同

Fig. 4 Metabolomic analysis of wild-type and CrbHLH59-overexpressing tobacco corollasA: Content profiles of anthocyanin-targeted metabolites by secondary classification. B: Content profiles of flavonoid-targeted metabolites by secondary classification. * indicates statistically significant differences; Student’s t-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1. The same below

图5 野生型和过表达CrbHLH59烟草花冠基因表达情况

Fig. 5 Gene expression in the corolla of wild-type and CrbHLH59-overexpressing tobacco plants

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