铁皮石斛葡甘聚糖合成关键酶基因Csl的筛选及功能研究

Dendrobium officinale is one of the most valuable Chinese medicinal herbs, the stem of which is used as a crude drug named "Fengdou". However, wild D. officinale resources are threathened by extinction due to its slow growth rate and overexploitation. Thus, large-scale artificial cultivation has been developed. Glucomannan (GM) is one of the main active polysaccharides in D. Officinale, which has important biological functions, e.g. immunomodulation and antitumor, etc. Previous studies showed that cellulose synthase-like (Csl) genes play important roles in GM backbone synthesis. By comparative analysis of the transcriptome databases genetrated from three cDNA libraries from two different growth stages and two tissues (stem and leaf) of D. officinale, 15 Csl unigenes have been identified. In this study, we will screen the Csl genes involved in the biosynthesis of Dendrobium glucomannan (DGM) by the assistance of plant chemistry, genetics, molecular biology and proteomics methods. Csl genes are aimed to be cloned by RT-PCR and RACE and their spatiotemporal expression patterns will be analyzed by qRT-PCR. Prokaryotic expression in E.coli system will be used to characterize functional enzymes. The correlation between Csl gene expression levels and the content of DGM will be studied by D. officinale protocorm transient expression and saccharide mapping analysis. This study will enable us to determine the best harvesting time by elucidating the mechanism in DGM biosynthesis. The study is also expected to provide a guidance for the quality control of D. officinale, and can be further used in molecular marker assisted breeding. In addition, it will set the foundation for the implementation of plant metabolic engineering and synthetic biology techniques for the genetic and quality improvement of D. officinale.

铁皮石斛是珍稀濒危保护植物，具有重要的药用、保健和经济价值。葡甘聚糖是铁皮石斛茎中主要的多糖类成分，具有调节免疫、抗肿瘤等活性。类纤维素合成酶基因（Csl）参与葡甘聚糖骨架合成。前期通过对不同生长时期不同多糖含量的铁皮石斛茎、叶进行转录组测序和差异基因表达分析，获得15个Csl基因。在此基础上本研究应用植物化学、遗传学、分子生物学和蛋白组学相结合的方法，拟筛选影响铁皮石斛葡甘聚糖（DGM）含量的Csl基因，通过RT-PCR和RACE技术克隆Csl基因序列，并利用荧光定量PCR、E.coli原核表达、蛋白结构与功能鉴定、铁皮石斛原球茎瞬时表达等技术和糖谱分析方法，研究Csl基因在铁皮石斛生长过程中的表达模式、对葡甘聚糖骨架合成的功能和作用，以及Csl基因表达水平和葡甘聚糖含量的相关性。研究结果可为铁皮石斛精准采收、质量控制、分子标记辅助育种、遗传改良和提高有效成分产量奠定理论基础。

铁皮石斛是珍稀濒危保护植物，具有重要的药用、保健和经济价值。葡甘聚糖（DGM）是铁皮石斛茎中主要的多糖类成分，具有调节免疫、抗肿瘤等活性。通过分离、纯化与结构鉴定，得到铁皮石斛中具有抗氧化、抑制心肌细胞凋亡的活性多糖DOP-GY。建立了建立“水提—α-淀粉酶处理—一次醇沉”处理方法，测定铁皮石斛甘聚糖为主的非淀粉多糖含量，以及综合高效凝胶色谱法、柱前衍生-HPLC法和红外光谱法的铁皮石斛糖谱综合分析方法，揭示铁皮石斛DGM含量受铁皮石斛栽培环境、采收时期、品种（家系）等影响的变异规律。类纤维素合成酶基因（Csl）参与DGM骨架合成，通过对铁皮石斛DcCSL基因家族成员的全基因组鉴定、系统进化、基因结构、蛋白质保守结构域和基序、启动子顺式元件、基因表达对胁迫响应的分析，挖掘出与DGM合成密切相关的DcCSLA家族基因，并利用荧光定量PCR、E.coli原核表达、蛋白结构与功能鉴定，研究DcCSLA家族基因在铁皮石斛生长过程中的表达模式、对葡甘聚糖骨架合成的功能和作用。研究结果可为铁皮石斛精准采收、质量控制、分子标记辅助育种、遗传改良和提高有效成分产量奠定理论基础。项目执行期间培养硕士研究生2名，发表论文9篇，其中SCI2篇，申请专利3项。