Genome-Wide Identification and Analysis on YUCCA Gene Family in Isatis indigotica Fort. and IiYUCCA6-1 Functional Exploration.

Auxin is one of the most critical hormones in plants. YUCCA (Tryptophan aminotransferase of Arabidopsis (TAA)/YUCCA) enzymes catalyze the key rate-limiting step of the tryptophan-dependent auxin biosynthesis pathway, from IPA (Indole-3-pyruvateacid) to IAA (Indole-3-acetic acid). Here, 13 YUCCA family genes were identified from Isatis indigotica, which were divided into four categories, distributing randomly on chromosomes (2n = 14). The typical and conservative motifs, including the flavin adenine dinucleotide (FAD)-binding motif and flavin-containing monooxygenases (FMO)-identifying sequence, existed in the gene structures. IiYUCCA genes were expressed differently in different organs (roots, stems, leaves, buds, flowers, and siliques) and developmental periods (7, 21, 60, and 150 days after germination). Taking IiYUCCA6-1 as an example, the YUCCA genes functions were discussed. The results showed that IiYUCCA6-1 was sensitive to PEG (polyethylene glycol), cold, wounding, and NaCl treatments. The over-expressed tobacco plants exhibited high auxin performances, and some early auxin response genes (NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6) were upregulated with increased IAA content. In the dark, the contents of total chlorophyll and hydrogen peroxide in the transgenic lines were significantly lower than in the control group, with NbSAG12 downregulated and some delayed leaf senescence characteristics, which delayed the senescence process to a certain extent. The findings provide comprehensive insight into the phylogenetic relationships, chromosomal distributions, and expression patterns and functions of the YUCCA gene family in I. indigotica.

Transcriptome sequencing of Salvia miltiorrhiza after infection by its endophytic fungi and identification of genes related to tanshinone biosynthesis.

Context: Salvia miltiorrhiza Bunge (Labiatae) is a traditional Chinese herb. Endophytic fungi, which are biotic elicitors, can induce accumulation of secondary metabolites in their host plants.Objective: To analyze the interaction mechanism between S. miltiorrhiza and endophytic fungi.Materials and methods: Endophytic fungi U104 producing tanshinone IIA were isolated from the healthy disease-free tissue of root of S. miltiorrhiza by conventional methods. The endophytic fungus U104 of S. miltiorrhiza was co-cultured with the sterile seedlings of S. miltiorrhiza for 20 d (temp:day/night = 26 °C/18 °C, photoperiod:12/12 h, illuminance:2000 Lx). Transcriptome sequencing of S. miltiorrhiza seedlings after 20 d of co-cultivation was performed using the Illumina platform.Results: A total of 3713 differentially expressed genes (DEGs) were obtained. These different expression genes, such as STPII, LTP2, MYB transcription factors, CNGC, CDPK, Rboh, CaM, MAP2K1/MEK1, WRKY33, SGT1/SGT and Hsp90/htpG, showed that host S. miltiorrhiza had biological defence response in the initial stage of interaction. Under the induction of endophytic fungi, 14 key enzyme genes were up-regulated in the tanshinone biosynthesis pathway: DXS, DXS2, DXR, HMGR3, AACT, MK, PMK, GGPPS2, GPPS, KSL, IDI, IPII, FDPS and CPS.Discussion and conclusions: A total of 14 key genes were obtained from the tanshinone component synthesis and metabolic pathways, providing a reasonable explanation for the accumulation of tanshinone components, an accumulation induced by endophytic fungi, in the host plants. The large amounts of data generated in this study provide a strong and powerful platform for future functional and molecular studies of interactions between host plants and their endophytic fungi.

Enhanced production of tanshinone IIA in endophytic fungi Emericella foeniculicola by genome shuffling.

CONTEXT: Tanshinone IIA, commercially produced from Salvia miltiorrhiza Bunge (C.Y.Wu) (Labiatae), has various biological benefits. Currently, this compound is mainly extracted from plants. However, because of the long growth cycle and the unstable quality of plants, the market demands can barely be satisfied. OBJECTIVE: The genomic shuffling technology is applied to screen the high-yield tanshinone IIA strain, which could be used to replace the plant S. miltiorrhiza for the production of tanshinone IIA. The change in the production of tanshinone IIA is clarified by comparing it with the original strain. MATERIALS AND METHODS: Tanshinone IIA was extracted from Strains cells, which was prepared through 0.5 mL protoplast samples by using hypertonic solution I from two different strains. Then, it was analyzed by high-performance liquid chromatography at 30 °C and UV 270 nm. Total DNA from the strains was extracted for RAPD amplification and electrophoresis to isolate the product. RESULTS: In this study, a high-yield tanshinone IIA strain F-3.4 was screened and the yield of tanshinone IIA was increased by 387.56 ± 0.02 mg/g, 11.07 times higher than that of the original strain TR21. DISCUSSION: This study shows that the genetic basis of high-yield strains is achieved through genome shuffling, which proves that genome shuffling can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining the strains with good traits and it is a useful method for the molecular breeding of industrial strains.

[Diversity of Endogeny Eumycetes in Gynostemma pentaphyllum and Its Correlation with Gypenoside XLIX].

