Gene Mining And Safety Evaluation Of Quinoa Saponin Biosynthesis

Quinoa(Chenopodium quinoa Willd.)has a high nutritional value and is rich in minerals,vitamins,polyphenols and flavonoids,so it has received more and more attention in recent years as a new functional food.Like quinoa seeds,quinoa leaves have the same excellent nutritional composition and biological activity and can be consumed as a vegetable,but the main reason affecting the taste of consumers is the presence of bitter substance saponins in quinoa.Quinoa saponins are important components of quinoa secondary metabolites,and their biological activities such as antibacterial and antioxidant have been widely reported,but the research on their biosynthetic mechanisms and food safety evaluation is still not deep enough,which hinders the development of quinoa industry.In this project,quinoa saponins were extracted from quinoa leaves by ultrasound-ethanol.Firstly,the quinoa species resources available in the laboratory were investigated through the laboratory established saponin assay method.Secondly,the metabolic changes of differential metabolites of terpene biosynthesis pathway in quinoa leaves during different growth periods were studied by modern analytical tools such as non-targeted metabolome,targeted metabolome and transcriptome,and the key genes of saponin biosynthesis pathway were mined to reveal the dynamic change process of saponin biosynthesis,which provides theoretical and practical guidance for low saponin quinoa cultivation from the source or future regulation of synthesis of related saponin molecular mechanisms.The results showed that the synthesis of saponin biosynthesis is a dynamic process,which can provide theoretical and practical guidance for the regulation of lowsaponin quinoa cultivation or the synthesis of related saponins in the future.Finally,the UPLC-Q-Exactive-MS/MS technique and animal testing were used to identify the sapogenins of quinoa saponin extracts and to analyze the plasma blood components of rats to elucidate the chemical composition and in vivo metabolic characteristics of quinoa triterpenoid saponins,providing a theoretical basis for the safe consumption and development of quinoa applications.The main studies are as follows.1.The ultrasonic-ethanol extraction conditions of quinoa saponin were determined,and the UV spectrophotometric method for the determination of crude saponin content in quinoa was established in the laboratory,and the method was examined and validated methodologically.The results showed that the highest saponin content in quinoa saponin extract was obtained at a material-to-liquid ratio of 1:30,an extraction time of0.5 h,and an ethanol concentration of 70%.The results of the methodological investigation showed that the maximum absorption was 545 nm and the method precision,recovery and detection limit were all in accordance with the requirements.The determination of saponin content of quinoa species resources at different growth periods revealed that there was a difference in saponin content between the filling and flowering stages,and quinoa leaves LL1 and ZL5 were selected as the materials for subsequent experiments.2.The abundance of metabolites in quinoa leaves was determined based on nontargeted metabolomics techniques,and the metabolite pathways of compounds and their biological significance were enriched and analyzed using principal component analysis,hierarchical cluster analysis,and statistical analysis.Based on the targeted metabolomics technique,the differential metabolites of saponin biosynthesis pathway were screened by constructing OPLS-DA model,and a total of six metabolites were identified as differential metabolites in the results.Finally,the relative content changes of quinoa leaf terpene metabolites were analyzed by correlation heat map and peak area ratio,and the intrinsic reasons for the differences in quinoa saponin content were investigated.3.Quinoa leaf transcriptional studies were performed by Illumina Hi Seq 4000 platform.Gene expression FPKM values were counted by HTSeq to screen for differential genes.Clustering analysis,GO functional classification analysis and KEGG Pathway significant enrichment analysis were performed on the differential genes to mine the key genes in quinoa saponin biosynthesis and to verify the expression of key genes in quinoa saponin biosynthesis by q RT-PCR.4.The mass spectrometric cleavage patterns of saponins and their glycosides were summarized based on the results of the qualitative analysis of quinoa seed saponin extract and rat plasma blood-entry components using ultra performance liquid chromatography-quadrupole-electrostatic field orbital ion trap coupled mass spectrometry(UPLC-Q-Exactive-MS/MS)technique.The changes in the relative contents of plasma blood entry components in rats were found by animal tests.The chemical composition as well as the in vivo metabolic characteristics of quinoa triterpenoid saponins were initially elucidated,and finally,the hemolytic effect of quinoa saponins on blood red blood cells was evaluated by hemolytic assay.In summary,this work is centered on quinoa saponin biosynthesis genes.The key genes in quinoa saponin biosynthesis were mined by metabolomics and transcriptomics techniques,and the gene expression results were verified by q PCR assays.Meanwhile,quinoa saponin glycosides were identified and explored by UPLC-Q-Exactive-MS/MS technique and animal experiments for the changes of quinoa saponin into blood components and saponin hemolytic activity in rats.The results of this study aim to provide a theoretical basis for quinoa saponin-related differentially expressed genes and physiological regulatory mechanisms,and to provide ideas and routes for future development and in-depth exploration of quinoa industry.