Abiotic stress treatment reveals expansin like A gene OfEXLA1 improving salt and drought tolerance of Osmanthus fragrans by responding to abscisic acid

Sweet osmanthus(Osmanthus fragrans) is a having general approval aromatic tree in China that is widely applied to landscaping and gardening. However, the evergreen tree adaptability is limited by many environmental stresses. Currently, limited information is available regarding the genetic analysis and functional identification of expansin genes in response to abiotic stress in sweet osmanthus. In this study, a total of 29 expansin genes were identified and divided into four groups by genome-wide analysis from the sweet osmanthus genome. Transcriptome and quantitative Real-time PCR analysis showed that the cell wall-localized protein expansin-like A(OfEXLA1) gene was significantly induced by salt and drought treatment. Histochemical GUS staining of transgenic Arabidopsis lines in which GUS activity was driven with the OfEXLA1 promoter, GUS activity was significantly induced by salt, drought, and exogenous abscisic acid(ABA). In yeast, we found OfEXLA1overexpression significantly improved the population of cells compared with wild-type strains after NaCl and polyethylene glycol(PEG)treatment. Additionally, OfEXLA1 overexpression not only promoted plant growth, but also improved the salt and drought tolerance in Arabidopsis. To gain insight into the role of ABA signaling in the regulation of OfEXLA1 improving abiotic tolerance in sweet osmanthus, four differentially expressed ABA Insensitive 5(ABI5)-like genes(OfABL4, OfABL5, OfABL7, and OfABL8) were identified from transcriptome, and dualluciferase(dual-LUC) and yeast one hybrid(Y1H) assay showed that OfABL4 and OfABL5 might bind to OfEXLA1 promoter to accumulate the OfEXLA1 expression by responding to ABA signaling to improve abiotic tolerance in sweet osmanthus. These results provide the information for understanding the molecular functions of expansin-like A gene and molecular breeding of sweet osmanthus in future.

Study on the molecular mechanism of Osmanthus fragrans Lour.on boosting immunity based on network pharmacology and molecular docking

Objective:To explore the molecular mechanism of Osmanthus Fragrans Lour.(OFL)in enhancing immunity.Methods:The compounds and action targets of OFL were collected from the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform.Protein targets of compounds were obtained from the UniProt database and relevant targets of boosting immunity were retrieved from the Genecards database.The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and Gene Ontology function enrichment analysis were performed through the DAVID analysis website,Visualization and Integrated Discovery.Finally,the results of the network analysis were validated by performing molecular docking using AutoDock vina.Results:A total of 7 active compounds and 167 potential active targets were identified in OFL.A total of 1549 genes with a correlation score of≥1 were retrieved from the Genecards website with the keyword“boost immunity”,and 107 genes were obtained by crossing the 167 genes of OFL with the 1549 genes of boosting immunity.A total of 4802 entries were obtained from Gene Ontology functional enrichment(P<0.05).A total of 234 signaling pathways were obtained through a Kyoto Encyclopedia of Genes and Genomes pathway analysis(P<0.05).Tumor necrosis factor(TNF)and interleukin 17(IL-17)signaling pathways were closely related to body immunity.The molecular docking results showed that all the core compounds in OFL the characteristics including low energy,a stable structure and high binding activity when bound to IL-17 and TNF-αprotein.Kaempferol showed the highest affinity with IL-17,and fucosterol showed the highest affinity with TNF-α.Conclusions:Through studies on network pharmacology and molecular docking,we have further demonstrated that OFL could enhance the immunity of the body through multi-component,multi-target and multi-pathway actions,and that IL-17/TNF-αsignalling pathway is the key molecular mechanism.

