Promoter activity analysis and transcriptional profile of Ginkgo biloba 1-Deoxy-D- Xylulose 5-Phosphate reductoisomerase gene (GbDXR) under abiotic stresses

Terpene trilactones (TTL) is a pharmacological ingredient in Ginkgo biloba and its content has become one of the key indices for medicinal value evaluation of ginkgo. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first step specific for isopentenyl diphosphate production in methylerythritol phosphate pathway, which provide the basic structure required for TTLs biosynthesis. To understand the mechanism controlling the GbDXR gene expression, the GbDXR promoter sequence was isolated and subjected to transient expression with the green fluorescent protein (GFP) in tobacco plants. Characteristic analysis revealed various cis-acting elements that related to light-regulated transcription, hormone signaling (auxin, ethylene), adversity stress and defense signaling (heat/dehydration stress) in the GbDXR promoter region. In transient expression assay, deletion of different portions of the upstream GbDXR promoter identified that the promoter region -3230bp to -865bp conserve the positive regulation function, which could promote the expression of GFP in the cytoplasm of tobacco leaf epidermal cells. The regulation function of the promoter region -865bp to -262bp remained to be elucidated. EMSA analysis suggested possible interactions of GbERF10 and GbERF17 with the ERF-binding elements in the upstream of GbDXR promoter. For abiotic stresses treatment, the expression of GbDXR gene could be significantly induced by UV-B and drought stress. In general, the GbDXR gene expressed differently in different ginkgo tissues but exhibited the highest transcriptional level in the root, with the maximum TTLs content simultaneously. The positive relationship between gene expression level and TTLs content indicated that the GbDXR is responsible for TTLs biosynthesis in G. biloba

Effects of 5-Aminolevulinic Acid on Chlorophyll, Photosynthesis, Soluble Sugar and Flavonoids of Ginkgo biloba

The flavonoid content determines the quality of Ginkgo biloba that can be increased by using of plant growth regulators. The objective of study was to observe the effect of 5-aminolevulinic acid (ALA), a key precursor in the biosynthesis of porphyrins and a new plant growth regulator, on photosynthetic rate, chlorophyll and soluble sugar content, flavonoid accumulation, and flavonoid enzyme activity in G. biloba leaves. The ginkgo seedlings were grown in greenhouse conditions under low levels (10 and 100 mg l-1) of foliar application of ALA. Photosynthetic rates of leaves increased significantly at day 4 in response to both ALA concentrations and remained elevated as compared to control for further 12 days. Chlorophyll and soluble sugar contents were significantly increased by day 4 and continued to increase by day 16; however, Chl a/b ratio remained unchanged. Total polyphenols, flavonoids, and anthocyanins, phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone isomerase (CHI) activities were increased from day 4 to 16 after ALA treatment. The increase in chlorophyll and soluble sugar contents, and activities of flavonoid enzymes (PAL, CHS and CHI) were likely to be closely associated with improvement of the accumulation of total polyphenols, flavonoids, anthocyanins and advance of leaf quality by ALA treatment. Foliar treatment with a low concentration of ALA therefore, might provide a useful means of improving pharmacological properties of G. biloba leaves

Extraction, Isolation and Identification of Antimicrobial Substances from Bacillus amyloliquefaciens CMN1308

Four separation methods of antimicrobial substances produced by CMN1308 (Bacillus amyloliquefaciens) were evaluated and selected according to number of antimicrobial substances and its activity in vitro. The results showed that extraction by acid precipitation of the fermentation supernatant of CMN1308 was the best with a diameter of inhibition zone of pathogen fungi P. expansum of 12.3 mm in a laboratory bioassay. Applying a silica thin layer chromatography (TLC), SDS-PAGE and other separation technologies we isolate antimicrobial substances, and the separated band were cut off for mass spectrometry analysis. The TLC of crude extract of CMN1308 show a topical band corresponding with the surfactin standard (Rf value =0.75), proved that the strain CMN1308 can produce this surface active compound. The mycoprotein extracted from CMN1308 was separated by Tricine-SDS-PAGE modified with the addition of urea in the separation gel. After mass spectrometric analysis and protein characterization, the isolated mycoprotein showed a maximum ion peak at M/Z of 2679 and molecular weight of 29.5 kDa, matching with protein flagellin. The extracellular antimicrobial protein of strain CMN1308 display four bands after urea-Tricine-SDS-PAGE, but after mass spectrometry analysis only two bands were identified. Band “A” with a maximum ion peak at M/Z of 1926 and molecular weight of 49.8 kDa, aligned with NCBI database, matching with DLDH (dihydrolipoamide dehydrogenase enzyme). Band “D” show the maximum ion peak at M/Z of 2936 and molecular weight of 22.4 kD, matching with a chitin binding protein. Thus, the strain CMN1308 has the potential to be developed as a commercial biological control agent for chestnut common pathogenic fungi

Transcriptome-based Discovery of AP2/ERF Transcription Factors Related to Terpene Trilactones Synthesis in Ginkgo biloba

