Solexa Sequencing Of Transcriptome And DGEs And Cloning Of Genes Related To Mogroside Ⅴ Biosynthesis In Siraitia Grosvenorii

SummaryThe fruit of Siraitia grosvenorii, which belongs to the family Cucurbitaceae, has long been used in traditional Chinese medicine as a pulmonary demulcent and emollient for the treatment of dry cough, sore throat, dire thirst and constipation. The main ingredient is mogroside V, it is one of the most sweet material in non-sugar sweet in the world, as300-400times the sweetness as that of sucrose, and it is a low-calorie, natural sweetener and an ideal health care products, in addition to have antioxidant, immunomodulatory, anticancer, lowering blood sugar effect. In order to reveal the mechanism of mogroside V biosynthesis from molecular level, Look for function genes in large-scale and full-length cDNA cloning, a transcriptom and three expression profiles with the high-throughput sequencing technology in Siraitia grosvenorii are studied. The major findings are as follows:1. The mainly content is mogroside â…¡ E between30d and50d. It sharp declined on70d, no mogroside â…¡E is detected. Mogroside â…¢ only appears in50d. Mogroside V is very low before50d, but it is dramatically increased25.9times from50d to70d, and it comes up to the highest levels till85d. The content of glucose did not change significantly in30d and50d, but decreased3.24times at70d. There shall be a certain degree of fluctuation relations between the content of glucose and that of mogroside â…¡E, and mogroside V.2. With Solexa high-throughput sequencing technology for transcriptome studying,3.41G of the raw data is obtained, and also obtained43,891Unigenes. There are739Unigenes, of which60Unigenes involve in the synthesis of terpenoid skeleton, associated with secondary metabolism. With the usage of transcriptome data, it found that almost all genes are related to the mogrosides V biosynthesis pathway. Also discovered80P450s,72glycosyltransferases, and90glucosyltransferases. These Unigenes are important genetic resources in the regulation for mogrosides biosynthesis in the future.3. In the3d,50d,70d expression profiles obtained more than3million clean tags, after removing the redundancy, more than85,000different types of tags were obtained. The analysis shows that mRNA, which is less than5.1-5.9%of the types of mRNA, accounts for nearly73-76%of the total ammount, but all which accounts for56-59%of types, is less than3.9-4.1%of total amount mRNA. Statistics on the differential expression genes found that, in the3d/50d genes, there are2119genes up-regulated,2642genes were down-regulated; in the3d/70d, the2150genes gone up-regulated,2797genes were down-regulated; in the50d/70d, the1078genes up-regulated,1422Genes down-regulated.4. Analysis on21genes expression levels involved in mogroside V skeleton biosynthesis through three expression profiles, it finds that there are no significant change on AACT, MVD, CMK, HDS, SQS gene expression in the three period, the remaining16genes are all have their own variation. In3d and50d, DXS, DXR, MCS, IDS come down, the remaining12genes go up-regulated. In50d and70d, except HMGR, MCS, IDS, IPP, GPS, FPS, CAS that come down, the other9genes go up. From3d to70d, the overall trend of each gene is:in addition to DXR, MCS, IDS, IPP-I come down in the expression, HMGS, HMGR, MK, PMK, MCT, IPI-II, GPS, FPS, SQS, CAS, and CS genes are up-regulated. Among these, SQE and CS genes changes mostly, the expression of70d increased8.89times and9.69times respectively than the3d.5. Studying on the gene expression of80cytochrome P450and90glucosyltransferases, it suggests that the four candidate P450genes:Unigene23541, Unigene24189, Unigene26598and Unigene43109and6candidate UDPG genes: Unigene4016, Unigene8672, Unigene13633, Unigene15400, Unigene35056and Unigene38974may be involved in mogroside V skeleton biosynthesis.6. Using RACE,12full-length cDNAs related to mogroside V biosynthesis were cloned for the first time, of which there are6in MVA pathway:AACT, HMGS, HMGR, MK, PMK, MVD, and6in common pathway:IPI, GPS, FPS, SQS, CAS, CS, etc, and the5'end of SQE. Also obtained MEP pathway of MCT and the IDS gene in full-length, the5'end of DXS and HDS, and3'end of MCS. Two full-length glycosyltransferase are obtained in another experiment.These full-lenth and partial genes were all first reported in Siraitia grosvenorii.By applying high-throughput Solexa sequencing technology on studying transcriptom and expression profiles in Siraitia grosvenorii firstly, a large number of genes information were abtained, and many genes related to secondary metabolite were cloned. This is a solid foundation for the study of Siraitia grosvenorii functional genomics and mogrosides V molecular mechanism of biosynthesis.