关键词：
Circadian expression;Differentially expressed genes;Early flowering;FLOWERING LOCUS T;Ginkgo biloba;Juvenile phase
摘要：
Ginkgo biloba has a long juvenile phase, that prevents efficient breeding and genetic improvement. The molecular mechanism underlying the transition from vegetative to reproductive phase is currently unknown in G. biloba. FLOWERING LOCUS T (FT) encodes a mobile signaling protein involved in regulating flowering and other aspects of plant development. Herein, a FT homolog (GbFT) was isolated and characterized from G. biloba. GbFT encodes a 1091 bp gene with a predicted protein of 173 amino acids. GbFT was specifically expressed in the male and female strobili of G. biloba. GbFT expression showed a pattern of circadian rhythm and daily oscillation. Overexpression of GbFT using the CaMV 35S promoter in transgenic Arabidopsis plants caused early flowering phenotype compared with wild-type (WT) plants, indicating that GbFT promotes flowering. RNA-seq analysis of GbFT transgenic Arabidopsis plants (GbFT-OE) and WT plants screened out 227 significant differentially expressed genes (DEGs). Of these DEGs, 23 flowering-related DEGs were up-regulated in GbFT-OE compared with WT plants. In addition, RNA-seq and qRT-PCR analysis results showed that ectopic overexpression of GbFT in Arabidopsis up-regulated other endogenous genes involved in flower organ development, transcription factors and ethylene pathway, which may explain early flowering phenotype in transgenic plants. Our results lay the foundation for clarifying the molecular regulatory mechanism of flowering and for developing methods to shorten the juvenile phase of G. biloba.
摘要：
Peach (Prunus persica) is one of the most economically important crops, its fruit quality is defined by various criteria, including external quality and fruit taste. Peach contain anthocyanins as the main pigment with important nutritional and medical usages. Therefore, increasing the anthocyanin content in peach skin can make the fruit appearance of bright and attractive. This study investigated the formation mechanism of the external quality of peach fruit and utilized RNA-seq technology to analyze the molecular mechanisms of anthocyanin biosynthesis regulation in P. persica fruit skin. Approximately 333,899,176 clean reads were obtained, 25,694 unigenes were annotated in five public databases. After the different expression genes of the compared sequences were quantitated by RPKM method and sifted by a limited term, in total, 1665, 3214, and 639 differentially expressed genes were screened among 120 DAF (days after flowering) vs 105 DAF, 135 DAF vs 105 DAF, and 135 DAF vs 120 DAF, respectively. We identified 15 structural genes, 16 transcription factors, and 17 transferase genes that may be involved in the anthocyanin biosynthesis from P. persica and analyzed their expression patterns by using real-time PCR. Our results presented comprehensive gene expression information about peach skin transcriptome; and enhanced our understanding of key genes regulating the biological pathways of anthocyanin biosynthesis. This study could help us understand the molecular mechanism of anthocyanin metabolism in peach skin and provide important technical support for regulating peach skin coloration.
摘要：
<jats:p>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.

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In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI).DOI link will become active after the article will be included in the complete issue.

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摘要：
Flavonoids are a group of metabolites in Ginkgo biloba thought to provide health benefits. R2R3-MYB transcription factors (TFs) play key roles in the transcriptional regulation of the flavonoid biosynthesis in plants. In this study, an R2R3-MYB transcription factor gene, GbMYBFL, was isolated from G. biloba and characterized. Results of bioinformatic analysis indicated that GbMYBFL is more closely related to the R2R3-MYB involved in flavonoid biosynthesis and displayed high similarity to MYB from other plants. The genmomic sequence of GbMYBFL had three exons and two introns, with its upstream sequence containing cis-acting regulatory elements Myb binding site, Myc recognition sites, and light, SA, MeJA responsive elements. Subcellular localization analysis indicates that GbMYBFL was located in the nucleus. Quantitative real-time PCR revealed that GbMYBFL was expressed in leaves, stems, roots, young fruits, male flower and female flower, and the level of transcription in male flower and leaves were higher than that in female flower, stems, roots, and young fruits. During G. biloba leaf growth, the transcription of GbMYBFL is positively correlated with the flavonoid content, suggesting that the GbMYBFL is involved in the flavonoid biosynthesis. Overexpression of GbMYBFL under the control of the CaMV35S promoter in Ginkgo callus notably enhanced the accumulation of flavonoids and anthocyanin compared with non-transformed callus. This finding suggested that GbMYBFL positively related to flavonoid biosynthesis, and the overexpression of GbMYBFL was sufficient to induce flavonoids and anthocyanin accumulation.