Ginkgo biloba is a unique tree in China with medicinally and phylogenetically important characteristics. Terpene trilactones (TTL) is a key active pharmaceutical ingredient in Ginkgo, so the content of TTL in Ginkgo has become one of the important indices for evaluating quality of the medicinal materials. By transcriptome sequencing on samples treated by chlormequat, ultraviolet (UV) and drought, totally 59820 contigs and 37564 unigenes were obtained. Furthermore, 18234 unigenes were annotated through COG, KEGG and GO analysis. There were 78 AP2/ERF transcription factors, 23 factors of up-regulation and 66 factors of down-regulation that were related with synthetic pathway of TTL in Ginkgo. Phylogenetic tree clustering analysis indicated that there were 42 AP2s could be clustered into ERF, DREB and RVA subfamilies. EMSA analysis demonstrated that GbERF13, GbERF25 and GbERF27 could bind with regulatory elements, such as E-box, in the upstream of GbMECPs promoter. Expression analysis showed that the expression level of GbERF25 was the highest in root, and GbERF25 and GbERF27 were expressed in relatively high transcription levels in leaf and other tissues. The results of qRT-PCR indicated that CCC treatment could significantly improve expression levels of ERF25 and ERF27, and UV and drought could induce transcription levels of ERF13 and ERF25, respectively. The results implied that ERF25 and ERF27 might involve in the induction and regulation of CCC treatment on synthesis of bilobalide in G. biloba. ERF13 might participate in the regulation of bilobalide synthesis induced by UV, and EFR25 might involve in the regulation of the synthesis induced by drought. During annual cycle of expression, the transcription levels of ERF13, ERF25 and ERF27 had significantly positive correlation with diterpene level with correlation coefficient 0.975. It implied that these transcription factors mainly acted on the MEP pathway that regulated synthesis of bilobalide. The aim of the research was to indicate the mechanism of environment or cultivation measure regulating target gene of TTL metabolic pathway by AP2/ERF, and establish metabolic network of AP2/ERF regulating TTL synthesis

Molecular Cloning and Expression Analysis of a MADS-Box Gene (GbMADS2) from Ginkgo biloba

As a kind of transcription factors gene family, MADS-box genes play an important role in plant development processes. To find genes involved in the floral transition of Ginkgo biloba, a MADS-box gene, designated as GbMADS2, was cloned from G. biloba based on EST sequences by RT-PCR. Sequence analysis results showed that the cDNA sequence of GbMADS2 contained a 663 bp length ORF encoding 221 amino acids protein, which displayed typical structure of plant MADS-box protein including MADS, I, and K domains and C terminus. The sequence of GbMADS2 protein was highly homologous to those of MADS-box proteins from other plant species with the highest homologous to AGAMOUS (CyAG) from Cycas revoluta. The phylogenetic tree analysis revealed that GbMADS2 belonged to AGAMOUS clade genes. Real-time PCR analysis indicated that expression levels of GbMADS2 gene in female and male flower were significantly higher than those in root, stem, and leaves, and that GbMADS2 expression level increased along with time of flower development. The spatial and time-course expression profile of GbMADS2 implied that GbMADS2 might be involved in development of reproductive organs. The isolation and expression analysis of GbMADS2 provided basis for further studying the molecular mechanism of flower development in G. biloba

High-Density Kinetic Analysis of the Metabolomic and Transcriptomic Response of Ginkgo biloba Flavonoids Biosynthesis to Selenium Treatments

As one of the rare and precious wood species since the ancient times, Gingko is also known as “living fossil”, which is a special plant resource of China. Gingko leaves, containing rich flavonoids, are valued with great medicinal significances. This paper treated Ginkgo seedlings by exogenous Sodium selenite (SS) in two ways: Foliage dressing (FD) and Root application (RA). Then transcriptome sequencing and metabolome test are performed. Results show that external SS has significant influence on the related gene expression level of flavonoids synthesis ways of Gingko, the FD can significantly induce gene expressions as CHS, FLS, FOMT, PAL, MYB1 and MYB2, and RA can significantly induce gene expressions as FOMT, MYB1 and MYB2. Compared with the control group, FA selenium application can help to accumulation of flavonoids, flavonols, flavonoids-C and isoflavones, especially quercetin and kaempferol that had a remarkable increase. This proved that a proper concentration of inorganic SS could promote the synthesis and accumulation of flavonoids in Gingko. qRT-PCR analysis also depicts that leaves treatment of sodium selenite can remarkably enhance the gene expression of CHS, FLS, FOMT and PAL, and RA selenium application can induce the gene expression of FLS and FOMT, but restrain the gene expression of CHS and PAL. Through the ways of FD and RA selenium application, this paper basically studied the regulatory effect of SS on ginkgo flavonoids synthesis and has laid a theoretical basis to improve flavonoids content in Ginkgo leaves through cultivation control means

Effect of exogenous gibberellin on endogenous hormone and ginkgolide content in Ginkgo leaves

Ginkgolide is one of the important secondary metabolites of Ginkgo biloba. The synthesis of plant secondary metabolites is influenced by exogenous phytohormones. In this study, ten-year-old ginkgo grafted seedlings were used as the test material, and the effects of 0, 2, 4, 6, 8, and 10 mmol L–1 gibberellin (GA3) on endogenous indole acetic acid (IAA), abscisic acid (ABA), GA3, and ginkgolide contents in ginkgo leaves were investigated. Results showed that exogenous GA3 treatment inhibited endogenous GA3, changed the contents of endogenous IAA and ABA in ginkgo leaves, and affected the accumulation of secondary metabolite ginkgolides. Among the given concentrations, the 4 mmol L–1 GA3 treatment could remarkably increase ginkgolide content. We found that the best harvesting period of ginkgo leaves were in late August and late September, in which the content of ginkgolides was the highest

Genome-Wide Identification of WRKY Family Genes and Analysis of Their Expression in Response to Abiotic Stress in Ginkgo biloba L.

