#METABOLOMICS WORKBENCH shijuan_20200313_021343 DATATRACK_ID:1941 STUDY_ID:ST001330 ANALYSIS_ID:AN002216 PROJECT_ID:PR000909
VERSION             	1
CREATED_ON             	March 22, 2020, 8:59 pm
#PROJECT
PR:PROJECT_TITLE                 	Rice panicle blast resistence
PR:PROJECT_SUMMARY               	Metabolomics studies of OsGF14b-mediated innate immunity against panicle blast
PR:PROJECT_SUMMARY               	in rice
PR:INSTITUTE                     	Guangdong Academy of Agricultural Sciences
PR:DEPARTMENT                    	Agro-biological Gene Research Center
PR:LAST_NAME                     	Yan
PR:FIRST_NAME                    	Shijuan
PR:ADDRESS                       	No. 20 Jinying Road, Tianhe District, Guangzhou City, Guangdong Province,
PR:ADDRESS                       	510640, China.
PR:EMAIL                         	shijuan@agrogene.ac.cn
PR:PHONE                         	+86-020-38213643
#STUDY
ST:STUDY_TITLE                   	Multi-omics of OsGF14b-mediated innate immunity against panicle blast in rice
ST:STUDY_SUMMARY                 	In the present study, we used a multi-omics approach to decipher the molecular
ST:STUDY_SUMMARY                 	mechanisms of OsGF14b in governing panicle resistance to Magnaporthe
ST:STUDY_SUMMARY                 	oryzae.Results revealed OsGF14b mediated panicle blast resistance was involved
ST:STUDY_SUMMARY                 	in the activation of auxin and JA signaling pathways, resulting in reprogramming
ST:STUDY_SUMMARY                 	of the phenylpropanoid and diterpenoid pathway.
ST:INSTITUTE                     	Agro-biological Gene Research Center , Guangdong Academy of Agricultural
ST:INSTITUTE                     	Sciences
ST:LAST_NAME                     	Yan
ST:FIRST_NAME                    	Shijuan
ST:ADDRESS                       	No. 20 Jinying Road, Tianhe District, Guangzhou City, Guangdong Province,
ST:ADDRESS                       	510640, China.
ST:EMAIL                         	shijuan@agrogene.ac.cn
ST:PHONE                         	+86-020-38213643
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Oryza sativa Japonica Group
SU:TAXONOMY_ID                   	39947
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-0h-1	Genotype:OsGF14b-overexpression line 2 | Treatment:0h	RAW_FILE_NAME=GF14b-2-0h-1
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-0h-2	Genotype:OsGF14b-overexpression line 2 | Treatment:0h	RAW_FILE_NAME=GF14b-2-0h-2
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-0h-3	Genotype:OsGF14b-overexpression line 2 | Treatment:0h	RAW_FILE_NAME=GF14b-2-0h-3
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-0h-4	Genotype:OsGF14b-overexpression line 2 | Treatment:0h	RAW_FILE_NAME=GF14b-2-0h-4
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-0h-5	Genotype:OsGF14b-overexpression line 2 | Treatment:0h	RAW_FILE_NAME=GF14b-2-0h-5
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-0h-1	Genotype:OsGF14b-overexpression line 4 | Treatment:0h	RAW_FILE_NAME=GF14b-4-0h-1
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-0h-2	Genotype:OsGF14b-overexpression line 4 | Treatment:0h	RAW_FILE_NAME=GF14b-4-0h-2
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-0h-3	Genotype:OsGF14b-overexpression line 4 | Treatment:0h	RAW_FILE_NAME=GF14b-4-0h-3
