Selection Of Mulberry Germplasm Resources With High Flavonoids Content And Research On Metabolic Accumulation Mechanism

Mulberry（Morus alba L）is widely planted in China,and mulberry leaves are rich in nutrients and secondary metabolites,which are not only the best feed for silkworm,but also the raw material of homology of medicine and food.As the use of mulberry leaves is recognized by more and more people,the demand for new varieties of mulberry with diversified uses is becoming more and more prominent.Flavonoids are one of the most abundant components in mulberry leaves,which have anti-cancer,anti-tumor,antioxidant and other effects.In this research,the mulberry germplasm resources preserved in Southwest University were used as materials to study the accumulation law of flavonoids in mulberry germplasm resources,evaluate the antioxidative and hypoglycemic effects of mulberry germplasm resources directly or indirectly,and excavate high-quality mulberry resources.The mulberry leaves with significant differences in contents of flavonoid were subjected to transcriptome sequencing analysis to explore key genes for flavonoid synthesis,and further study the correlation between the expression of related genes and the content of flavonoids in different mulberry germplasm resources,and to explore the marker genes of high-quality resources.The main results are as follows:1.The contents of total flavonoids in mulberry leaves of 324 mulberry germplasm resources were determined by ultrasonic assisted ethanol extraction and Na NO₂-Al（NO₃）₃-Na OH method.It was found that there were significant differences among resources,and the content ranged from 6.81to 55.6 mg/g,among which Hunan huasang No.1 had the highest content;The content of total flavonoids in Hunan resources was higher than that in other regions,and the content in Tibet resources was the lowest;The content of total flavonoids in tender leaves was significantly higher than that in mature leaves.2.Through the target metabolomic analysis,it was found that the flavonoids in mulberry leaves were mainly rutin,isoquercetin and astragalin.In this study,the HPLC system was optimized to detect these three substances,and the mulberry resources with high yield and mulberry leaves with high flavonoids content per unit mass were identified and evaluated.The results showed that the top three mulberry resources with high yield were Xiangyu NO.3,Yongsheng NO.1 and Tianquan NO.53;The top three mulberry resources with high flavonoids content per unit mass were Pingshan NO.1,Hunan huasang NO.2 and Hulusang.The identification and evaluation results of the antioxidant capacity of total flavonoids from mulberry leaves to scavenge DPPH,ABTS and hydroxyl radicals showed that the top three high-yielding mulberry resources with antioxidant capacity were 731,Yongsheng NO.1 and Xiangyu NO.10;The results of the evaluation of the ability of inhibitingα-glucosidase activity showed that the top three mulberry resources with hypoglycemic effect were Qiancha NO.1,Srilanka NO.1 and Hanyuan NO.10.3.Through the transcriptome sequencing analysis of young leaves and mature leaves with significant differences in flavonoid content,the differential genes related to flavonol synthesis（PAL1,C4H,4CL2,4CL6,CHS,CHI,F3H,F3’H）and the key genes and transcription factors of flavonoid synthesis reported in the literature（FLS,F3GT,MYB8,MYB12）were selected as the candidate genes of this study.Their expression differences in different mulberry resources and leaf positions were analyzed by q RT-PCR,and their correlation with the contents of total flavonoids,rutin and isoquercetin was analyzed.There were significant differences in the expression of 12 genes in different resources;4CL2,4CL6,CHS,F3H,F3’H,MYB8 and MYB12 were highly expressed in young leaves,while C4H and F3GT were highly expressed in mature leaves.Correlation analysis showed that the relative expression of 4CL2 was significantly positively correlated with flavonoids,the relative expression of C4H,CHI,FLS,F3GT and MYB12 was significantly negatively correlated with flavonoids,and the genes with the most closely related gene expression and flavonoid content were 4CL2,CHI and F3GT.4.After mulberry seedlings were stressed with different UV-B irradiation time（3 h,6 h,9 h）,flavonoids in leaves accumulated significantly,and their content increased with the increase of UV-B irradiation time.Since 9 h of UV-B irradiation every day seriously affects the leaf health of mulberry leaves,the experimental results showed that in order to increase the content of flavonoids in mulberry leaves,UV-B irradiation for 6 h a day for 7 days was the best irradiation time.According to the expression level of candidate marker genes 4CL2,CHI and F3GT in response to UV-B radiation,it was found that the expression of 4CL2 would be inhibited by UV-B,and the expression of CHI and F3GT would be promoted by UV-B.