Differences in leaf functional traits between red and green leaves of two evergreen shrubs Photinia×fraseri and Osmanthus fragrans

Leaf functional traits are adaptations that enable plants to live under different environmental conditions. This study aims to evaluate the differences in leaf functional traits between red and green leaves of two evergreen shrubs Photinia × fraseri and Osrnanthus fragrans. Specific areas of red leaves are higher than that of green leaves in both species. Thus, the material investment per unit area and per lamina of red leaves is significantly lower than that of green leaves, implying an utmost effort of red leaves to increase light capture and use efficiency because of their low leafchlorophyll concentration. The higher petiole length of green leaves compared with that of red leaves indicates that adult green leaves may have large fractional biomass allocation to support the lamina structures in capturing light with maximum efficiency and obtaining a high growth rate. The high range of the phenotypic plasticity of leaf size, leaf thickness, single-leaf wet and dry weights, and leaf moisture of green leaves may be beneficial in achieving efficient control of water loss and nutrient deprivation. The high range of phenotypic plasticity of leaf chlorophyll concentration of red leaves may be advantageous in increasing resource （especially light） capture anduse efficiency because this leaf type is juvenile in the growth stage and has low leaf-chlorophyll concentration.

Identification and quantification of the bioactive components in Osmanthus fragrans roots by HPLC-MS/MS

The roots of O. fragrans are also a valuable resource in addition to its flowers and fruits. In this study, the HPLC-MS/MS method used for analyzing the chemical constituents in O. fragrans roots extract was developed, which showed high sensitivity for both qualitative and quantitative analyses. Thirty-two compounds were first discovered in O. fragrans roots, one compound of which was reported for the first time. The simultaneous determination method for acteoside, isoacteoside, oleuropein and phillyrin was validated to be sensitive and accurate. Then it was applied to determine the content of bioactive components in O. fragrans roots from different cultivars. The content of oleuropein and phillyrin in the twelve batches was relatively stable, while the content of acteoside and isoacteoside varied greatly.Moreover, the therapeutic material basis and mechanism of O. fragrans roots exerting its traditional pharmacodynamics were analyzed by network pharmacology. The results showed that O. fragrans roots might be effective for the treatment of inflammation, cardiovascular diseases, cancer, and rheumatoid arthritis, which is consistent with the traditional pharmacodynamics of O. fragrans roots. This work can provide an analytical method for the comprehensive development of O. fragrans roots.

CPTA treatment reveals potential transcription factors associated with carotenoid metabolism in flowers of Osmanthus fragrans

Osmanthus fragrans is one of the top ten traditional flowers in China.It is divided into three different groups according to its color.α-Carotene and β-carotene are the main determinants to distinguish the color differences between three groups.However,the dominant genes and transcription factors involved in carotenoid metabolism remain unclear.CPTA treatment(0.7mmol·L−1)remarkably promoted lycopene,α-carotene and β-carotene contents in flowers.Transcriptome sequencing analysis revealed that CPTA treatment could trigger chain reactions in carotenoid metabolism pathway genes.Four up-regulated and 10 down-regulated transcription factors which have close association with carotenoid variation were significantly induced by CPTA treatment.The up-regulated TFs such as MYB43,MYB123,HSF,were further subjected to transcript expression determination in different cultivars with drastic colors.Among them,transcript expression of four up-regulated TFs coincided with the carotenoid accumulation in different cultivars.We selected up-regulated OfMYB43 to verify its function,which is related to stress tolerance and transcriptional regulation.Transient overexpression of OfMYB43 in O.fragrans flowers showed that it could remarkably promote the expression of PDS,ZISO,LCYE and CCD4,leading to increased accumulation of β-branch carotenoids.OfMYB43 was a potential positive regulator of carotenoid biosynthesis in O.fragrans flowers.This study provides insight into the molecular mechanism of carotenoid metabolism in O.fragrans.

cDNA Cloning and Expression Analysis of the Chalcone Synthases (CHS) in <i>Osmanthus fragrans</i>