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-0h-4	Genotype:OsGF14b-overexpression line 4 | Treatment:0h	RAW_FILE_NAME=GF14b-4-0h-4
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-0h-5	Genotype:OsGF14b-overexpression line 4 | Treatment:0h	RAW_FILE_NAME=GF14b-4-0h-5
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-0h-1	Genotype:OsGF14b-overexpression line 6 | Treatment:0h	RAW_FILE_NAME=GF14b-6-0h-1
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-0h-2	Genotype:OsGF14b-overexpression line 6 | Treatment:0h	RAW_FILE_NAME=GF14b-6-0h-2
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-0h-3	Genotype:OsGF14b-overexpression line 6 | Treatment:0h	RAW_FILE_NAME=GF14b-6-0h-3
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-0h-4	Genotype:OsGF14b-overexpression line 6 | Treatment:0h	RAW_FILE_NAME=GF14b-6-0h-4
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-0h-5	Genotype:OsGF14b-overexpression line 6 | Treatment:0h	RAW_FILE_NAME=GF14b-6-0h-5
SUBJECT_SAMPLE_FACTORS           	-	Nip-0h-1	Genotype:Wild-type | Treatment:0h	RAW_FILE_NAME=Nip-0h-1
SUBJECT_SAMPLE_FACTORS           	-	Nip-0h-2	Genotype:Wild-type | Treatment:0h	RAW_FILE_NAME=Nip-0h-2
SUBJECT_SAMPLE_FACTORS           	-	Nip-0h-3	Genotype:Wild-type | Treatment:0h	RAW_FILE_NAME=Nip-0h-3
SUBJECT_SAMPLE_FACTORS           	-	Nip-0h-4	Genotype:Wild-type | Treatment:0h	RAW_FILE_NAME=Nip-0h-4
SUBJECT_SAMPLE_FACTORS           	-	Nip-0h-5	Genotype:Wild-type | Treatment:0h	RAW_FILE_NAME=Nip-0h-5
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-24h-1	Genotype:OsGF14b-overexpression line 2 | Treatment:24h	RAW_FILE_NAME=GF14b-2-24h-1
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-24h-2	Genotype:OsGF14b-overexpression line 2 | Treatment:24h	RAW_FILE_NAME=GF14b-2-24h-2
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-24h-3	Genotype:OsGF14b-overexpression line 2 | Treatment:24h	RAW_FILE_NAME=GF14b-2-24h-3
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-24h-4	Genotype:OsGF14b-overexpression line 2 | Treatment:24h	RAW_FILE_NAME=GF14b-2-24h-4
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-2-24h-5	Genotype:OsGF14b-overexpression line 2 | Treatment:24h	RAW_FILE_NAME=GF14b-2-24h-5
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-24h-1	Genotype:OsGF14b-overexpression line 4 | Treatment:24h	RAW_FILE_NAME=GF14b-4-24h-1
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-24h-5	Genotype:OsGF14b-overexpression line 4 | Treatment:24h	RAW_FILE_NAME=GF14b-4-24h-5
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-24h-2	Genotype:OsGF14b-overexpression line 4 | Treatment:24h	RAW_FILE_NAME=GF14b-4-24h-2
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-24h-3	Genotype:OsGF14b-overexpression line 4 | Treatment:24h	RAW_FILE_NAME=GF14b-4-24h-3
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-4-24h-4	Genotype:OsGF14b-overexpression line 4 | Treatment:24h	RAW_FILE_NAME=GF14b-4-24h-4
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-24h-1	Genotype:OsGF14b-overexpression line 6 | Treatment:24h	RAW_FILE_NAME=GF14b-6-24h-1
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-24h-2	Genotype:OsGF14b-overexpression line 6 | Treatment:24h	RAW_FILE_NAME=GF14b-6-24h-2
