Synthesis of Crocin I and Crocin II by Multigene Stacking in Nicotiana benthamiana.

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer's disease. Crocins are mainly biosynthesized in the stigmas of food-medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco.

Post-Ripening and Key Glycosyltransferase Catalysis to Promote Sweet Mogrosides Accumulation of Siraitia grosvenorii Fruits.

Sweet mogrosides are not only the primary bioactive ingredient in Siraitia grosvenorii fruits that exhibit anti-tussive properties and expectorate phlegm, but they are also responsible for the fruit's sweetness. Increasing the content or proportion of sweet mogrosides in Siraitia grosvenorii fruits is significant for improving their quality and industrial production. Post-ripening is an essential step in the post-harvest processing of Siraitia grosvenorii fruits, but the underlying mechanism and condition of post-ripening on Siraitia grosvenorii quality improvement need to be studied systematically. Therefore, this study analyzed the mogroside metabolism in Siraitia grosvenorii fruits under different post-ripening conditions. We further examined the catalytic activity of glycosyltransferase UGT94-289-3 in vitro. The results showed that the post-ripening process of fruits could catalyze the glycosylation of bitter-tasting mogroside IIE and III to form sweet mogrosides containing four to six glucose units. After ripening at 35 °C for two weeks, the content of mogroside V changed significantly, with a maximum increase of 80%, while the increase in mogroside VI was over twice its initial amount. Furthermore, under the suitable catalytic condition, UGT94-289-3 could efficiently convert the mogrosides with less than three glucose units into structurally diverse sweet mogrosides, i.e., with mogroside III as the substrate, 95% of it can converted into sweet mogrosides. These findings suggest that controlling the temperature and related catalytic conditions may activate UGT94-289-3 and promote the accumulation of sweet mogrosides. This study provides an effective method for improving the quality of Siraitia grosvenorii fruits and the accumulation of sweet mogrosides, as well as a new economical, green, and efficient method for producing sweet mogrosides.

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides.

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides.

Heterologous Biosynthesis of Health-Promoting Baicalein in Lycopersicon esculentum.

Baicalein is a valuable flavonoid isolated from the medicinal plant Scutellaria baicalensis Georgi, which exhibits intensive biological activities, such as anticancer and antiviral activities. However, its production is limited in the root with low yield. In this study, In-Fusion and 2A peptide linker were developed to assemble SbCLL-7, SbCHI, SbCHS-2, SbFNSII-2 and SbCYP82D1.1 genes driven by the AtPD7, CaMV 35S and AtUBQ10 promoters with HSP, E9 and NOS terminators, and were used to engineer baicalein biosynthesis in transgenic tomato plants. The genetically modified tomato plants with this construct synthesized baicalein, ranging from 150 ng/g to 558 ng/g FW (fresh weight). Baicalein-fortified tomatoes have the potential to be health-promoting fresh vegetables and provide an alternative source of baicalein production, with great prospects for market application.

Systematic characterization of flavonoids from Siraitia grosvenorii leaf extract using an integrated strategy of high-speed counter-current chromatography combined with ultra high performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry.

The chemical constituents of the Siraitia grosvenorii leaf extract were studied. Firstly, high-speed counter-current chromatography was applied to the one-step separation of four compounds from S. grosvenorii leaf extract with the solvent system composed of 0.01% acetic acid water/n-butanol/n-hexane/methanol (5:3:1:1, v/v/v/v). In this work, 270 mg of crude sample yielded four compounds, a new kaempferol O-glycoside derivative, kaempferol 3-O-α-L-[4-O-(4-carboxy-3-hydroxy-3-methylbutanoyl)]-rhamnopyranoside-7-O-α-L-rhamnopyranoside, named kaempferitrin A (2.1 mg, 90%), and three known compounds, grosvenorine (3.4 mg, 93%), kaempferitrin (14.4 mg, 99%) and afzelin (4 mg, 98%), and the structures of these compounds were identified by NMR spectroscopy and mass spectrometry. Then, ultra high performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry was used to illustrate the dominant flavonoids in S. grosvenorii leaf extract. 34 flavonoids including 19 kaempferol O-glycosides, 4 quercetin O-glycosides, 6 flavanone derivatives, and 5 polymethoxyflavones, were accurately or tentatively identified by carefully comparing their retention times, UV data, precise masses, the typical fragments of the standards and literature data. Most of these compounds were reported for the first time. This study establishes a foundation for the further development and utilization of S. grosvenorii leaves in future.

