基于转录组的黄精多糖代谢关键酶基因的筛选与验证

Transcriptome-based Screening and Validation of Key Enzyme Genes for Polygonatum Polysaccharide Metabolism

目的:通过湖北黄精、滇黄精、多花黄精根茎的转录组学分析,对影响不同品种黄精多糖含量的关键酶基因进行筛选和验证,并进行氨基酸序列的深入分析,丰富黄精属植物的转录组数据并为其多糖生物合成机制和遗传改良提供参考。方法:使用了Illumina NovaSeq高通量测序平台对黄精转录组进行测序分析,根据Nr、基因本体(GO)、京都基因与基因组百科全书(KEGG)数据库的注释分析比较3种黄精转录组的差异,筛选出多糖代谢途径中的关键酶,对关键酶基因表达量进行聚类分析并与多糖含量进行相关性分析,然后对筛选出的8个关键酶基因进行实时荧光定量聚合酶链式反应(Real-time PCR)验证,结合测序结果进一步筛选出多糖生物合成关键酶基因进行同源基因序列分析,构建系统进化树,预测模体(motif)、保守结构域、蛋白序列等电点与相对分子质量,并使用同源建模法构建三维蛋白结构。结果:通过Nr数据库的注释发现3种黄精与芦笋的基因同源性最高,GO数据库注释结果表明这3种黄精在结合功能、催化活性、代谢过程和细胞组分上差异显著,而KEGG通路注释结果说明这3种黄精在淀粉与蔗糖代谢通路和半乳糖代谢通路上差异显著,根据聚类分析、相关性分析、Real-time PCR验证实验、表达量情况与氨基酸序列的结构与功能预测推测出显著影响不同品种黄精多糖含量的关键酶为β-果糖苷酶(sacA)。结论:sacA可能是黄精多糖含量差异的主要影响因素,也是黄精多糖主要为果聚糖的重要原因。

Objective:To screen and validate key enzyme genes affecting the polysaccharide content in different Polygonatum species and perform in-depth amino acid sequence analysis by transcriptomic analysis of P.zanlanscianense,P.kingianum,and P.cyrtonema rhizomes to enrich the transcriptome data of Polygonatum plants and provide references for polysaccharide biosynthesis mechanism and genetic improvement.Method:The Polygonatum transcriptome was sequenced and analyzed using the Illumina NovaSeq high-throughput sequencing platform,and the differences in the transcriptomes of the three Polygonatum species were compared and according to the annotations of Nr,Gene Ontology(GO),and Kyoto Encyclopedia of Genes and Genomes(KEGG) databases.The key enzymes in the polysaccharide metabolism pathway were screened,and the expression of key enzyme genes was clustered and correlated with the polysaccharide content.Finally,Real-time polymerase chain reaction(Real-time PCR) was performed to validate the eight key enzyme genes,and the key genes of polysaccharide biosynthesis were further screened for homologous gene sequence analysis in combination with sequencing results,followed by constructing phylogenetic trees,predicting motifs,conserved structural domains,protein sequence isoelectric points,and molecular weights,and constructing 3D protein structures by using homology modeling method.Result:The annotation of the Nr database revealed that three Polygonatum species had the highest gene homology with Asparagus officinalis.GO database annotation results showed that three Polygonatum species differed significantly in binding,catalytic activity,metabolic processes,and cellular components,while the KEGG pathway annotation results indicated that three Polygonatum species differed significantly in the starch and sucrose metabolic pathway and galactose metabolic pathway.According to clustering analysis,correlation analysis,Real-time PCR,expression profiles,and structural and functional predictions of amino acid sequences,the key enzyme significantly affecting the polysaccharide content in different Polygonatum species was inferred to be β-fructofuranosidase(sacA).Conclusion:SacA may be the main influencing factor for the difference in polysaccharide content of Polygonatum,and is also an important reason why Polygonatum polysaccharides are mainly fructans.