Project description:This study analyzed the differences in karyotype and genetic variation between a mutant and wild-type Siraitia grosvenorii. Genetic variation included changes in genome and gene expression by SRAP molecular markers. Results showed that wild-type S. grosvenorii was diploid, with a chromosome number of 2n = 2x = 28, whereas the mutant was tetraploid with a chromosome number of 2n = 4x = 56. 4573 DNA bands were obtained using 189 different primer combinations, 577 of which were polymorphic, averaging 3.1 bands for each primer pair, while 1998 pairs were identical. There were no apparent differences on bands amplified by most primer pairs. After comparing the diploid and tetraploid strains, the data generally indicated that the polymorphism would be quite low. 2917 cDNA bands were generated using 133 primer combinations, and stable and clearly differential fragments were sorted out, cloned and sequenced. Ninety-two differentially expressed fragments were successfully sequenced. Sequence analysis showed that most fragments had significant homologous nucleotide sequences with resistant to stress and photosynthesis genes, including ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxykinase, pyruvate kinase, peroxisomal membrane transporter, NBS-LRR type resistance protein, protein phosphatase and others. The results revealed that the tetraploid strain has more resistant and photosynthesis ability than its diploid relatives, which providing reference information and resources for molecular breeding and seedless Luohanguo.
Project description:Continuous phytochemical studies of the crude extract of Luo Han Guo (Siraitia grosvenorii) furnished three additional new cucurbitane triterpene glycosides, namely 11-deoxymogroside V, 11-deoxyisomogroside V, and 11-deoxymogroside VI. The structures of all the isolated compounds were characterized on the basis of extensive NMR and mass spectral data as well as hydrolysis studies. The complete ¹H- and ¹³C-NMR spectral assignments of the three unknown compounds are reported for the first time based on COSY, TOCSY, HSQC, and HMBC spectroscopic data.
Project description:CYP450 plays an essential role in the development and growth of the fruits of Siraitia grosvenorii. However, little is known about the SgCYP450-4 gene in S. grosvenorii. Here, based on transcriptome data, a full-length cDNA sequence of SgCYP450-4 was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) strategies. SgCYP450-4 is 1677 bp in length (GenBank accession No. AEM42985.1) and contains a complete open reading frame (ORF) of 1422 bp. The deduced protein was composed of 473 amino acids, the molecular weight is 54.01 kDa, the theoretical isoelectric point (PI) is 8.8, and the protein was predicted to possess cytochrome P450 domains. SgCYP450-4 gene was highly expressed in root, diploid fruit and fruit treated with hormone and pollination. At 10 days after treatment with pollination and hormones, the expression of SgCYP450-4 had the highest level and then decreased over time, which was consistent with the development of fruits of S. Grosvenorii. Hormonal treatment could significantly induce the expression of SgCYP450-4. These results provide a reference for regulation of fruit development and the use of parthenocarpy to generate seedless fruit, and provide a scientific basis for the production of growth regulator application agents.
Project description:Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS) and cycloartenol synthase (CAS) are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR) approach. The SgSQS cDNA has a 1254 bp open reading frame (ORF) encoding 417 amino acids, and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression.