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目的克隆中国红豆杉altered phloem development(APL)基因,研究APL在中国红豆杉剥皮后树皮再生过程中的表达状况,以揭示其可能的调控作用机制。方法利用反转录PCR(RT-PCR)方法克隆得到3个中国红豆杉APL(TcAPLs)基因的cDNA全长序列,分别命名为TcAPL1、TcAPL2和TcAPL3,进一步利用生物信息学工具对其进行分析,最后利用半定量RT-PCR对中国红豆杉不同组织和qRT-PCR技术对其剥皮再生不同时期的TcAPLs进行表达分析。结果系统进化树结果显示,TcAPL1与TcAPL2编码的蛋白与川桑的APL蛋白亲缘关系最近,聚为一类;TcAPL3处在APL另一个大的独立分支上。组织表达谱分析表明TcAPL1和TcAPL22个基因主要在根、茎、叶片和韧皮部(含形成层)中均高表达;TcAPL3在叶片中表达量较高,而在根和木质部(含形成层)表达较低。在中国红豆杉剥皮再生过程中,TcAPL1和TcAPL22个基因的表达水平随着时间的延长表现为先上升后下降的趋势,均在36 d下降最明显;TcAPL3的表达则在整个组织再生过程中被抑制。结论克隆了3个TcALs基因,3个基因在中国红豆杉剥皮再生过程中均有响应,推测它们在中国红豆杉植物剥皮后组织再生中起一定的调控作用。
Objective To clone the gene of altered phloem development (APL) of Taxus chinensis to study the expression of APL in bark regeneration process of Taxus cuspidata, and to reveal its possible regulation mechanism. Methods TcAPL1 cDNA sequences were cloned by reverse transcription PCR (RT-PCR) and named as TcAPL1, TcAPL2 and TcAPL3, respectively, and further analyzed by bioinformatics tools. Finally, semi-quantitative RT-PCR was used to analyze the expression of TcAPLs from different tissues of Taxus chinensis and qRT-PCR at different stages of their regeneration. Results The phylogenetic tree showed that the proteins encoded by TcAPL1 and TcAPL2 were closest to the APL protein of Chuan Sang and clustered into one group; TcAPL3 was located on another large independent branch of APL. The TcAPL1 and TcAPL22 genes were mainly expressed in roots, stems, leaves and phloem (including the formation layer). TcAPL3 expression was higher in leaves and more in roots and xylem low. During the process of Taxus chinensis regeneration, the expression levels of TcAPL1 and TcAPL22 genes increased firstly and then decreased with time, and they all decreased most significantly at 36 days. The expression of TcAPL3 was up-regulated during the whole tissue regeneration process inhibition. Conclusion Three TcALs genes were cloned and all three genes responded to the regeneration process of Taxus cuspidata. It is speculated that they play a regulatory role in tissue regeneration after Taxus chinensis plantation.