Establishment Of High Efficient Extraction Technology Of Sesamin And Discovery Of Synthetic Regulatory Genes

Sesamin is the main lignan in sesame seeds,with the deepening of the research and application of sesamin physiological function and medicinal value.Accordingly,higher requirements are put forward to extract and refine sesamin from sesame and its products.Thus,the present study was initiated to establish an accurate,reliable,simple,and efficient microextraction and detection technology of sesamin in sesame seeds and use it to analyze the variation characteristics of sesamin content in specific sesame germplasm.Moreover,this study aimed to explore the molecular mechanisms involved in sesamin biosynthesis and identify some related candidate genes.We used the response surface methodology（RSM）to set an efficient extraction method of sesamin from sesame seeds,with Zhongfengzhi No.1 as the experimental material.In addition,we constructed an F₂population from Zhongtezhi No.1 and Fenzhi No.2 as parents,and the sesamin-related candidate genes were identified via the BSA-seq method.The main results are as follows:（1）Quantitative analysis was performed using high-performance liquid chromatography（HPLC）in combination with single factor test and response surface methodology（RSM）.Next,the significance test,the lack of fit test,and correlation analysis of the established response surface model were accessed.The results showed that the influencing factors of sesamin content were:crusher amplitude>diameters of steel balls>crusher time>seeds weight.Furthermore,the interaction between diameters of steel balls and crusher amplitude was highly significant（P=0.0002）,indicating it is the primary factor that influences sesamin content.Finally,through response surface methodology analysis and verification test,we found that the optimal parameters for sesamin microextraction technology were:steel ball diameter of 6.5 mm,crushing time of 225 s,crushing amplitude of 1335 r/min,and seeds weight of 0.20 g.（2）Phenotypic analysis and statistical analysis of the F₂ population constituted of1095 individuals,were achieved using the established sesamin extraction method.The content of sesamin in the population varied from 4.02 to 14.68 g?kg^-1,with an average of8.784 g?kg^-1.Seventy（70）lines exhibited a sesamin content greater than 12.0 g?kg^-1,which has not ever been reported.The coefficient of variation was 22.29%,indicating obvious variation.The sesamin content in the population showed a normal distribution,which is typical for quantitative traits.Fifty（50）plants with extreme differences in sesamin content were selected to construct high and low sesamin mixed pool,and sequencing was performed by determining the phenotype of the F₂ population.The results showed that the effective data obtained from the parents and the two mixed samples were abundant.Based on SNP-index and Euclide distance（ED）analysis,the target interval was finally located on chromosome 6,which contained 237 potential candidate genes.We examined the expression of these genes using previously released transcriptome data in the laboratory and found that twelve（12）of them were expressed differentially in seeds.By comparing the expression patterns of these differentially expressed genes at different developmental stages in the two parents,three genes（SIN＿1018449,SIN＿1018439,and SIN＿1015517）were selected as candidate genes for future studies.These candidate genes might be involved in controlling sesamin biosynthesis in sesame seeds.