毛竹PeIRX10基因在木聚糖合成中的功能  

Functions of PeIRX10 Gene from Phyllostachys edulis in Xylan Synthesis

作　　者：王杰[1,2] 李军 仝婷婷[4] 邵香君 宋丽丽[1,2] 吴蔼民[4] 

机构地区：[1]南京农业大学食品科技学院,南京210095 [2]浙江农林大学亚热带森林培育国家重点实验室培育基地,临安311300 [3]浙江省临海市林木种子苗木管理站,临海317000 [4]华南农业大学广东省森林植物种质创新与利用重点实验室,广州510642 [5]浙江省临安市种苗管理总站,临安311300

出　　处：《林业科学》2016年第11期79-87,共9页Scientia Silvae Sinicae

基　　金：浙江省杰出青年基金项目(LR15C160001)

摘　　要：【目的】克隆毛竹木聚糖合成关键酶基因Pe IRX10,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Pe IRX10;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Pe IRX10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Pe IRX10基因的功能。【结果】从毛竹中克隆得到Pe IRX10(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,Pe IRX10基因在毛竹笋和茎中高度表达。过量表达Pe IRX10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,Pe IRX10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�【Objective】Pe IRX10 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 Pe IRX10. The nucleotide sequence and encoded amino acid sequence of Pe IRX10 were analyzed using bioinformatics methods. The expression pattern of Pe IRX10 in different organs（root,stem,leaf,shoot and flower） was detected by qRT-PCR. The binary vector of Pe IRX10 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 Pe IRX10 in the xylan synthesis was done by phenotype observation,stem section,sugar composition analysis and xylan immunolocalization. 【Result】An ORF sequence of Pe IRX10（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 p I of about 6. 26. The protein sequence encoded by Pe IRX10 exhibited relatively high similarity to those of other plants（〉 80%）,indicating that it belongs to GT47 family. qRT-PCR showed that Pe IRX10 was highly expressed in shoots and culms of P. edulis. After overexpression of Pe IRX10,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 dete

关 键 词：毛竹 木聚糖 次生细胞壁 PeIRX10 

分 类 号：S718.46[农业科学—林学] Q943.2[生物学—植物学]