薰衣草花粒数调控相关LaCKX1基因的克隆及功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2026-0310

摘要/Abstract

摘要：

目的细胞分裂素氧化/脱氢酶（cytokinin oxidase/dehydrogenase, CKX）能不可逆地降解细胞分裂素（cytokinin， CK），在植物花芽分化中起关键调控作用。探究薰衣草LaCKX1基因在花穗发育中的功能，为解析细胞分裂素通路对薰衣草花粒数形成的调控机制提供理论依据。方法以薰衣草‘新农薰1号’花穗cDNA为模板，克隆LaCKX1基因，并运用生物信息学方法分析其蛋白理化性质及系统进化关系；通过烟草瞬时表达明确该蛋白的亚细胞定位；利用逆转录实时定量聚合酶链式反应（RT-qPCR）检测LaCKX1基因在薰衣草不同品种、不同组织及花穗发育时期中的表达水平；通过遗传转化拟南芥和薰衣草，验证LaCKX1基因的功能及对内源CK含量的影响。结果成功克隆LaCKX1基因（CDS区1 575 bp，编码524个氨基酸），定位于细胞膜。LaCKX1具有明显的组织特异性，在花穗中高表达，尤其在花穗发育初期表达水平较高，且在少花粒品种S1时期的表达量显著高于多花粒品种。此外，过表达LaCKX1基因导致拟南芥内源CK含量下降，引发根系变长、株高降低及小花数量减少等表型变化；过表达LaCKX1同样会使薰衣草叶片中CK含量降低，而通过基因沉默抑制该基因表达则使新生叶片中CK含量升高。结论 LaCKX1基因可能通过负调控薰衣草细胞分裂素的生物合成，参与调控其花穗发育进程。

关键词: 薰衣草, LaCKX1基因, 细胞分裂素, 细胞分裂素氧化/脱氢酶, 花粒数, 基因克隆, 功能验证

Abstract:

Objective Cytokinin oxidase/dehydrogenase (CKX) irreversibly degrades cytokinin (CK) and plays a key regulatory role in plant flower bud differentiation. This study explored the function of the LaCKX1 gene during Lavandula angustifolia spike development, providing a theoretical basis for revealing the regulatory mechanism of the cytokinin signaling pathway on floret number formation in L. angustifolia. Method The LaCKX1 gene was cloned from the flower spike cDNA of L. angustifolia cultivar ‘Xinnongxun No.1’. Its protein physicochemical properties and phylogenetic relationships were analyzed using bioinformatics methods. Subcellular localization of the LaCKX1 protein was determined by transient expression in Nicotiana benthamiana. The expression levels of LaCKX1 in different L. angustifolia varieties, tissues, and spike developmental stages were examined by real-time quantitative PCR (RT-qPCR). The function of LaCKX1 and its effect on endogenous CK content were verified through genetic transformation in Arabidopsis thaliana and L. angustifolia. Result The LaCKX1 gene (coding sequence of 1 575 bp, encoding 524 amino acids) was successfully cloned, and its encoded protein was localized to the cell membrane. LaCKX1 exhibited a distinct tissue-specific expression pattern, with high transcript levels in flower spikes, particularly during the early stages of flower spike development. Its expression was significantly higher in the low-grain cultivar than in the high-grain cultivar at the S1 stage. Furthermore, LaCKX1 overexpression in Arabidopsis resulted in decreased endogenous CK content, leading to phenotypic changes including longer roots, reduced plant height, and fewer flowers. Similarly, LaCKX1 overexpression reduced CK content in L. angustifolia leaves, whereas gene silencing increased CK content in newly formed leaves. Conclusion LaCKX1 may negatively regulate CK biosynthesis in L. angustifolia, thereby participating in the regulation of its flower spike development.

Key words: Lavandula angustifolia, LaCKX1 gene, cytokinin, cytokinin oxidase/dehydrogenase, floret number, gene cloning, functional verification

岳丽萍, 王爱凡, 王姗, 李雪龙, 陈宇翔, 苏秀娟. 薰衣草花粒数调控相关LaCKX1基因的克隆及功能验证[J]. 生物技术通报, 2026, 42(9): 238-247.

