毛竹PeIRX10基因在木聚糖合成中的功能

doi:10.11707/j.1001-7488.20161110

摘要/Abstract

摘要： [目的] 克隆毛竹木聚糖合成关键酶基因PeIRX10，并对其进行表达分析和功能初探，为毛竹中木聚糖合成分子机制的探究提供理论依据。[方法] 以毛竹为研究对象，根据毛竹基因组数据库中序列信息设计引物，通过RT-PCR克隆得到1个在模式植物拟南芥木聚糖合成中起关键作用的同源基因，命名为PeIRX10；运用生物信息学的方法对其核苷酸序列及其编码的氨基酸序列进行分析；通过qRT-PCR对毛竹的根、茎、叶、花和笋5个部位进行组织表达特异性分析。利用Gateway克隆技术构建PeIRX10 的植物表达载体，通过蘸花法转化拟南芥irx10l（-/-）irx10（+/-）双突植株，经BASTA筛选及PCR植株表型鉴定获得具有irx10l（-/-）irx10（-/-）双突背景的转基因植株；通过表型观察、茎部切片甲苯胺蓝染色观察、细胞壁单糖成分分析以及木聚糖免疫定位观察探讨该植株中PeIRX10基因的功能。[结果] 从毛竹中克隆得到PeIRX10（PH01004923G0080，http：//www.bamboogdb.org/）基因的ORF序列，长度为1 251 bp，编码416个氨基酸，蛋白质的理论分子质量为46 821 Da，等电点为6.26；其氨基酸序列与其他植物的IRX10基因具有很高的相似性（>80%），属于GT47家族；qRT-PCR结果表明，PeIRX10基因在毛竹笋和茎中高度表达。过量表达PeIRX10 基因的转基因拟南芥植株可以互补于拟南芥IRX10双突植株irx10l（-/-）irx10（-/-），表现出正常的株高、茎粗以及叶片大小和数量；转基因拟南芥植株的茎部切片甲苯胺蓝染色观察发现，其维管束间纤维和木质部导管细胞的细胞壁厚度与野生型拟南芥相近；细胞壁单糖分析发现，互补植株木糖含量以及其他单糖（例如阿拉伯糖、半乳糖和葡萄糖等）的含量与野生型相近；木聚糖免疫定位分析表明，PeIRX10基因的过表达可以使irx10l（-/-）irx10（-/-）双突植株中木质部和束间纤维中LM10信号几乎完全恢复。[结论] 在irx10l（-/-）irx10（-/-）双突拟南芥植株中过量表达PeIRX10基因可以使植株的表型和次生细胞壁缺陷恢复，表明毛竹PeIRX10基因与拟南芥IRX10基因具有相似的功能，都在木聚糖的合成过程中发挥重要作用。

关键词: 毛竹, 木聚糖, 次生细胞壁, PeIRX10

Abstract: [Objective] PeIRX10 gene was cloned and functionally analyzed for further understanding of the molecular mechanism of xylan synthesis in Phyllostachys edulis.[Method] According to the sequence information from the bamboo genome database,primers were designed. A homologous gene from P.edulis playing critical role in xylan synthesis in Arabidopsis thaliana was cloned and named as PeIRX10. The nucleotide sequence and encoded amino acid sequence of PeIRX10 were analyzed using bioinformatics methods. The expression pattern of PeIRX10 in different organs (root, stem, leaf, shoot and flower) was detected by qRT-PCR. The binary vector of PeIRX10 overexpression was constructed using Gateway technology and then transformed into the A.thaliana irx10l(-/-) irx10(+/-) mutant plants by floral dip. Transgenic plants with double mutant background were selected by BASTA and PCR identification. Finally, the conserved function of PeIRX10 in thexylan synthesis was done by phenotype observation, stem section, sugar composition analysis and xylan immunolocalization. [Result] An ORF sequence of PeIRX10(PH01004923G0080,http://www.bamboogdb.org/)from P. edulis was cloned with a length of 1 251 bp,encoding 416 amino acids, with a predicted molecular weiqht of about 46 821 Da and pI of about 6.26. The protein sequence encoded by PeIRX10 exhibited relatively high similarity to those of other plants (>80%), indicating that it belongs to GT47 family. qRT-PCR showed that PeIRX10 was highly expressed in shoots and culms of P. edulis. After overexpression of PeIRX10, complementation of irx10l(-/-)irx10(-/-) double mutant can restore the phenotype with evidence of normal plant height, stem diameter, leaf numbers and sizes. Moreover, the analysis of monosaccharides indicated that the content of xylose and other monosaccharides, such as arabinose, fucose, galactose and glucose in the complementation plants was not significantly different to those of the wild type. Immunostaining of cross sections detected by LM10 labeling revealed strong signals in xylem cells in the irx10l(-/-)irx10(-/-) double mutant when overexpressedby PeIRX10. [Conclusion] Overexpression of PeIRX10 in the irx10l(-/-)irx10(-/-) double mutant rescued the plant growth phenotype and restored the secondary wall thickness, which suggested that the PeIRX10 gene have a function similar to the IRX10 geneof A. thaliana and play an important role in the synthesis of xylan.

Key words: Phyllostachys edulis, xylan, secondary wall synthesis, PeIRX10

中图分类号:

王杰, 李军, 仝婷婷, 邵香君, 宋丽丽, 吴蔼民. 毛竹PeIRX10基因在木聚糖合成中的功能[J]. 林业科学, 2016, 52(11): 79-87.

Wang Jie, Li Jun, Tong Tingting, Shao Xiangjun, Song Lili, Wu Aimin. Functions of PeIRX10 Gene from Phyllostachys edulis in Xylan Synthesis[J]. Scientia Silvae Sinicae, 2016, 52(11): 79-87.

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