摘要：
Plant cell walls primarily comprise lignin, which performs functions of mechanical support, water transport, and stress responses. Lignin biosynthesis pathway proceeds through metabolic grid featuring complexity and diversity in enzymatic reaction. Cinnamate-4-hydroxylase (C4H, EC 1.14.13.11) is the gene encoding enzyme that catalyzes the second step of phenylpropanoid pathway responsible for biosynthesis of lignin. A full-length cDNA of C4H (designated as GbC4H), which spanned 1816-bp with a 1518-bp open reading frame encoding a 505-amino-acid protein, was cloned from Ginkgo biloba. A GbC4H genomic DNA fragment, spanning 3249-bp, was cloned and found to contain two exons and one intron. GbC4H protein showed high similarities with other plant C4Hs to include conserved domains of cytochrome P450 family. GT-1, W-box, and Myb/Myc recognition sites involved in stress response were detected in a 1265-bp upstream promoter region of GbC4H. Phylogenetic analysis suggested the common evolutionary ancestor shared by plant C4Hs including the gymnosperm enzyme. pET-28a-GbC4H plasmid was constructed and expressed in Escherichia coli strain BL21. Enzymatic assay revealed that recombinant GbC4H protein catalyzes conversion of trans-cinnamic acid to p-coumaric acid. Expression analyses in different organs showed high expression of GbC4H in stems and roots, whereas low expressions was found in fruits, carpopodium, and petioles. Further analysis indicated linear correlation of lignin contents with transcript levels of GbC4H among different tissues. GbC4H transcription was increased by treatments with UV-B, cold, salicylic acid, and abscisic acid, indicating the possible role of GbC4H in response to stresses and hormonal signal. Understanding of GbC4H function could benefit molecular breeding and reinforcement of defense mechanisms in Ginkgo.
摘要：
<jats:p>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.</jats:p>
摘要：
Fungal diseases of plants continue to contribute to heavy crop losses in spite of the best control efforts of plant pathologists. Breeding for disease-resistant varieties and the application of synthetic chemical fungicides are the most widely accepted approaches in plant disease management. CMN1308 strain of Bacillus amyloliquefaciens was isolated from healthy Chinese chestnut fruit, which has antibiosis and induced resistance to the prevention mechanism of pathogenic bacterium after picking. In order to evaluate the antibiosis mechanism, CMN1308 was fostered with the method of confront culture. The antimicrobial components were also isolated from the culture of CMN1308, and their stability and antimicrobial activity was tested under different treatments such as temperature, pH and UV. The results showed that CMN1308 displays advantages in regard to spatial competition against the major pathogens of chestnut, Rhizopus stolonifer, Fusarium solani, Stachybotrys chartarum, Cryphonectria parasitica, Lasiodiplodia theobromae, Penicillium expansum and Aspergillus niger. Among this, CMN1308 had the best antimicrobial activity against P. expansum, with the inhibition zone diameter of 27.1 mm. The antimicrobial material isolated from CMN1308 culture showed a strong inhibition to the growth of P. expansum hyphal and also had a good stability to high temperature, alkali media and UV, but was sensitive to acidic conditions. Furthermore, CMN1308 increased the peroxidase, polyphenol oxidase activity and reduced the MDA content in chestnuts after infecting by pathogenic fungi. Thus, producing antibiotic compounds and inducible resistance are the main factors that may explain the antibacterial mechanism of CMN1308 on chestnut pathogenic. The results of this study might help to optimize the practical use of CMN1308 in the biological control of chestnut rot or other fruit rot infected by pathogenic fungi.
摘要：
Recently, there has been an increasing interest among researchers in using combinations of biological control agents to exploit potential synergistic effects among them. In the present study, there were investigated commercially acceptable formulations of Bacillus cereus CE3 wetting powder with long storage life and retained efficacy to control chestnut and other fruit rot caused by Endothia parasitica (Murr) and Fusarium solani. The study sought to develop a new B. cereus formulation that would be more effective and better suited to the conditions of field application. By a series of experiments, the formulation was confirmed as follows: 60% B. cereus freeze-dried powder, 28.9% diatomite as carrier, 4% sodium lignin sulfonate as disperser, 6% alkyl naphthalene sulfonate as wetting agent, 1% K2HPO4 as stabilizer, 0.1% β-cyclodextrin as ultraviolet protectant. The controlling experiments showed that the diluted 100 times of 60% B. cereus wetting powder had 79.47% corrosion rate to chestnut pathogens; and this result is comparable to the diluted 1,000 times of 70% thiophanatemethyl. Safety evaluation results showed that rats acute oral lethal dose 50% was 5,000.35, therefore application of B. cereus wettable powder could not cause a person or animal poisoning. This work illustrated that 60% B. cereus wetting powder had commercial potential; however, to apply this formulation as a biological pesticide in the field, masses production processes need to be further studied.
摘要：
The terpene trilactones (TTLs) are believed to be important for the pharmacological properties of Ginkgo biloba leaves extract. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a critical enzyme involved in the biosynthetic pathway of TTLs. In this study, an 1.2-kb fragment of 5' flanking region of the HMGR gene (GbHMGR), was isolated from G. biloba by genome walking. Extensive sequence analysis revealed the presence of evolutionarily conserved and over-represented putative cis-acting elements in light-regulated transcription, hormone signaling (gibberellic acid, jasmonate and salicylic acid), elicitor and stress responses (cold/dehydration responses), and plant defense signaling (W-box/WRKY) that are common to the promoter region of GbHMGR. EMSA analysis suggested possible functionality of W-box in GbHMGR promoter region. The behavior of gene transcripts in ginkgo callus upon light, low temperature, MeJA and SA treatments further verified the regulatory function of GbHMGR promoter. A significant positive relationship between gene expression level and total TTL contents suggested that GbHMGR might be one of key genes involved in TTL biosynthesis in G. biloba.
