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第3类亮氨酸拉链蛋白(ClassⅢHomeodomain Leucine Zipper,HD-ZipⅢ)主要参与维管组织形成、顶端分生组织的分化、胚胎形态发生及侧生器官极性建立等调控过程。本研究基于转录组测序数据采用RACE技术克隆杉木(Cunninghamia lanceolata)HD-Zip Ⅲ基因,分析序列特征并检测在不同器官和组织中的表达模式,以期了解其在杉木木材发育过程中的作用。结果获得了HD-ZipⅢ家族的4个基因全长c DNA序列,分别命名为ClHDZ1(KF931396)、ClHDZ2(KF931397)、ClHDZ3(KF931398)和ClHDZ4(KF931399),相应编码蛋白分别由857、841、851和842个氨基酸残基组成,都含有同源异型域(homeodomain,HD)、亮氨酸拉链(leucine zipper,LZ)结构域、类固醇敏感的脂质调节蛋白(steroidogenic acute regulatory protein-related lipid transfer domain,START)结构域,丝氨酸/苏氨酸蛋白(START adjacent domain,SAD)结构域,以及保守的甲硫氨酸-谷氨酸-赖氨酸-组氨酸-亮氨酸-丙氨酸(Met,Glu,Lys,His,Leu,Ala,MEKHLA)结构域。分子进化树显示来自裸子植物、被子叶植物的HD-ZipⅢ分别聚类在不同的分支,其中ClHDZ1和ClHDZ3是在进化历程中较早出现的一类,ClHDZ2和ClHDZ4归属裸子植物的C8亚类。4个ClHDZs在不同器官中都有不同程度的表达,且在根中表达量皆为最低。ClHDZ1主要在针叶中表达;ClHDZ4基因主要在雌球花中表达;ClHDZ2跟ClHDZ3表达模式相似,主要在茎中表达,且表达量均随着木质化程度的提高而增强,暗示可能参与杉木木质部的发育。进一步的定量PCR分析显示,ClHDZ2主要在韧皮部和木质部中表达,相应数值分别是形成层的2.0和2.2倍;ClHDZ3在木质部中的表达量是韧皮部和形成层的8倍以上。这两个基因在响应应压木诱导处理中呈现出不同的表达模式,ClHDZ2在对应木中优势表达,而ClHDZ3则呈现出与木质素变化一致的模式,即应压木中的ClHDZ3基因表达量显著高于对应木中的ClHDZ3基因表达量,推测ClHDZ2和ClHDZ3这两个基因可能参与调控杉木木材形成过程。杉木HD-Zip Ⅲ基因的分子生物学研究为揭示杉木木材形成提供了新的科学依据,并为杉木木材形成分子机制的深入研究打好了基础。
ClassⅢHomeodomain Leucine Zipper (HD-ZipⅢ) is mainly involved in the regulation of vascular tissue formation, apical meristem differentiation, embryogenesis and lateral organ polarity establishment. In this study, RACE was used to clone the HD-Zip Ⅲ gene of Cunninghamia lanceolata based on the transcriptome sequencing data. The sequence characteristics were analyzed and the expression patterns in different organs and tissues were examined in order to understand its role in the development of Chinese fir wood. Results The full length c DNA sequences of four genes of HD-Zip Ⅲ family were obtained and named as ClHDZ1 (KF931396), ClHDZ2 (KF931397), ClHDZ3 (KF931398) and ClHDZ4 (KF931399) respectively. The corresponding encoded proteins were encoded by 857,841,851 And 842 amino acid residues all contain the homeodomain (HD), the leucine zipper (LZ) domain, the steroidogenic acute regulatory protein-related lipid transfer domain , START) domain, the START adjacent domain (SAD) domain, and the conserved methionine-glutamic acid-lysine-histidine-leucine-alanine Met, Glu, Lys, His, Leu, Ala, MEKHLA) domains. Molecular phylogenetic tree showed that HD-Zip Ⅲ from gymnosperm and angiosperm plants clustered in different branches respectively. Among them, ClHDZ1 and ClHDZ3 were the earlier ones in the evolutionary history. ClHDZ2 and ClHDZ4 belonged to C8 subclasses of gymnosperms. The four ClHDZs expressed differently in different organs, and the lowest in roots. ClHDZ1 was mainly expressed in needles; ClHDZ4 gene was mainly expressed in female flowers; ClHDZ2 was similar to ClHDZ3 in expression pattern, and was mainly expressed in stems. The expression level of ClHDZ1 increased with the increase of lignification, Development. Further quantitative PCR analysis showed that ClHDZ2 was mainly expressed in phloem and xylem, with corresponding values of 2.0 and 2.2 times that of cambium, respectively. ClHDZ3 expressed more than 8 times in phloem and phloem than in phloem and xylem. The two genes showed different expression patterns in response to stress-induced wood processing, ClHDZ2 was predominantly expressed in the corresponding wood, while ClHDZ3 exhibited a pattern consistent with the change in lignin, that is, the expression level of ClHDZ3 in stress wood Which was significantly higher than that of ClHDZ3 gene in the corresponding wood. It is speculated that the two genes ClHDZ2 and ClHDZ3 may be involved in the regulation of fir wood formation. The molecular study of HD-Zip Ⅲ gene in Chinese fir has provided a new scientific basis for revealing the formation of Chinese fir wood and laid the foundation for the further study on the molecular mechanism of Chinese fir wood.