YUE Li-ping, WANG Ai-fan, WANG Shan, LI Xue-long, CHEN Yu-xiang, SU Xiu-juan. Cloning and Functional Analysis of LaCKX1 Gene Related to Regulation of Floret Number in Lavandula angustifolia[J]. Biotechnology Bulletin, 2026, 42(9): 238-247.

图/表 7

图1 薰衣草花穗不同发育时期S1：穗发育初期；S2：第一茎节开始发育；S3：第一茎节发育至一半；S4：第一茎节发育完全。下同

Fig. 1 Different flowering stages of L. angustifoliaS1: Early stage of panicle development. S2: First stem node begins to develop. S3: First stem node is half-developed. S4: First stem node is fully developed. The same below

图2 LaCKX1蛋白多重序列比对（A）及系统进化树（B）分析黑色、红色、蓝色部分分别表示同源性=100%、≥75%、≥50%；绿色框为FAD结构域，红色框为细胞分裂素结合结构域

Fig. 2 Multiple sequence alignment (A) and phylogenetic tree (B) of the LaCKX1 protein with CKX proteins from other speciesThe black, red, and blue parts indicate homology = 100%, ≥75%, and ≥50%, respectively. Green boxes are the FAD-binding domain, and red boxes are the cytokinin-binding domain

图3 LaCKX1的亚细胞定位

Fig. 3 Subcellular localization of LaCKX1

图4 LaCKX1在不同组织与不同花穗发育阶段的表达水平不同字母表示在0.05水平上差异显著（P<0.05）。下同

Fig. 4 Expression levels of LaCKX1 in different tissues and at different flower spike developmental stagesDifferent letters indicate significant differences at the 0.05 level (P<0.05). The same below

图5 LaCKX1转基因拟南芥PCR阳性鉴定与表型差异比较及CK含量变化A：转基因拟南芥PCR阳性鉴定（M：Marker 2000；1-3：LaCKX1基因过表达拟南芥抗性苗；4：阳性对照；5：阴性对照）；B：LaCKX1过表达拟南芥植株与野生型（WT）幼苗培养14 d根长对比；C：培养35 d株高表型；D：培养35 d株高统计分析；E：植物细胞分裂素（CK）含量测定

Fig. 5 PCR identification of positive transgenic LaCKX1Arabidopsis, comparison of phenotypic differences, and changes in CK contentA: PCR identification of positive transgenic Arabidopsis thaliana (M: 2000 DNA marker. 1-3: Resistant seedlings of LaCKX1-overexpressing Arabidopsis. 4: Positive control. 5: Negative control). B: Comparison of root length between LaCKX1-overexpressing Arabidopsis lines and wild-type (WT) seedlings at 14 d of culture. C: Plant height phenotype at 35 d of culture. D: Statistical analysis of plant height at 35 d of culture. E: Determination of plant cytokinin (CK) content

图6 LaCKX1基因过表达（A、B）与基因沉默（C、D）薰衣草的基因表达水平及CK含量检测A：LaCKX1过表达薰衣草株系与野生型（WT）中LaCKX1的表达水平；B：LaCKX1过表达薰衣草株系与野生型（WT）中CK含量；C：基因沉默薰衣草目的基因表达水平；D：基因沉默薰衣草CK含量

Fig. 6 Detection of gene expression and CK content in LaCKX1-overexpressed (A, B) and gene-silenced (C, D) L. angustifoliaA: Expression levels of LaCKX1 in LaCKX1-overexpressing L. angustifolia lines and wild-type (WT). B: Cytokinin (CK) contents in LaCKX1-overexpressing L. angustifolia lines and wild-type (WT). C: Expression level of the target gene in L. angustifolia gene-silenced lines. D: CK content in L. angustifolia gene-silenced lines

图7 LaCKX1调控薰衣草花粒数的作用模式图：促进作用；：抑制作用；：CK

Fig. 7 Model diagram of LaCKX1 regulating floret grain number in L. angustifolia: Promotional effect. : Inhibitory effect. : CK

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