In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach was used to clone a Chalcone synthases cDNA from flower of sweet osmanthus “Chenghong Dangui” and it was designated as OfCHS (O. fragrans, CHS). The cDNA was 1383 bp long and a coding sequence (CDS) of 1173 bp encoding a polypeptide of 391 amino acids with an estimated molecular mass of 39.9 kDa. The theoretical isoelectric point was 6.23. Phylogenetic analysis demonstrated that OfCHS clustered with Olea europaea, Solenostemon scutellarioides, Perilla frutescens, Antirrhinum majus and Digitalis lanata. We also detected the expression of OfCHS in different tissues in “Dangui” and in two cultivars with varied coloration, “Zi Yingui” and “Chenghong Dangui” at different floral stages using quantitative real-time PCR. We observed that OfCHS transcript was higher in leaves than in petals in “Dangui”. The transcripts of OfCHS in “Zi Yingui” petals were higher than those in “Dangui” at three stages especially at xianyan stage and there was no significant difference between the two cultivars in the full flowering stage. “Chenghong Dangui” has a relatively high anthocyanin content compared to “Zi Yingui”. The relative amount of anthocyanin of “Chenghong Dangui” initially increases, and then decreases during the bloom period. However, the expression of CHS is the highest at the initial flowering stage. These data suggest that the OfCHS does not play a key role in the accumulation of total flavonoid in this cultivar. These data could contribute to explain the different accumulation of flavonoids in petals of the two cultivars.

Study on Prediction of the First Flowering Date of Osmanthus fragrans

[ Objective] The study aimed to forecast the first flowering date of Osmanthus fragrans. [ Method] Based on the data about flowering stage of O. fragrans in Guilin City from 1999 -2012, the correlation between meteorological factors and the first flowering date of O. fragrans was analyzed, and a forecast model for the first flowering date of O. fragrans was established. EResult] Among meteorological factors, precipitation, humidity, temperature and sunshine hours could obviously affect the first flowering date of O. fragrans, especially humidity in August, temperature and sunshine hours in September. The forecast model could exactly forecast the first flowering date in the partial early or too late year, and the av- erage error of date was 6 days. The factors influencing the first flowering date of O. fragrans are complex, and there is an artificial error in record data of the flowering stage, so using a single model to forecast the first flowering date have some disadvantages. When forecasting the first flower- ing date, we should consider all influencing factors to get a good forecast result. [ Conclusion] The research could provide a new method to forecast the first flowednq date of O. fraclrans.

Development Prospects of Osmanthus fragrans Mixed Wine Based on Liquor and Spirits

As a plant resource, Osmanthus fragrans has both medicinal and edible effects. This paper reviews the active components and health-care functions of O. fragrans, and introduces the current research situation of O. fragrans mixed wine. In addition, combined with the development of new products of plant-based flower wine, it analyzes the development prospects of O. fragrans mixed wine based on liquor and spirits.

First Report of Botryosphaeria dothidea Causing Sweet Osmanthus Leaf Dieback in China

Sweet osmanthus is one of the ten traditional famous flowers in China.The occurrence of the diseases caused by fungi other than Botryosphaeria spp.has been reported mainly from China on sweet osmanthus.A leaf dieback of sweet osmanthus caused by Botryosphaeria sp.was found for the first time in 2007 in Nanning City,Guangxi,China.The objectives of the present study were to isolate and characterize the causal organism of sweet osmanthus leaf dieback.The fungus was isolated from the lesions of affected sweet osmanthus leaves and its pathogenicity to sweet osmanthus was confirmed using a detached-leaf-inoculation method.The identification of the pathogen was carried out mainly based on the morphological characters and molecular analysis of internal transcribed spacer （ITS） sequences.The morphological characters of the pathogenic isolate GHX6 were agreed with that of Botryosphaeria dothidea.The ITS sequence of the isolate was amplified with primers ITS1 and ITS4,and submitted to GenBank （accession no.GQ368251）.Molecular analysis based on ITS sequence comparison between the isolate GHX6 and the other related fungi derived from GenBank supported that the causal agent of the sweet osmanthus leaf dieback belonged to Botryosphaeria dothidea.This is the first report of Botryosphaeria dothidea causing leaf dieback on sweet osmanthus in China.