Modification of isoprene synthesis to enable production of curcurbitadienol synthesis in Saccharomyces cerevisiae.

Cucurbitane-type triterpenoids such as mogrosides and cucurbitacins that are present in the plants of Cucurbitaceae are widely used in Asian traditional medicine. Cucurbitadienol is the skeleton of cucurbitane-type triterpenoids. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the eventual transformation of cucurbitadienol. The synthetic pathway of cucurbitadienol was constructed in Saccharomyces cerevisiae by introducing the cucurbitadienol synthase gene from different plants, resulting in 7.80 mg cucurbitadienol from 1 L of fermentation broth. Improving supplies of isoprenoid precursors was then investigated for increasing cucurbitadienol production. Cucurbitadienol production increased to 21.47 mg/L through the overexpression of a global regulatory factor (UPC2) gene of triterpenoid synthase. In addition, knockout of the ERG7 gene increased cucurbitadienol production from 21.47 to 61.80 mg/L. Finally, fed-batch fermentation was performed, and 63.00 mg/L cucurbitadienol was produced. This work is an important step towards the total biosynthesis of valuable cucurbitane-type triterpenoids and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for triterpenoids.

Rapid Evaluation of Chemical Consistency of Artificially Induced and Natural Resina Draconis Using Ultra-Performance Liquid Chromatography Quadrupole-Time-of-Flight Mass Spectrometry-Based Chemical Profiling.

Resina Draconis is a highly valued traditional medicine widely used in Arabia since ancient times, and it has been commonly used as an antidiarrheic, antimicrobial, antiulcer, blood circulation promoter as well as an anti-inflammatory agent. The tree source from which this medicine orignates grows extremely slowly, producing a very low yield of Resina Draconis. To meet the increasing market demand, artificial methods for stimulating Resina Draconis formation have been developed and applied. However, the chemical differences between artificially induced Resina Draconis (AIRD) and natural Resina Draconis (NRD) have been rarely studied. The aim of this research was to explore and identify the chemical constituents of AIRD and NRD using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS) based chemical profiling. A total of 56 chromatographic peaks were detected in AIRD, of these, 44 peaks have had their structures tentatively characterized based on high-resolution mass spectra (HRMS) data, fragmentation ions information, reference standards data and literature review. In total, 40 peaks were found both in AIRD and NRD. The potential chemical transformation mechanisms active in Resina Draconis during formation were explored. To the best of our knowledge, this is the first evaluation of the chemical profiles of both AIRD and NRD. Furthermore, these findings are expected to provide a rational basis for the quality assessment of AIRD and the use of AIRD as a substitute for NRD.

[Prospects of molecular breeding in medical plants].

The molecular-assisted breeding, transgenic breeding and molecular designing breeding are three development directions of plant molecular breeding. Base on these three development directions, this paper summarizes developing status and new tendency of research field of genetic linkage mapping, QTL mapping, association mapping, molecular-assisted selections, pollen-mediated transformations, agrobacterium-mediated transformations, particle gun-mediated transformations, genome editing technologies, whole-genome sequencing, transcriptome sequencing, proteome sequencing and varietal molecular designing. The objective and existing problem of medical plant molecular breeding were discussed the prospect of these three molecular breeding technologies application on medical plant molecular breeding was outlooked.

[Study on exogenous hormones inducing parthenocarpy fruit growth and development and quality of Siraitia grosvenorii].

To explore the growth and development and analyze the quality of the parthenocarpy fruit induced by exogenous hormones of Siraitia grosvenorii. the horizontal and vertical diameter, volume of the fruit were respectively measured by morphological and the content of endogenous hormones were determined by ELISA. The size and seed and content of mogrosides of mature fruit were determined. The results showed that the fruit of parthenocarpy was seedless and its growth and development is similar to the diploid fruit by hand pollination and triploid fruit by hand pollination or hormones. But the absolute value of horizontal and vertical diameter, volume of parthenocarpy fruit was less than those of fruit by hand pollination, while triploid was opposite. The content of IAA, ABA and ratio of ABA/GA was obviously wavy. At 0-30 d the content of IAA and ABA of parthenocarpy fruit first reduced then increased, content of IAA and GA parthenocarpy fruit was higher than that of fruit by hand pollination. Mogrosides of parthenocarpy fruit was close to pollination fruit. Hormones can induce S. grosvenorii parthenocarpy to get seedless fruit and the fruit shape and size and quality is close to normal diploid fruit by hand pollination and better than triploid fruit by hormone or hand pollination.

