#METABOLOMICS WORKBENCH zoukai3412085_20190505_051408 DATATRACK_ID:1718 STUDY_ID:ST001194 ANALYSIS_ID:AN001990 PROJECT_ID:PR000806 VERSION 1 CREATED_ON June 17, 2019, 12:12 am #PROJECT PR:PROJECT_TITLE Flavonoid study of Ginkgo leaves facing to different elevation and plant age PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Flavonoid study of Ginkgo leaves facing to different elevation and plant age PR:INSTITUTE Central South University, China PR:DEPARTMENT School of Minerals Processing and Bioengineering PR:LABORATORY Key Laboratory of the Ministry of Education PR:LAST_NAME Zou PR:FIRST_NAME Kai PR:ADDRESS Central South University, 932 Lushan South Road, Yuelu District, Changsha City, PR:ADDRESS Hunan Province PR:EMAIL zoukai3412085@hotmail.com PR:PHONE +8615273119784 #STUDY ST:STUDY_TITLE Flavonoid study of Ginkgo leaves facing to different elevation and plant age ST:STUDY_SUMMARY Ginkgo biloba leaves are always resources for flavonoids pharmaceutical ST:STUDY_SUMMARY industry. Thus, artificial planting and industrial harvesting become the vital ST:STUDY_SUMMARY aspect to get higher drug yields. In this research, we performed de novo ST:STUDY_SUMMARY transcriptome sequencing of Ginkgo leaves coupled with high-performance liquid ST:STUDY_SUMMARY chromatography/quadrupole-time-of-flight mass spectrometry analyses to obtain a ST:STUDY_SUMMARY comprehensive understanding of the influence of elevation and plant age on ST:STUDY_SUMMARY flavonoid synthesis. A total of 557,659,530 clean reads were assembled into ST:STUDY_SUMMARY 188,155 unigenes, of which 135,102 (71.80%) were successfully annotated in seven ST:STUDY_SUMMARY public databases. The differentially expressed genes analysis indicated DFR, LAR ST:STUDY_SUMMARY and ANR were significantly up-regulated with the increase of elevation in young ST:STUDY_SUMMARY Ginkgo trees leaves. With less strict saliency, the relative concentration of ST:STUDY_SUMMARY flavonoid derivatives with high parent ion signal intensity was likely to ST:STUDY_SUMMARY support this conclusion. Complex gene variations were observed with the plant ST:STUDY_SUMMARY age change. However, flavonoid derivatives analysis predicted the potential ST:STUDY_SUMMARY possibility that the rise of plant age is more likely to be detrimental to the ST:STUDY_SUMMARY biosynthesis of Ginkgo flavonoids in leaves. From the overall DEGs involved in ST:STUDY_SUMMARY flavonoid biosynthesis, DFRs seemed to show more considerable variability ST:STUDY_SUMMARY towards the variation of elevation and plant age. Furthermore, our research ST:STUDY_SUMMARY effectively expanded the functional genomic library of Ginkgo and provided a ST:STUDY_SUMMARY reference for artificial planting and industrial harvesting. ST:INSTITUTE Central South University, China ST:LAST_NAME Zou ST:FIRST_NAME Kai ST:ADDRESS Central South University, 932 Lushan South Road, Yuelu District, Changsha City, ST:ADDRESS Hunan Province ST:EMAIL zoukai3412085@hotmail.com ST:PHONE +8615273119784 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Ginkgo Biloba SU:TAXONOMY_ID 3311 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - LY-1 Elevation:Low | Relative Age:Young SUBJECT_SAMPLE_FACTORS - LY-2 Elevation:Low | Relative Age:Young SUBJECT_SAMPLE_FACTORS - LY-3 Elevation:Low | Relative Age:Young SUBJECT_SAMPLE_FACTORS - HY-1 Elevation:High | Relative Age:Young SUBJECT_SAMPLE_FACTORS - HY-2 Elevation:High | Relative Age:Young SUBJECT_SAMPLE_FACTORS - HY-3 Elevation:High | Relative Age:Young SUBJECT_SAMPLE_FACTORS - LO-1 Elevation:Low | Relative Age:Old SUBJECT_SAMPLE_FACTORS - LO-2 Elevation:Low | Relative Age:Old SUBJECT_SAMPLE_FACTORS - LO-3 Elevation:Low | Relative Age:Old SUBJECT_SAMPLE_FACTORS - HO-1 Elevation:High | Relative Age:Old SUBJECT_SAMPLE_FACTORS - HO-2 Elevation:High | Relative Age:Old SUBJECT_SAMPLE_FACTORS - HO-3 Elevation:High | Relative Age:Old #COLLECTION CO:COLLECTION_SUMMARY The Ginkgo plants grow wildly in Pot bottles of mountain Nature Reserve in Hunan CO:COLLECTION_SUMMARY Province, China. A total of twelve trees (26°55'13'' N to 30°6'44'' N, CO:COLLECTION_SUMMARY 110°36'16'' E to 110°49'2'' E) were chosen to collect mature leaf samples. CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY All samples were divided into four groups – low elevation and young age (LY), TR:TREATMENT_SUMMARY high elevation and young age (HY), low elevation and older age (LO), high TR:TREATMENT_SUMMARY elevation and older age (HO). Each group involved three tree individuals. Each TR:TREATMENT_SUMMARY sample was mixed by three copies of ten similarly sized and healthy leaves, TR:TREATMENT_SUMMARY which were cut off from the sunny side five meters above the ground of the same TR:TREATMENT_SUMMARY tree. After harvesting and short-time surface cleaning by 75% ethanol and TR:TREATMENT_SUMMARY sterile water, these leaves were frozen in liquid nitrogen immediately until TR:TREATMENT_SUMMARY used. