山苍子TCP基因鉴定及其调控萜类合成的研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0740

摘要/Abstract

摘要：

目的筛选并验证可能参与调控山苍子萜类合成的TCP转录因子。方法基于山苍子全基因组及果实不同发育时期的转录组数据，采用生物信息学方法对TCP转录因子的理化性质、染色体定位、基因结构、保守基序、顺式作用元件及物种内共线性关系进行系统分析，并利用RT-qPCR检测CIN亚家族成员在不同发育时期及组织中的表达模式，对关键转录因子进行亚细胞定位，并结合酵母单杂和瞬时转化技术验证其功能。结果共鉴定出30个TCP基因家族成员（LcTCPs），分布于9条染色体，命名为LcTCP1-LcTCP30，开放读码框长度504-1 755 bp，分子量17 711.98-63 421.45 Da。系统进化将其划分为PCF（17个）、CIN（7个）和CYC/TB1（6个）3个亚家族，保守基序分析结果与分组一致。基因扩张主要受全基因组复制事件驱动，各亚家族间成员高度保守。RT-qPCR结果显示，CIN亚家族中LcTCP6与LcTCP11在精油合成高峰期及果实中的表达量较高，提示其与萜类合成相关。其中LcTCP11与关键基因LcDXS5在果实发育过程中呈现相似的表达模式，并定位于细胞核。功能验证表明LcTCP11能够调控LcDXS5的表达。结论从山苍子基因组中共鉴定30个TCP转录因子，其中LcTCP11与萜类合成关键基因LcDXS5呈协同表达，且在果实中表达显著高于其他组织，推测其可能通过调控LcDXS5参与山苍子萜类合成。

关键词: 山苍子, TCP转录因子, LcTCP11, LcDXS5, 萜类合成, 转录调控, 表达模式

Abstract:

Objective To screen and validate TCP transcription factors potentially involved in regulating terpenoid biosynthesis in Litsea cubeba. Method Based on the whole-genome sequence of L. cubeba and transcriptome data from fruits at different developmental stages, bioinformatics approaches were employed to systematically analyze the physicochemical properties, chromosomal distribution, gene structures, conserved motifs, cis-acting elements, and collinearity of TCP transcription factors. RT-qPCR was applied to examine the expression patterns of CIN subfamily members in different tissues and fruit developmental stages. Subcellular localization of key transcription factors was performed, and their functions were further validated through yeast one-hybrid and transient expression assays. Result A total of 30 TCP gene family members (LcTCPs) were identified, unevenly distributed across nine chromosomes and designated LcTCP1 to LcTCP30. Their open reading frames ranged from 504 to 1 755 bp, with predicted molecular weights of 17 711.98–63 421.45 Da. These genes were classified into three subfamilies via phylogenetic analysis: PCF (17 members), CIN (7 members), and CYC/TB1 (6 members), consistent with the conserved motif patterns. Gene family expansion was mainly driven by whole-genome duplication events, and members within each subfamily had high conservation. RT-qPCR analysis revealed that LcTCP6 and LcTCP11 of the CIN subfamily showed relatively high expression in the fruits and during the peak stage of essential oil biosynthesis, suggesting their potential involvement in terpenoid metabolism. Notably, LcTCP11 showed an expression pattern similar to that of the key terpenoid biosynthetic gene LcDXS5 during fruit development and was localized in the nucleus. Functional assays confirmed that LcTCP11 regulated the expression of LcDXS5. Conclusion Thirty TCP transcription factors were identified from the L. cubeba genome. Among them, LcTCP11 showed co-expression with the key terpenoid biosynthetic gene LcDXS5 and was highly expressed in fruits compared with other tissues, suggesting that LcTCP11 may participate in regulating terpenoid biosynthesis in L. cubeba through regulating LcDXS5.

Key words: Litsea cubeba, TCP transcription factors, LcTCP11, LcDXS5, terpenoid biosynthesis, transcriptional regulation, expression pattern

倪飞飞, 陈益存, 高暝, 张盛剿, 彭方有, 陈涛梅, 赵耘霄, 汪阳东. 山苍子TCP基因鉴定及其调控萜类合成的研究[J]. 生物技术通报, 2026, 42(4): 227-238.

NI Fei-fei, CHEN Yi-cun, GAO Ming, ZHANG Sheng-jiao, PENG Fang-you, CHEN Tao-mei, ZHAO Yun-xiao, WANG Yang-dong. Identification of Litsea cubebaTCP Genes and Their Roles in the Regulation of Terpenoid Biosynthesis[J]. Biotechnology Bulletin, 2026, 42(4): 227-238.

图/表 9

图1 山苍子LcTCPs基因的染色体定位蓝色表示PCF亚家族，黄色表示CYC/TB1亚家族，红色表示CIN亚家族

Fig. 1 Chromosomal localization of LcTCP genesBlue indicates the PCF subfamily, yellow indicates the CYC/TB1 subfamily, and red indicates the CIN subfamily

图2 山苍子及拟南芥TCPs家族蛋白的系统发育进化树

Fig. 2 Phylogenetic tree of TCP proteins from L. cubeba and A. thaliana

图3 LcTCPs蛋白的保守基序及基因结构

Fig. 3 Conserved motif and gene structure prediction of the LcTCPs proteins

图4 LcTCPs基因的共线性关系粉色框表示染色体，蓝色线表示PCF亚家族基因共线性，红色线表示CIN亚家族基因共线性，黄色线表示CYC/TB1亚家族基因共线性，灰色背景是山苍子基因组所有基因中存在的片段重复基因

Fig. 4 Synteny relationship of LcTCP genesPink boxes indicate chromosomes, blue lines indicate syntenic relationships among PCF subfamily genes, red lines indicate those of the CIN subfamily, and yellow lines indicate those of the CYC/TB1 subfamily. The gray background indicates segmentally duplicated genes across the L. cubeba genome

图5 LcTCPs转录因子启动子顺式元件分析

Fig. 5 Cis-acting element analysis of LcTCP transcription factor promoters

图6 不同组织的表达模式及果实不同发育时期表达相关性A：6个山苍子CIN亚家族成员在果实不同发育时期及不同组织的表达模式；B：LcTCP6、LcTCP11、LcTCP19、LcTCP30与LcDXS5在果实不同发育阶段的表达相关性；F1：花后30 d；F2：花后60 d，精油合成高峰期；F3：花后90 d；F4：花后120 d。不同字母表示差异显著（P<0.05）

Fig. 6 Expression patterns in different tissues and correlation of expression at different fruit developmental stagesA: Expression patterns of 6 CIN subfamily members from L. cubeba at different fruit developmental stages and in various tissues. B: Expression correlation among LcTCP6, LcTCP11, LcTCP19, LcTCP30, and LcDXS5 at different fruit developmental stages. F1: 30 d after flowering; F2: 60 d after flowering, the peak period of essential oil synthesis; F3: 90 d after flowering; F4: 120 d after flowering. Different lower letters indicate significant differences (P<0.05)

图7 瞬时转化山苍子叶片LcTCP11及LcDXS5的表达水平OE1、OE2、OE3代表3个过表达株系，数据以均值±标准差（SD）表示，来自3次重复实验（*P<0.05，**P<0.01，***P<0.001，****P<0.000 1）

Fig. 7 Expression analysis of LcTCP11 and LcDXS5 in transiently transformed L. cubeba leavesOE1, OE2, and OE3 refer to three overexpression lines. Data are expressed as mean±SD from three replicates (*P<0.05, **P<0.01, ***P<0.001,and ****P<0.000 1)

图8 LcTCP11亚细胞定位A：载体示意图；B：空载亚细胞定位；C：LcTCP11-GFP融合表达定位

Fig. 8 Subcellular localization of LcTCP11A: Schematic diagram of the vector. B: Subcellular localization of the empty vector. C: Localization of LcTCP11-GFP fusion expression

图9 酵母单杂交试验验证LcTCP11对LcDXS5启动子TCP元件的识别作用图中红色字体分别代表启动子上的结合元件序列以及突变之后的元件序列。将同时表达pGADT7-LcTCP11和pAbAi-3×gtgggtcc的酵母细胞在含有AbA的选择培养基（SD/-Trp/-Ura）上于30 ℃培养48 h。待长出单个菌落后，将单个菌落用水按10¹‒10⁴倍稀释后，每个稀释度取10 μL点种到新的含有AbA的选择培养基（SD/-Trp/-Ura）上，继续培养48 h

Fig. 9 Y1H assays verifying the recognition of LcTCP11 to the TCP element in LcDXS5 promoterThe red text in the figure indicates the binding element sequences on the promoter and the corresponding mutated element sequences, respectively. Yeast cells co-expressing pGADT7-LcTCP11 and pAbAi-3×gtgggtcc were cultured on selective medium containing AbA (SD/-Trp/-Ura) at 30 ℃ for 48 h. Once single colonies had grown, they were diluted in water to 10¹-10⁴ fold, and 10 μL of each dilution was spotted onto fresh selective medium containing AbA (SD/-Trp/-Ura) for a further 48 h of cultivation

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