Sweet Osmanthus

ＳｗｅｅｔＯｓｍａｎｔｈｕｓＳｗｅｅｔＯｓｍａｎｔｈｕｓ，ａｆｌｏｗｅｒｎａｔｉｖｅｔｏＣｈｉｎａ，ｉｓｃｏｎｓｉｄｅｒｅｄｏｎｅｏｆｔｈｅｔｅｎｍｏｓｔｆａｍｏｕｓＣｈｉｎｅｓｅｆｌｏｗｅｒｓ．Ｔｈｅｓｔｒｏｎｇｓｃｅｎｔｏｆｔｈｅｆｌｏｗｅｒｔｒ...

桂花(Osmanthus fragrans Lour.)4-香豆酸辅酶A连接酶(4CL)基因克隆与表达分析

本研究运用RT-PCR和RACE方法,从‘橙红丹桂’花瓣中克隆出了桂花4-香豆酸辅酶A连接酶(4CL)基因。生物信息学分析和实时定量分析表明,本实验所克隆的基因(全长2 054 bp)是4CL基因家族的成员之一;时空表达分析发现,4CL基因在花组织中特异性表达,营养器官根、叶中表达量均很低;4CL基因在开花过程中的时间表达趋势是"低-高-低",其表达水平变化趋势与花青素和类胡萝卜素含量变化趋势类似。我们推测本研究克隆出的4CL基因与桂花花色代谢相关。

桂花(Osmanthus fragrans)转录组SSR特征分析

为解析桂花转录组数据中SSR的特点及潜在利用价值,本研究基于‘早银桂’和‘橙红丹桂’转录组数据信息,利用MISA软件从桂花的117 595个Unigenes中进行SSR位点的查找、分析,共检测到16 015个SSR位点,平均密度是1/5.2 kb。桂花SSR基序97.73%的重复次数在5～10次之间,重复次数最多的是6次(28.12%);SSR长度分布在12～36 bp之间,其中12～16 bp的最多,占66.8%,其总体变化趋势是随着重复次数增加重复次数急速下降。基于已知的SSR位点,利用primer3设计引物并从中挑选出196对引物,其中92对引物(占46.94%)已被注释到参与信号的转录翻译、苯丙氨酸代谢、植物激素信号转导等功能。综上所述‘,早银桂’和‘橙红丹桂’转录组SSR位点出现频率高,类型较丰富、多态性潜能较高,为今后桂花分子育种提供了理论依据。

Analysis of Fragrance Composition in Three Cultivars of Osmanthus fragrans Albus Group Flower by Gas Chromatography-Mass Spectrometry

With supercritical CO2 fluid extraction（SCFE）, essential oil was extracted from three cultivars of Xianning osmanthus. The fresh osmanthus flower was processed with a petroleum ether digestion method to produce the extractum. The yields of essential oil and extractum were 0.19 % and 0.13 % （m/m） respectively. The essential oil and fragrance composition and content extracted were analyzed with gas chromatography-mass spectrometer （GC-MS）. The result showed that essential oil contained 36.99%（area/total area） of ionone, ionol and 13.11% of linalool; ionone and ionol contained in extractum were as high as up to 33.33%, while linalool up to 21.92%. Whether essential oil or extractum contains only about 40% fat acid and other ester matters. None of environmental estrogen （phthalic ester） was found in fragrance ingredients. The result also showed that the quality of O. fragrans Albus group fragrance in Xianning is better than that produced in Hangzhou and Anhui districts.