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-24h-3	Genotype:OsGF14b-overexpression line 6 | Treatment:24h	RAW_FILE_NAME=GF14b-6-24h-3
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-24h-4	Genotype:OsGF14b-overexpression line 6 | Treatment:24h	RAW_FILE_NAME=GF14b-6-24h-4
SUBJECT_SAMPLE_FACTORS           	-	OXGF14b-6-24h-5	Genotype:OsGF14b-overexpression line 6 | Treatment:24h	RAW_FILE_NAME=GF14b-6-24h-5
SUBJECT_SAMPLE_FACTORS           	-	Nip-24h-1	Genotype:Wild-type | Treatment:24h	RAW_FILE_NAME=Nip-24h-1
SUBJECT_SAMPLE_FACTORS           	-	Nip-24h-2	Genotype:Wild-type | Treatment:24h	RAW_FILE_NAME=Nip-24h-2
SUBJECT_SAMPLE_FACTORS           	-	Nip-24h-3	Genotype:Wild-type | Treatment:24h	RAW_FILE_NAME=Nip-24h-3
SUBJECT_SAMPLE_FACTORS           	-	Nip-24h-4	Genotype:Wild-type | Treatment:24h	RAW_FILE_NAME=Nip-24h-4
SUBJECT_SAMPLE_FACTORS           	-	Nip-24h-5	Genotype:Wild-type | Treatment:24h	RAW_FILE_NAME=Nip-24h-5
#COLLECTION
CO:COLLECTION_SUMMARY            	The panicles at the initial heading stage of the wild-type Nipponbare (Nip) and
CO:COLLECTION_SUMMARY            	OsGF14b-overexpressing plants were harvested before (Nip-0h; OXGF14b-2-0h;
CO:COLLECTION_SUMMARY            	OXGF14b-4-0h; OXGF14b-6-0h) and after M. oryzae 24-hour inoculation (Nip-24h;
CO:COLLECTION_SUMMARY            	OXGF14b-2-24h; OXGF14b-4-24h; OXGF14b-6-24h) respectively. They were immediately
CO:COLLECTION_SUMMARY            	frozen in liquid nitrogen, with each biological replicate containing panicle
CO:COLLECTION_SUMMARY            	pooled from 10 individual plants.
CO:SAMPLE_TYPE                   	Seeds
#TREATMENT
TR:TREATMENT_SUMMARY             	Wild-type japonica rice (Oryzae sativa cv. Nipponbare) and three OsGF14b gene
TR:TREATMENT_SUMMARY             	overexpressing lines, including transgenic line 2 (OXGF14b-2), transgenic line 4
TR:TREATMENT_SUMMARY             	(OXGF14b-4), transgenic line 6 (OXGF14b-6) were used in this study. Rice seeds
TR:TREATMENT_SUMMARY             	were surface-sterilized and transferred to 1/2 MS medium and incubated in a
TR:TREATMENT_SUMMARY             	growth chamber for germination under light of 200 μmol/m2/s with a 12-h
TR:TREATMENT_SUMMARY             	photoperiod at 26℃. Subsequently, rice seedlings were transplanted into soil
TR:TREATMENT_SUMMARY             	and kept in a greenhouse. M. oryzae GD08-T13 was used for rice blast
TR:TREATMENT_SUMMARY             	inoculation.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The rice panicle pre-cooled in liquid nitrogen were ground using a Mixer/mill
SP:SAMPLEPREP_SUMMARY            	(MM400; Retsch) with steel ball for 30 seconds at 30 HZ. Fifty milligram of rice
SP:SAMPLEPREP_SUMMARY            	panicle powder of each sample was extracted with a fixed volume (1 ml) of
SP:SAMPLEPREP_SUMMARY            	pre-cooled (−20 °C) extraction solvent (methanol: chloroform: water = 5: 2:
SP:SAMPLEPREP_SUMMARY            	2) was added to homogenized tissues. After adding the extraction solvent, the
SP:SAMPLEPREP_SUMMARY            	vials/tubes were thoroughly vortexed for 1 min and then incubated on an orbital
SP:SAMPLEPREP_SUMMARY            	shaker (200 rpm) for 10 min at 4 °C followed by a 15 min sonication step.
