| The high cost of producing mogroside V and its low contents in fruits restrict its application. Increasing the contents of mogroside Ⅴ in S. grosvenorii fruits and reducing the cost of production have become a research hotspot. However, traditional cultivation and breeding methods are difficult to increase the contents of mogroside Ⅴ in fruits. All mogrosides have common aglycone, but different in the numbers and connection mode of glucose groups in C-3, C-24 and the function of oxyl in C-7, C-11. Genetic engineering breeding and biosynthesis have become a new way to increase mogroside Ⅴ contents directly in fruits at lower cost. To achieve this goal, isolation and identification of key enzymes involved in mogroside Ⅴ biosynthesis is needed. In this research, we studied metabolic regularity of mogrosides in S. grosvenorii fruits, the relation between mogroside Ⅴ accumulation and its substrate glucose under different control conditions, sequenced transcriptome of mogrol, mogroside ⅡE, mogroside Ⅲ metabolic enzyme genes, cloned and expressed UDP-glucosyltransferase genes in E. coli. The results show:1、From 10 d to 90 d after pollination during ripening of S. grosvenorii NyuanB6 fruits, the main components were mogroside HE and Ⅲ before 30 d. From 30 d to 50 d mogroside ⅡE, Ⅲ, Ⅳ, Ⅴ coexisted in the fruits and after 50 d the major component was mogroside V, which implied that mogroside ⅡE could serve as a precursor of mogroside Ⅴ which could be formed from mogroside Ⅲ,Ⅳ.2、Content changes of mogroside and sugar in S. grosvenorii were measured under different parameter such as varieties, shading, and storing in air or carbon dioxide. In all varieties, shading or not, stored in the air or carbon dioxide, the mogroside ⅡE, Ⅲ decreased gradually and mogroside V accumulated dramatically as the contents of glucose in fruits decline; In all varieties, shading or not, from 30 d after pollination to ripening, starch accumulation decreased gradually and glucose contents kept above 10%. In high mogroside Ⅴ content variety, photosynthesis rate, glucose contents and mogroside ⅡE, Ⅲ, Ⅴ contents were high. After 30 d, shading decreased leaf photosynthesis rate and accumulations of soluble sugar and sucrose, but ultimately increased contents of glucose and mogroside Ⅴ. These results indicated that during the accumulation of mogroside V, the fruits would consume glucose. However, sufficient supply of glucose in S. grosvenorii implies that glucose is not the limiting factor of mogroside V accumulation.3、Mogrol contents were measured in different tissues and development stages of S. grosvenorii and other 6 cucurbitaceous plants. Mogrol is only synthesized in S. grosvenorii among Cucurbitaceae family. The contents of mogrol remained around 3% before 30 d, but decreased dramatically after 30 d till almost disappeared. Mogroside HE and Ⅲ accumulation have similar pattern. This indicated that mogrol、mogroside ⅡE and Ⅲ were the major limiting factors of mogroside V accumulation.4、Gene expression at 0 d,3 d,15 d and 30 d of S. grosvenorii during mogrol synthesis was analyzed through high-throughput sequencing.81940 unigenes and a total of 57107 (69.69%) unique sequences were annotated.31436 unigenes were compared to Kyoto Encyclopedia of Genes and Genomes (KEGG) and 2543 were related to secondary metabolism and involved in 21 metabolic pathways. All known key enzyme genes were found in terpenoids and steroids metabolic pathways. Among them, oxidosqualene cyclase (OSC)、cytochrome P450 (CYP450) and UDP-glycosyltransferase genes, which located at the end of terpenoids and steroids synthesis, were differentially expressed at different growing stages. Expression of SgCS gene, which encoded the precursor of mogroside, increased sharply from 0 d to 15 d and decreased dramatically from 15 d to 30 d. Expression of SgCAS gene, which encode the precursor of steroids, increased from 3 d to 15 d and kept in high level from 15 d to 30 d.11 candidate CYP450 genes expressed coordinately and their expression patterns were consistent with the accumulation pattern of mogrol.6 candidate UDP-glucosyltransferase genes expression were in accordance with mogroside HE accumulation.2 candidate UDP-glucosyltransferase genes expression were in accordance with mogroside III accumulation. These abundant information from transcriptome analysis helps to isolate and clone genes involved in the mogroside V synthesis.5、3 UDP-glucosyltransferase genes SgUGT4、SgUGT6'SgUGT7 were cloned using Rapid Amplification of cDNA Ends (RACE) technology from 5 d and 70 d pulps. SgUGT4 gene, which contained the PSPG-box domain of UDP-glucosyltransferase family, was the closest relatives of UGT73 family of glycosylated mogrol. It mainly expressed at around 50 d when mogroside V was quickly synthesized. Its expression level is higher in high mogroside V contents variety, which indicated that it might involved in the synthesis of mogroside V. SgUGT4、SgUGT6'SgUGT7 genes were expressed with E. coli and pichia yeast system and corresponding protein were obtained, which benefits further in vitro functional studies and structure analysis of those genes. |
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