彩叶桂(Osmanthus fragrans)品种‘虔南桂妃’叶片红色消退过程中的转录组分析

彩叶桂(Qsmanthus fragrans)叶片发育不同时期色泽变化具有显著差异,但目前对于其内在的分子调控机制尚不清楚。本研究利用彩叶桂品种‘虔南桂妃’红色(R)、黄绿色(YG)和绿色(G)叶片进行高通量测序,以期在转录组水平上解析叶色变化的潜在原因。通过系统分析转录组数据,共得到36.66 Gb的碱基数据,检测出32252个新转录本。在R-vs-G、R-vs-YG和YG-vs-G比较组中共筛选到23954个差异表达基因(differentially expressed genes,DEGs),其中包含57类共3308个转录因子家族成员,且下调表达的差异基因数目大于上调表达的基因数目。KEGG和GO功能分析表明,差异表达基因主要与“催化活性”、“细胞”和“代谢”等生物学功能相关。此外,筛选到了6个类黄酮生物合成途径上显著差异表达的基因,分别编码查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、二氢黄酮醇4-还原酶(DFR)、UDP-糖基转移酶(UGT)、黄酮醇合成酶(FLS)和花青素合成酶(ANS)。与红色叶片相比,这些基因的表达量在黄绿色和绿色叶片中显著降低。推测下调表达的6个类黄酮生物合成途径基因可能促进了‘虔南桂妃’叶片红色的消退。本研究结果对深入理解彩叶桂叶色变异的分子机制具有重要的理论价值,获得的基因序列为今后桂花叶色遗传改良提供了宝贵的基因资源。

桂花OfWRKY120基因鉴定及盐胁迫响应功能研究

【目的】根据前期基因家族鉴定分析得到OfWRKY120,对该基因盐胁迫响应的功能进行分析,为桂花盐胁迫下的调控机制研究奠定基础。【方法】利用生物信息学方法对OfWRKY120保守结构域、系统进化进行分析,并采用qRT-PCR技术研究盐胁迫下OfWRKY120在桂花叶片中的相对表达量。构建OfWRKY120超表达载体,使用亚细胞定位、酵母自激活检测等探究其特性,将该基因瞬时转化本氏烟草后,对烟草进行盐处理,利用DAB、NBT染色和相关生理指标的测定,解析瞬时转化OfWRKY120对本氏烟草耐盐性的影响。【结果】OfWRKY120基因全长为1422 bp,编码474个氨基酸,存在WRKY superfamily保守结构域。盐胁迫能够激活桂花叶片中OfWRKY120的表达,在72 h时相对表达量最高,与对照有显著差异。亚细胞定位结果表明,OfWRKY120定位于细胞核。酵母自激活结果显示OfWRKY120无自激活活性。盐胁迫后,OfWRKY120瞬时过表达植株的超氧阴离子(O-·2)和脯氨酸含量显著高于空载对照,且DAB和NBT染色表现出更深的颜色。本氏烟草中的qRT-PCR结果显示,OfWRKY120显著降低了NbCAT的表达,而显著提高了NbP5CS1、NbP5CS2和NbP5CR的表达。【结论】OfWRKY120过表达增加了盐胁迫下本氏烟草中活性氧(ROS)的含量,导致植物对盐胁迫的敏感性提高。

GC-MS结合化学计量学方法分析桂花净油分子蒸馏馏分的挥发性成分

为了探究分子蒸馏技术纯化桂花净油的不同馏分挥发性成分与香气差异,采用气相色谱-质谱(Gas chromatography-mass spectrometry,GC-MS)结合化学计量学方法中的正交偏最小二乘判别分析法(Orthogonal partial least-squares discrimination analysis,OPLS-DA)、P-value与变量投影重要性(Variableimportant in the projection,VIP)与相对气味活度值(Relative odor activity value,ROAV)对桂花净油的轻组分、重组分和残留物组分挥发性成分进行分析。结果表明:三种馏分中共检测到8大类挥发性成分共73种,其中醇类22种、醛类5种、酸类10种、酚类3种、酮类5种、酯类17种、烷烃类5种、烯萜类6种。各馏分中醇类物质的种类与相对含量最高,是桂花净油挥发性成分的主要贡献物质,且各馏分的挥发性成分种类与相对含量具有显著性差异(P<0.05);建立的三种馏分挥发性成分判别模型,分类准确率为98.2%;并在分析确定出三种馏分27种差异性挥发物成分(VIP>1)后,对轻组分14种、重组分9种和残留物组分11种的关键香气贡献成分(ROAV>1)进行香气轮廓分析,轻组分与重组分、残留组分相比,表现出的花香味、甜味以及松木香味更为强烈。轻组分的挥发性成分种类与相对含量最多,香气特征中的桂花花香味最为浓郁强烈。本研究采用GC-MS结合化学计量学方法分析桂花净油各馏分挥发性成分差异与香气特征,为桂花净油分子蒸馏各馏分综合利用提供理论依据。

