北美鹅掌楸LtCHS基因的克隆及生物信息学与组织表达特征分析

doi:10.11707/j.1001-7488.20150505

摘要/Abstract

摘要：

【目的】鹅掌楸属花色种间差异较大,是研究木本植物花色形成与调控机制的较理想材料。查尔酮合成酶基因(CHS)是黄酮类化合物生物合成途径中的关键基因,与植物花色形成密切相关,同时又是研究科以下系统发育的理想基因,种间进化差异较大,因此CHS基因用于研究鹅掌楸属花色差异形成机制有较强的可行性。本研究克隆北美鹅掌楸的查尔酮合成酶基因,并对其进行生物信息学及组织表达分析,以期为鹅掌楸属树种花色的形成和调控机制研究提供参考。【方法】以北美鹅掌楸的花芽为材料,采用同源克隆和RACE技术克隆查尔酮合成酶基因(LtCHS),并进一步扩增该基因的基因组序列。对该基因进行生物信息学分析。采用半定量RT-PCR方法分析LtCHS基因在鹅掌楸属种间及不同组织中的表达水平。【结果】获得2个查尔酮合成酶基因:LtCHS 1与LtCHS2。LtCHS 1基因的cDNA全长875 bp,包含1个702 bp的ORF,其基因组DNA (gDNA)只含有1个外显子,没有内含子,编码233个氨基酸,蛋白质分子量为71.88 kDa,等电点为5.09。LtCHS2基因的cDNA全长1 457 bp,包括1个1 185 bp的ORF,其gDNA含有2个外显子和1个内含子,编码394个氨基酸,蛋白质分子量为120.0 kDa,等电点为 4.97。2个基因的组织表达分析结果显示,LtCHS基因不仅在同一树种不同组织间存在差异,而且在种间也存在表达差异。LtCHS 1基因仅在北美鹅掌楸中表达,LtCHS2基因在鹅掌楸中表达,在杂交鹅掌楸中二者均有表达。【结论】获得的2个LtCHS基因属于同一基因家族,但执行不同功能。二者在种间表达存在差异可能与鹅掌楸属花色形成有关。结合鹅掌楸属种间花色特征,可初步推测,LtCHS 1基因与LtCHS2基因可能分别调控不同类型花青素的合成。

关键词: 北美鹅掌楸, 查尔酮合成酶, 基因克隆, 半定量RT-PCR, 组织表达

Abstract:

【Objective】 With obvious inter-specific variation, Liriodendron trees are ideal woody plants for studying gene regulation on flower color. Chalcone synthase genes (CHS) play key roles in the pathway of flavonoid metabolism, and are closely related with plant flower. CHS genes are also ideal for phylogeny study within family due to its large differences between species. Therefore, CHS genes can be used to study the underlying causes for the differences in flower color. In the study, CHS genes were cloned from L. tulipifera, and its bioinformatics and tissue expression were analyzed to provide understanding of the mechanisms of formation and regulation of flower color in Liriodendron. 【Method】 CHS genes (LtCHS) from the flower buds of L. tulipifera were cloned using RACE (rapid amplification cDNA ends) method, and their corresponding genome DNA (gDNA) were also amplified. Based on bioinformatics analysis, semi-quantitative RT-PCR was used to analyze the expression levels of LtCHS genes among different species and different tissues.【Result】The full-length cDNA of LtCHS 1 and LtCHS 2 genes are 875 bp and 1 457 bp, with the open reading frame(ORF)of 702 bp and 1 185 bp respectively. LtCHS 1 gene contains only one exon and encodes 233 amino acids. Its encoding protein has a molecular weight of 71.88 kDa, with theoretical isoelectric point of 5.09. While LtCHS 2 gene consists of two exons and one intron, encoding 394 amino acids. The protein has a molecular weight of 120.0 kDa, with theoretical isoelectric point of 4.97. Analysis of tissue expression of the two genes showed that LtCHS genes had differences not only among tissues of the same species, but also among species. The LtCHS 1 gene was expressed only in L. tulipifera and LtCHS 2 gene was expressed only in L. chinense, while in L. chinense × tulipifera, both genes were expressed.【Conclusion】 The two LtCHS genes obtained in this paper belong to the same family but perform different functions. The expression differences of the LtCHS genes may be associated with color formation of Liriodendron. Considering the characteristics of flower color between species, LtCHS 1 and LtCHS 2 genes may regulate the synthesis of different types of anthocyanins.

Key words: Liriodendron tulipifera, chalcone synthase, gene cloning, semi-quantitative RT-PCR, tissue expression

中图分类号:

S718.46

罗群凤, 胥猛, 冯源恒, 徐嘉娟, 李火根. 北美鹅掌楸LtCHS基因的克隆及生物信息学与组织表达特征分析[J]. 林业科学, 2015, 51(5): 37-45.

Luo Qunfeng, Xu Meng, Feng Yuanheng, Xu Jiajuan, Li Huogen. Cloning and Bioinformatics of Chalcone Synthase Gene (CHS) in Liriodendron tulipifera and Characterization of Its Tissue Expression[J]. Scientia Silvae Sinicae, 2015, 51(5): 37-45.

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