杉木木糖合成酶基因ClUAXS2的全长克隆与分析

Full-length cDNA Cloning and Analysis of UDP-D-Apiose/UDP-D-Xylose Synthase Gene ClUAXS2 in Cunninghamia lanceolata

木糖合成酶UAXS基因是植物体内调控细胞壁中UDP-D-葡萄糖醛酸脱羧反应生成UDP-D-木糖/芹菜糖的关键性核苷糖合成酶,对植物细胞壁多糖物质的稳定合成和转换有重要意义。从杉木转录组中挑选获得木糖合成酶基因UAXS的中间序列,使用全长克隆技术和PCR技术克隆得到1个杉木木糖合成酶基因ClUAXS2。结果表明,ClUAXS2基因全长为1 753bp,包含的1 158bp的开放阅读框,可编码长度为386个氨基酸残基的蛋白质,其蛋白质分子式为C1936H3026N524O578S19,分子质量为43.90ku,等电位点为5.60。ClUAXS2基因为亲水性蛋白,没有信号肽结构域,编码1个疑似跨膜区域。蛋白质二级结构分析表明其蛋白主要骨架为Loop环和α-螺旋,预测亚细胞定位于细胞质中。通过对比分析发现ClUAXS2基因属于UAXS家族基因,编码1个UDP-D-Apiose/UDP-D-Xylose synthase 2(UAXS2)木糖酶蛋白。进化树分析结果显示,杉木ClUAXS2基因与无油樟(AtUAXS2)和可可树(TcUAXS2)的氨基酸序列相似度达到85%、86%,与拟南芥(AtAXS2),葡萄(VvUAXS)、毛果杨(PtAXS1)的氨基酸序列相似度为84%。实时荧光定量PCR结果表明,ClUAXS2基因在杉木的根、茎、叶中均有表达,但在1年生杉木幼苗茎中的表达量最高;ClUAXS2基因在不同杉木无性系茎中的表达量差异不显著,在根和叶中的表达量存在差异。

The xylose synthase UAXSgene is a key nucleoside carbohydrate synthase in regulating the decarboxylation of UDP-D-glucuronic acid in the cell wall to produce UDP-D-xylose/apigenin,which is important for the stable synthesis and transformation of polysaccharide from plant cell wall.In this study,the intermediate sequence of xylose synthase gene UAXS was selected from the transcriptome of Chinese fir,a xylose synthase gene ClUAXS2 was cloned from Chinese fir cDNA by RT-PCR and RACE techniques.The results showed that the full-length ClUAXS2 gene was 1 753 bp,containing an open reading frame of 1 158 bp,encoding aprotein with a length of 386 amino acid residues.Its protein molecular formula was C_（1936）H_（3026）N_（524）O_（578）S_（19） with a molecular weight of 43.90 ku,and a isoelectric point of 5.60.The ClUAXS2 gene is a hydrophilic protein with no signal peptide domain,encoding a suspected transmembrane domains.The analysis of protein two level structure showed that the main skeleton of the protein was loop ring and alpha he-lix,and subcellular localization was predicted in cytoplasm.Through comparative analysis,we found that the ClUAXS2 gene belonged to the UAXSfamily gene,encoding a UDP-D-Apiose/UDP-D-Xylose synthase 2（UAXS2）xylose protein.Phylogenetic tree analysis showed that ClUAXS2 genes amino acid sequence similarity between Amborella trichopodaand Theobroma cacao reached 85% and 86%,and the amino acid sequence similarity of Arabidopsis thaliana,Vitis viniferaand Populus trichocarpawas 84%.Real time quantitative PCR results showed that the ClUAXS2 gene was expressed in Chinese fir root,stem and leaf,while the expression was the highest in one year old Chinese fir stem.The ClUAXS2 gene relative expression level was no significant difference in the stems of different Chinese fir clones,but there was a certain difference in the expression level of ClUAXS2 gene in roots and leaves.