SP:SAMPLEPREP_SUMMARY            	For phase separation, a volume of 500 µl of solvent (methanol: water = 1: 3),
SP:SAMPLEPREP_SUMMARY            	was added to each vial/tube and the samples were again thoroughly vortexed for
SP:SAMPLEPREP_SUMMARY            	1 min. After that, the samples are centrifuged at a speed of 14000rpm for
SP:SAMPLEPREP_SUMMARY            	10 min at 4 °C. Two fixed volume of 200 μL of the polar phase (the lower
SP:SAMPLEPREP_SUMMARY            	phase) were transferred into pre-labeled 1.5 ml microcentrifuge tube
SP:SAMPLEPREP_SUMMARY            	respectively. Then the samples were dried in a SpeedVac concentrator without
SP:SAMPLEPREP_SUMMARY            	heating. Two dried 200 μL aliquots of the polar phase in each sample were
SP:SAMPLEPREP_SUMMARY            	analyzed using gas chromatography tandem mass spectrometry (GC-MS) and liquid
SP:SAMPLEPREP_SUMMARY            	chromatography tandem mass spectrometry (LC-MS) for metabolomics study. The
SP:SAMPLEPREP_SUMMARY            	dried 200 µl aliquots of the polar phase for GC-MS analysis were re-suspended
SP:SAMPLEPREP_SUMMARY            	in methoxyamine-hydrochloride/pyridine solution for methoxymization of carbonyl
SP:SAMPLEPREP_SUMMARY            	groups followed by heating at 37 °C for 2 h. The samples were further
SP:SAMPLEPREP_SUMMARY            	derivatized with N-methyl-N-trimethylsilyltrifloracetamide (MSTFA) for 30 min
SP:SAMPLEPREP_SUMMARY            	at 37 °C. then one µl of the derivatized sample mixture was injected onto the
SP:SAMPLEPREP_SUMMARY            	GC-column and measured. Another dried 200 µl aliquots of the polar phase were
SP:SAMPLEPREP_SUMMARY            	re-suspended in 150 µl UPLC-grade methanol: water (1:1, vol/vol) and subjected
SP:SAMPLEPREP_SUMMARY            	to LC-MS analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	One µl was taken from each sample and injected into GC-MS at 270°C in a split
CH:CHROMATOGRAPHY_SUMMARY        	mode (50: 1) with helium carrier gas (> 99.999% purity) flow set to 1 ml/min,
CH:CHROMATOGRAPHY_SUMMARY        	and separated by a DB-35MS UI (30 m × 0.25 mm, 0.25 µm) capillary column. The
CH:CHROMATOGRAPHY_SUMMARY        	temperature was isothermal for 4 min at 90°C, followed by an 8°C per min ramp
CH:CHROMATOGRAPHY_SUMMARY        	up to 205°C, then held for 2 min, and finally ramped up at a rate of 15°C per
CH:CHROMATOGRAPHY_SUMMARY        	min to 310°C, held for 2 min.
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890A
CH:COLUMN_NAME                   	Agilent DB35-MS (30×0.25mm, 0.25µm)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 5975
MS:INSTRUMENT_TYPE               	Single quadrupole
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The transfer line temperature was set to 300°C, and the ion source temperature
MS:MS_COMMENTS                   	was set to 230°C. The mass range analyzed was from m/z 85 to 700. The Agilent
MS:MS_COMMENTS                   	MassHunter Qualitative Analysis (version B06.00) software and the Agilent
MS:MS_COMMENTS                   	MassHunter Quantitative Analysis (version B.07.01) were jointly used for GC-MS
MS:MS_COMMENTS                   	data analyses. NIST library and in-house database established using authentic
MS:MS_COMMENTS                   	standards were used together for metabolite identification.
MS:MS_RESULTS_FILE               	ST001330_AN002216_Results.txt	UNITS:Relative content	Has m/z:No	Has RT:Yes	RT units:Minutes
#END