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Andrographolide was dissolved in acetonitrile as an internal standard solution SP:SAMPLEPREP_SUMMARY with the terminal concentration of 133μM. Of each leaf sample, 200mg liquid SP:SAMPLEPREP_SUMMARY nitrogen-grinded powder was homogenized in 30ml 70% ethanol solution (v: v= 70: SP:SAMPLEPREP_SUMMARY 30) followed by 1 min vortex and 1 h ultrasound extraction as previously SP:SAMPLEPREP_SUMMARY described with minor modifications (Yu, Lai et al. 2003, Tohge, Nishiyama et al. SP:SAMPLEPREP_SUMMARY 2005, Zhou, Yao et al. 2014). After a 13000rpm centrifugation for 10min at 4℃, SP:SAMPLEPREP_SUMMARY 1ml supernatant of the solution was transferred and evaporated to dryness under SP:SAMPLEPREP_SUMMARY nitrogen gas at 37℃ (Li, Guo et al. 2017). The residue was re-dissolved in 1ml SP:SAMPLEPREP_SUMMARY acetonitrile and centrifuged at 13000rpm for 10 min at 4℃. 950μL supernatant SP:SAMPLEPREP_SUMMARY was transferred to mix with 50μL internal standard solution to be the final SP:SAMPLEPREP_SUMMARY sample for further UPLC-QTOF/MS analysis. SP:PROCESSING_STORAGE_CONDITIONS Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation was completed on a Shimadzu LC-30AD Series UHPLC CH:CHROMATOGRAPHY_SUMMARY system (Shimadzu, Duisburg, Germany) equipped with supplementary SIL-30AC CH:CHROMATOGRAPHY_SUMMARY injector and (PAD) SPD-M20A detector. ACQUITY UPLCTM BEH C18 column (100 mm × CH:CHROMATOGRAPHY_SUMMARY 2.1 mm, 1.7 μm, Waters, Milford, USA) was attached to the whole analyses at CH:CHROMATOGRAPHY_SUMMARY 35℃. A flow rate of 0.3 mL/min was chosen to use while 0.1% formic acid water CH:CHROMATOGRAPHY_SUMMARY (A) and acetonitrile (B) comprising the mobile phase. The gradient elution CH:CHROMATOGRAPHY_SUMMARY conditions were optimized as follows:0-3 min, 5% B → 5% B; 3-25 min, 5% B → CH:CHROMATOGRAPHY_SUMMARY 95% B; 25-28 min, 95% B → 95% B; 28-28.1 min, 95% → 5% B, followed by 4 min CH:CHROMATOGRAPHY_SUMMARY re-equilibration. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu LC-30AD Series UPLC system CH:COLUMN_NAME Waters Acquity BEH C18 (100 x 2mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5600+ TripleTOF MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS AB SCIEX TripleTOF 5600+ system (AB SCIEX Technologies, USA), equipped with MS:MS_COMMENTS electrospray ionization (ESI) source, was coupled to the UHPLC system and used MS:MS_COMMENTS to scan parent ion molecular weight from 100 to 1500. Other MS parameters were MS:MS_COMMENTS set as below: electrospray ionization temperature (℃): 500 (ESI-); nebulizer MS:MS_COMMENTS gas pressure (psi): 60 (ESI-); ion spray voltage (KV): 4.5 (ESI-); collision MS:MS_COMMENTS energies (V): 35 (ESI-). The raw data outputted from LC-MS was pretreated by MS:MS_COMMENTS MarkerView (version 1.2.1.1, AB SCIEX Technologies, USA), including peak MS:MS_COMMENTS recognition (retention time 2 – 28 min, noise threshold 100), alignment, MS:MS_COMMENTS calibration of the internal standard, filtering and normalization to total area. MS:MS_COMMENTS A three-dimensional data set contained sample information, peak retention time MS:MS_COMMENTS (RT), peak relative intensities and mass-to-charge ratio (m/z) was obtained to MS:MS_COMMENTS perform a series of statistical analysis. PeakView (version 1.2.0.3, AB SCIEX MS:MS_COMMENTS Technologies, USA) was recommended to visualize raw data of target components in MS:MS_COMMENTS two-stage mass-to-charge ratio map. Then, based on fragment ion information, MS:MS_COMMENTS such components were identified by comparing to HMDB (http://www.hmdb.ca/), MS:MS_COMMENTS PubChem (https://pubchem.ncbi.nlm.nih.gov/), NIST (https://www.nist.gov/), MS:MS_COMMENTS MassBank (http://www.massbank.jp/) and METLIN (https://metlin.scripps.edu/) MS:MS_COMMENTS databases. MS:MS_RESULTS_FILE ST001194_AN001990_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END