气相色谱-质谱结合相对气味活性值分析不同干燥方式对桂花浸膏与净油香气成分的影响

为分析与比较不同干燥方式对桂花浸膏与净油挥发性成分及香气特征的影响,采用气相色谱-质谱(gas chromatography-massspectrometry,GC-MS)联用技术对桂花浸膏与净油的挥发性成分进行检测,通过建立偏最小二乘判别分析(partial least squares-discriminant analysis,PLS-DA)模型,并结合相对气味活性值(relative odor activity value,ROAV)、香气雷达图与主成分分析(principal component analysis,PCA)分析不同干燥方式对挥发性香气成分的影响。结果表明,在不同干燥方式下,桂花浸膏与净油样品中共鉴定挥发性成分有113种,不同样品的挥发性成分种类和含量具有显著性差异(P<0.05),通过PLS-DA筛选出36种变量投影重要性大于1的标志性挥发性成分,对不同干燥方式浸膏和净油的区别判断预测准确率为99.6%。ROAV和香气雷达图结果表明,二氢-β-紫罗兰醇、香叶基香叶醇、壬醛、β-紫罗兰酮、二氢-β-紫罗兰酮对桂花浸膏和净油香气贡献较大,上述挥发性成分提供了花香、木香、果香气息,尤其是冷冻干燥提取的浸膏香气更为浓郁,PCA表明冷冻干燥有利于桂花浸膏和净油香味品质的提升。本研究结果揭示了不同干燥方式桂花浸膏与净油的挥发性成分及香气特征差异,为其产品的工艺优化和品质提升提供理论支撑。

中式桂花啤酒的研究进展

桂花花香馥郁,在啤酒酿造过程中添加桂花进行增味,不仅可以缩短传统桂花酒的酿造周期,而且可以将桂花内黄酮、多糖、多酚、萜烯等多种活性成分浸取到啤酒中。该文章介绍桂花的生物活性成分、桂花酿酒的功效,阐明中式桂花啤酒酿造工艺,并对中式桂花啤酒酿造时桂花品种选择、添加方式和添加量进行探讨。

氮磷钾配方施肥对丹桂苗木叶片内源激素含量的影响

采用L9(3^(3))设置3因素(氮、磷和钾肥)3水平(氮单株施用量分别为0、5、10 g,磷单株施用量分别为0、2、4 g,钾单株施用量分别为0、3、6 g)正交实验,对‘浦城丹桂’幼苗间隔30 d进行2次氮磷钾配施处理,采用酶联免疫吸附分析法(ELISA)测定第2次施肥后60、120、180、240 d后叶片4种激素[生长素(IAA)、脱落酸(ABA)、赤霉素(GA_(3))、玉米素核苷(ZR)]含量。结果表明:N_(5)P_(0)K_(3)处理可以显著提高IAA、ABA和ZR的含量,于施肥240 d达到最高值(分别为139.90、157.79、14.86 ng/g),相比CK分别提高了101.52%、36.79%、73.08%;N_(5)P_(2)K_(6)处理可以显著提高GA_(3)的含量,于施肥240 d达到最高值(13.57 ng/g),相比CK提高了97.71%;N_(5)P_(0)K_(3)处理显著提高ABA/GA_(3)和ZR/GA_(3)值,显著降低ABA/IAA和ZR/IAA值;N和K因素对IAA、GA_(3)、ABA和ZR含量的影响均有显著差异,P因素仅对GA_(3)含量的影响有显著差异;氮磷钾配施对丹桂苗木叶片内源激素含量及其之间的稳态有显著影响,综合方差和极差分析,得出提高叶片IAA、GA_(3)和ZR含量的最佳配施方案为N_(5)P_(2)K_(6),提高叶片ABA含量的最佳配施方案为N_(5)P_(0)K_(3)。