太子参多糖合成关键酶基因的克隆、序列分析及对赤霉素的响应

Cloning and Sequence Analysis of Key Enzyme Genes for Polysaccharide Synthesis of Pseudostellaria heterophylla and the Response to Gibberellin

目的:从太子参块根中克隆得到类纤维素合成酶(Cellulose synthase-like protein,Csl)基因,分析该基因及太子参多糖在赤霉素处理下的响应。方法:基于Csl的同源性和太子参转录组数据库,利用克隆技术得到太子参Csl基因(PhCslG)序列,以不同浓度赤霉素及其抑制剂多效唑处理的太子参块根作为材料,采用实时荧光定量PCR技术(qRT-PCR)和多糖含量检测技术进行定量,并分析赤霉素对该基因和多糖含量的影响。结果:PhCslG含2205 bp的开放阅读框,编码734个氨基酸,该基因编码的蛋白具有2个纤维素合成酶蛋白家族的cellulose_synt保守结构域(pfam03552.14)和2个D-D-D-QXXRW模体,qRT-PCR和多糖含量测定结果表明,中、低浓度的赤霉素可促进PhCslG上调表达,多糖含量增加,高浓度赤霉素作用反之。结论:本研究首次成功克隆得到PhCslG基因序列,为进一步阐明该基因的功能和后续综合开展太子参多糖合成和积累的调控机制等研究提供了前期基础。

Objective:To obtain the cellulose synthase-like protein(Csl)gene in tuber of Pseudostellaria heterophylla,to analyze the response of gene and Pseudostellaria heterophylla polysaccharide to gibberellin treatment.Methods:Based on the homology of Csl and the transcriptome database of Pseudostellaria heterophylla,the Csl gene sequence(PhCslG)of Pseudostellaria heterophylla was obtained by cloning.The tuber of Pseudostellaria heterophylla treated with gibberellin and its inhibitor dotriazole with different concentrations was used as material,Real-time fluorescence quantitative PCR(qRT-PCR)and the polysaccharide content determination methods were used for quantitative analysis,the effects of gibberellin on the gene and polysaccharide content were analyzed.Results:The PhCslG sequence had a open reading frame(2205 bp),encoding 734 amino acids,the protein coded by the gene had two cellulose_synt conservative structure domain(pfam03552.14)of cellulose synthase gene encoding protein family and 2 D-D-D-QXXRW motifs.The results of qRT-PCR and polysaccharide content detection showed that the gibberellin with low or medium concentration promoted the up-regulation of PhCslG expression,the content of polysaccharide increased,the effect of high concentration gibberellin was opposite.Conclusion:The PhCslG gene sequence is cloned successfully for the first time,which provides a preliminary basis for further elucidation of the function of this gene and subsequent comprehensive research on the regulation mechanism of polysaccharide synthesis and accumulation of Pseudostellaria heterophylla.