[Selection of reference genes of Siraitia grosvenorii by real-time PCR].

Siraitia grosvenorii is a traditional Chinese medicine also as edible food. This study selected six candidate reference genes by real-time quantitative PCR, the expression stability of the candidate reference genes in the different samples was analyzed by using the software and methods of geNorm, NormFinder, BestKeeper, Delta CT method and RefFinder, reference genes for S. grosvenorii were selected for the first time. The results showed that 18SrRNA expressed most stable in all samples, was the best reference gene in the genetic analysis. The study has a guiding role for the analysis of gene expression using qRT-PCR methods, providing a suitable reference genes to ensure the results in the study on differential expressed gene in synthesis and biological pathways, also other genes of S. grosvenorii.

[Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii].

Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD260/280, OD260/230, RNA integrity (RIN) and yield of the total RNA with this method were 2.01, 2.02, 9.50 and 260 mirog.g-1, respectively. The open reading frame (ORF) of dehydroascorbate reductase (DHAR), named as SgDHAR, was cloned by rapid amplification of cDNA ends (RACE) and RT-PCR method from S. grosvenorii. The GenBank accession number for this gene is KC907731. The SgDHAR gene contains a full-length cDNA of 1,252 bp including ORF of 819 bp and encodes a predicted protein of 272 amino acids. The molecular mass is 30.217 7 kD and the isoelectric point is 8.76. Homology comparison showed that it shared 87% nucleotide sequence homology with Cucumis sativus. Expression patterns using qRT-PCR analysis showed that SgDHAR was mainly expressed in fruit and stem, followed by flower, and was lowest in root, while the expression level was 6.83 times in triploid. T than that in diploid. Therefore, SgDHAR gene may be involved in abortion of triploid seedless S. grosvenorii.

[Reasons that seedless fruits of Siraitia grosvenorii become small].

OBJECTIVE: To study the reasons that the seedless fruits of Siraitia grosvenorii developed to smaller ones. METHODS: The differences of fruit expanding, gene expressing and cell development were investigated between the triploid and diploid fruits from two strains F050 and F049. RESULTS: The results showed the expanding of triploid fruits was stopped about 20 days after artificial pollination, 10 days earlier than the diploid fruits. Meanwhile, it was also investigated that aux expressing level in the triploid fruits was greatly higher than that in diploid fruits, while its ipt, cyt-p450, spds, cycB, cycD1, cycD3, cdkA, cdkB, exp and xth expressing level were greatly lower than that in diploid fruits. The majority of sarcocarp cells of the triploid fruits kept in the stage of small ones comparing with the diploid fruits. CONCLUSION: The expression of the genes of the proteins involving cell division and expansion was inhibited as the significant reduction of endogenous IAA, CTK, GA and SPDS after bad fertilization and the embryo abortion, which resulted in the end of division and extension of sarcocarp cells. Above reasons induce small fruits of triploid seedless Siraitia grosvenorii.

Root Metabolism and Effects of Root Exudates on the Growth of Ralstonia solanacearum and Fusarium moniliforme Were Significantly Different between the Two Genotypes of Peanuts.

Wild peanut species Arachis correntina (A. correntina) had a higher continuous cropping tolerance than peanut cultivars, closely correlating with the regulatory effects of its root exudates on soil microorganisms. To reveal the resistance mechanism of A. correntina to pathogens, we adopted transcriptomic and metabolomics approaches to analyze differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) between A. correntina and peanut cultivar Guihua85 (GH85) under hydroponic conditions. Interaction experiments of peanut root exudates with Ralstonia solanacearum (R. solanacearum) and Fusarium moniliforme (F. moniliforme) were carried out in this study. The result of transcriptome and metabolomics association analysis showed that there were fewer up-regulated DEGs and DEMs in A. correntina compared with GH85, which were closely associated with the metabolism of amino acids and phenolic acids. Root exudates of GH85 had stronger effects on promoting the growth of R. solanacearum and F. moniliforme than those of A. correntina under 1 and 5 percent volume (1% and 5%) of root exudates treatments. Thirty percent volume (30%) of A. correntina and GH85 root exudates significantly inhibited the growth of two pathogens. The exogenous amino acids and phenolic acids influenced R. solanacearum and F. moniliforme showing concentration effects from growth promotion to inhibition as with the root exudates. In conclusion, the greater resilience of A. correntina) to changes in metabolic pathways for amino acids and phenolic acids might aid in the repression of pathogenic bacteria and fungi.