Ginkgo biloba is widely planted, and the extracts of leaves contain flavonoids, terpene esters and other medicinal active ingredients. WRKY proteins are a large transcription factor family in plants, which play an important role in the regulation of plant secondary metabolism and development, as well as the response to biotic and abiotic stress. In our study, we identified 40 genes with conserved WRKY motifs in the G. biloba genome and classified into groups I (groups I-N and -C), II (groups IIa, b, c, d, and e), and III, which include 12, 26, and 2 GbWRKY genes, respectively. Meanwhile, the expression patterns of 10 GbWRKY (GbWRKY2, GbWRKY3, GbWRKY5, GbWRKY7, GbWRKY11, GbWRKY15, GbWRKY23, GbWRKY29, GbWRKY31, GbWRKY32) under different tissue and abiotic stress conditions were analyzed. Under stress treatment, the expression patterns of 10 WRKY genes were changed. 10 ginkgo WRKY transcription factors were induced by ETH and SA, but there are two different induced response modes. The expression of 10 WRKY genes was inhibited under low temperature, high temperature and MeJA hormone induction. Most WRKY genes were up-regulated under the induction of high salt and ABA. GbWRKYs were differentially expressed in various tissues after abiotic stress and plant hormone treatments, thereby indicating their possible roles in biological processes and abiotic stress tolerance and adaptation. Our results provided insight into the genome-wide identification of GbWRKYs, as well as their differential responses to stresses and hormones. These data can also be utilized to identify potential molecular targets to confer tolerance to various stresses in G. biloba

Biocontrol of Pear Postharvest Decay by Kombucha

Kombucha is sweetened, slightly alcoholic, and lightly effervescent tea drink. Its acidity inhibits the growth of harmful bacteria. Here, we studied effect of kombucha on post-harvest preservation of pear. The Hosui pears were soaked with the kombucha for 15 minutes, and in distilled water as the control, respectively. Superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), hydrogen peroxide (H2O2), proline content, electric conductivity, weight loss rate and good fruit rate in pear were measured during storage at room temperature. The results showed that the kombucha could effectively prolong the storage time of pear fruit. The fruit quality of the treated group was 1.5 times higher than that of the control pear fruit when stored at room temperature for 18 days. The weight loss rate, MDA content and electric conductivity of the treatment group were lower than those of the control group during the whole storage period, indicating that kombucha can inhibit the transformation of polysaccharides such as starch and pectin, and delay the degradation of nutrients in the fruit, resulting in a decrease in weight loss rate, inhibition of membrane lipid peroxidation, reducing MDA content, electric conductivity, maintaining cell membrane stability, delaying pear fruit senescence. Kombucha can inhibit the content of H2O2 and proline, increase the activity of POD and SOD increase the storage stability of fruits. The purpose of this study was to elucidate the physiological mechanism of post-harvest preservation of pear by kombucha. The use of kombucha can prolong the supply period of pear, increase economic benefits and expand the market of pear

Characterization and expression analysis of four members genes of flavanone 3-hydroxylase families from Chamaemelum nobile

Chamaemelum nobile is a traditional Chinese herbal medicine, whose secondary metabolites used in the pharmacology of Chinese medicine. Among them, the flavonoids have great research value. Flavanone 3-hydroxylase (F3H) is one of the core enzymes in the early steps of flavonoid biosynthesis. This study aimed to elucidate the structures, functions, and expression levels of F3H families from C. nobile. Four members of the F3H family were screened from C. nobile transcriptome data and performed bioinformatics analysis. Results showed that CnF3H1~4 had a high similarity with the other F3H plants, and all genes contained two conserved isopenicillin N synthase-like and oxoglutarate/iron-dependent dioxygenase domains. Further analysis revealed that the four CnF3H proteins contained some differences in binding sites. The results of secondary and 3-D structures displayed that the composition and proportion of the four CnF3H secondary structures were basically the same, and their 3D structures were consistent with the secondary structures. The phylogenetic tree displayed that CnF3H2, CnF3H3, and CnF3H4 were grouped with Asteraceae. The expression patterns of CnF3Hs in the roots, stems, leaves, and flowers of C. nobile were evaluated using the value of RPKM. The results indicated that CnF3Hs had significant difference in the expression of different tissues. Especially, CnF3H1~3 and CnF3H4 had the highest expression levels in the flowers and roots, respectively. Hence, CnF3Hs played a significant role in the flavonoid metabolism