OBJECTIVE: To study the correlation between active components and endogeny eumycetes in Gynostemma pentaphyllum of different types from different habitats. METHODS: Endogeny eumycetes from different parts of Gynostemma pentaphyllum were isolated by general isolation methods. Insert method and point planting method were used for identification. The content of gypenoside XLIX were determined by HPLC. RESULTS: 125 endogeny eumycetes inhabiting in Gynostemma pentaphyllum were isolated from roots, rhizomes and leaves. By colony morphology and microscopic characteristics, 22 genera from 10 families, 7 orders, 2 classes were identified. Fusarium was the most abundant endogeny eumycetes in Gynostemma pentaphyllum with the account of 22. 4%. Penicillium and Leptosphaeria was 12. 8% and 9. 6% respectively. The correlation between Gypenoside XLIX and endogeny eumycetes in Gynostemma pentaphyllum was revealed. CONCLUSION: Endogeny eumycetes are diverse in species and quantity. The endogeny eumycetes species is related to the quality of Gynostemma pentaphyllum.

Gypenoside biotransformation into ginsenoside F2 by endophytic Aspergillus niger from Gynostemma pentaphyllum.

Ginsenoside F2 is a protopanaxadiol saponin compound with various biological activities, including antioxidant, anti-inflammatory, and anticancer properties. Ginsenoside F2 can be found in ginseng, but in low quantities. Therefore, ginsenoside F2 production predominantly relies on the biotransformation of various ginsenosides, such as ginsenosides Rb1 and Rd. In this study, we reported the production of ginsenoside F2 by gypenoside biotransformation with Aspergillus niger JGL8, isolated from Gynostemma pentaphyllum. Ginsenoside F2 could be produced by two different biotransformation pathways, namely Gyp-V-Rd-F2 and Gyp-XVII-F2. The product exhibited antioxidant activity against free radicals (DPPH) with IC50 value of 29.54 µg/mL. Optimal biotransformation conditions were a pH of 5.0, temperature of 40 °C, and 2 mg/mL of substrate. Enzyme kinetic parameters revealed that the hydrolysis rate of Gyp-V, Rd, and Gyp-XVII was 0.625, 0.588, and 0.417 mM/h, respectively. In conclusion, we demonstrated that gypenoside is a substitutable substrate for ginsenoside F2 biotransformation.

Multi-integrated approach for unraveling small open reading frames potentially associated with secondary metabolism in Streptomyces.

IMPORTANCE: Due to their small size and special chemical features, small open reading frame (smORF)-encoding peptides (SEPs) are often neglected. However, they may play critical roles in regulating gene expression, enzyme activity, and metabolite production. Studies on bacterial microproteins have mainly focused on pathogenic bacteria, which are importance to systematically investigate SEPs in streptomycetes and are rich sources of bioactive secondary metabolites. Our study is the first to perform a global identification of smORFs in streptomycetes. We established a peptidogenomic workflow for non-model microbial strains and identified multiple novel smORFs that are potentially linked to secondary metabolism in streptomycetes. Our multi-integrated approach in this study is meaningful to improve the quality and quantity of the detected smORFs. Ultimately, the workflow we established could be extended to other organisms and would benefit the genome mining of microproteins with critical functions for regulation and engineering useful microorganisms.

Phillyrin produced by Colletotrichum gloeosporioides, an endophytic fungus isolated from Forsythia suspensa.

To investigate the phillyrin-producing endophytic fungi from the medicinal plant Forsythia suspensa, a total of 24 strains of endophytic fungi were isolated from the healthy stems, leaves and fruits of the plant, 9 from stems, 5 from leaves, and 10 from fruits respectively. All fungal isolates were fermented in liquid PDA medium and their extracts were preliminary analyzed by TLC. One isolated strain G10, which was from the fruit of F. suspensa, had the same R(f) value as authentic phillyrin. The potential phillyrin-producing fungus G10 was further analyzed by HPLC and HPLC-MS, and the results showed that the isolate G10 possessed of a retention time and ion peaks identical with the authentic compound phillyrin. The isolate G10 was identified as Colletotrichum gloeosporioides based on the morphological characteristics and ITS sequence analysis. The current research indicates that the numerous endophytic fungi inside the medicinal plants are precious resource for the pharmaceutical natural products that are originally from the plants.

mTOR complex component Rictor interacts with PKCzeta and regulates cancer cell metastasis.

Epidermal growth factor (EGF) mediates breast cancer cell chemotaxis and metastasis through mechanisms that involve the growth-regulatory mammalian target of rapamycin (mTOR) complex mTORC2, but the mechanisms involved remain obscure. Here, we report that the rapamycin-insensitive mTORC2 component protein Rictor is a critical mediator of metastasis in breast cancer cells. In patients with ductal carcinoma, Rictor expression was associated with increased lymph node metastasis. EGF induced translocation and colocalization of Rictor with protein kinase Cζ (PKCζ), a pivotal molecule in chemotaxis signaling. Further, Rictor coimmunoprecipitated with PKCζ in the absence of the mTORC2 complex. Small interfering RNA-mediated knockdown of Rictor inhibited EGF-induced PKCζ phosphorylation and translocation along with phosphorylation of the key F-actin binding protein cofilin. In parallel, Rictor knockdown reduced cellular chemotactic capacity and ablated pulmonary metastasis in a xenograft mouse model of breast cancer. Our findings identify Rictor as an important mediator of chemotaxis and metastasis in breast cancer cells.