Heterologous mogrosides biosynthesis in cucumber and tomato by genetic manipulation.

Mogrosides are widely used as high-value natural zero-calorie sweeteners that exhibit an array of biological activities and allow for vegetable flavour breeding by modern molecular biotechnology. In this study, we developed an In-fusion based gene stacking strategy for transgene stacking and a multi-gene vector harbouring 6 mogrosides biosynthesis genes and transformed it into Cucumis sativus and Lycopersicon esculentum. Here we show that transgenic cucumber can produce mogroside V and siamenoside I at 587 ng/g FW and 113 ng/g FW, respectively, and cultivated transgenic tomato with mogroside III. This study provides a strategy for vegetable flavour improvement, paving the way for heterologous biosynthesis of mogrosides.

Genome-Wide Identification and Expression Analysis of WRKY Transcription Factors in Siraitia siamensis.

WRKY transcription factors, as the largest gene family in higher plants, play an important role in various biological processes including growth and development, regulation of secondary metabolites, and stress response. In this study, we performed genome-wide identification and analysis of WRKY transcription factors in S. siamensis. A total of 59 SsWRKY genes were identified that were distributed on all 14 chromosomes, and these were classified into three major groups based on phylogenetic relationships. Each of these groups had similar conserved motifs and gene structures. We compared all the S. siamensis SsWRKY genes with WRKY genes identified from three diverse plant species, and the results implied that segmental duplication and tandem duplication play an important roles in the evolution processes of the WRKY gene family. Promoter region analysis revealed that SsWRKY genes included many cis-acting elements related to plant growth and development, phytohormone response, and both abiotic and biotic stress. Expression profiles originating from the transcriptome database showed expression patterns of these SsWRKY genes in four different tissues and revealed that most genes are expressed in plant roots. Fifteen SsWRKY genes with low-temperature response motifs were surveyed for their gene expression under cold stress, showing that most genes displayed continuous up-regulation during cold treatment. Our study provides a foundation for further study on the function and regulatory mechanism of the SsWRKY gene family.

Karyotype analysis and genetic variation of a mutant in Siraitia grosvenorii.

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.

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis.

BACKGROUND: Siraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway. RESULTS: In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis. CONCLUSION: A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven CYP450s and five UDPGs were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from S. grosvenorii.

The first genetic linkage map of Luohanguo (Siraitia grosvenorii ) based on ISSR and SRAP markers.

In this study, the first genetic map of Luohanguo (Siraitia grosvenorii (Swingle) C. Jeffrey) was constructed with 150 F2 population individuals using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. A total of 100 ISSRs and 196 SRAP primer combinations generated 51 and 222 polymorphic markers, respectively. Among the 273 markers obtained, 199 markers (29 ISSRs and 170 SRAPs) were mapped to 25 linkage groups. The map covered 1463.3 cM with a mean map distance of 7.35 cM between adjacent markers and a maximum map distance of 52.6 cM between two markers. The markers were distributed randomly in 25 groups except for minor clusters in the distal region of linkage groups. All 25 linkage groups consisted of 2-36 loci ranging in length from 19.5 to 152.6 cM and accounted for 59.8% of the total map distance. This map provides reference information for future molecular breeding work on Luohanguo.

[Effects of controlled atmosphere storage on fruit quality of Siraitia grosvenorii during storage].

UNLABELLED: To explore the condition of CA storage for fruit of Siraitia grosvenorii. METHOD: The CA storage of 5% CO2, 15% CO2, 30% O2 under normal temperature was applied in the experiments, and the results were compared. RESULT: The results showed that CA storage condition could alleviate the reduction of weight loss and water content, the best CA storage condition were 5% CO2, which alleviates the reduction of firmness and increases of soluble solid content during the 45 day period.

[Growth curve of Siraitia grosvenorii and correlative analysis of seed and growth of fruit].

OBJECTIVE: To establish the growth curve of Siraitia grosvenorii and analyze the correlation between seed and growth of fruit. METHOD: The growth curve fitting function was applied for the study of correlation between seed and fruit growth. RESULT: The significant positive correlation existed between seed and horizontal diameter x vertical diameter, not between seed and flesh weight of single fruit. CONCLUSION: The growth curve is a reciprocal function, and seed is one of major factors to influencing the size of fruits and the shape of fruit.