谷子萌发吸水期关键代谢途径的筛选与分析

doi:10.3864/j.issn.0578-1752.2020.15.002

Abstract

Abstract:

【Objective】 Foxtail millet (Setaria italica L.) is a hardy cereal and known for its tolerance against drought and barrenness, which originated in China. In this study, the transcriptome of foxtail millet was analyzed in germinating seeds during different uptake of water phases, which is to get a lot of differently expressed genes, and find the important metabolic pathways, key genes and metabolites of regulating germination in foxtail millet. 【Method】The cDNA libraries of Jingu 20 were constructed in germinating seeds during rapid initial uptake of water, plateau phase, further increase in water uptake for the transcriptome analysis. The cluster analysis of gene expression was performed by K-Means. Differential expression analysis used DESeq. The functional annotations of differentially expressed genes were obtained by using COG, GO, KEGG, and so on. The key metabolism pathways and genes that regulated germination in foxtail millet were found during different water uptake phase through KEGG enrichment of DEGs. The reliability of sequencing results was confirmed by qRT-PCR. The contents of metabolites were assayed by HPLC.【Result】The genome-wide gene expression profile was obtained by RNA-sequencing during rapid initial uptake of water, plateau phase and further increase in water uptake. A total of 33 643 genes were expressed. Nine co-expression clusters with distinctive patterns were identified. There were 3 893, 4 612 and 8 472 DEGs in rapid initial uptake of water and plateau phase, plateau phase and further increase in water uptake, rapid initial uptake of water and further increase in water uptake, respectively. The KEGG pathway enrichment analysis showed that these DEGs of the three comparisons were significantly associated with phenylpropanoid biosynthesis,phenylalanine metabolism, starch and sucrose metabolism. The DEGs between rapid initial uptake of water and plateau phase, between rapid initial uptake of water and further increase in water uptake were also enriched in plant hormone signal transduction. And the number of genes enriched in the metabolism pathway of phenylpropanoid biosynthesis and phenylalanine metabolism was the largest in the three comparisons. The peroxidase genes had the highest proportion among these genes of the two pathways. The results obtained from four phenylpropanoids-related genes tested by qRT-PCR were consistent with the trend of regulation identified by gene expression profile. In dormant seeds of foxtail millet, there was the preformed mRNAs of 4-coumarate-CoA ligase 3, which displayed firstly decreasing, then increasing and finally declining patterns during the uptake of water stage. The contents of phenylpropanoids-related metabolites in germination seeds of foxtail millet indicated that large amounts of sinapic acid were stored in dormant seeds. During uptake of water by a dry seed, the content of sinapic acid gradually decreased, but those of p-coumaric acid, caffeic acid and ferulic acid increased first and then decreased. 【Conclusion】DEGs of foxtail millet were significantly related with phenylpropanoid biosynthesis and phenylalanine metabolism in germinating seeds during different uptake of water stages. The upstream and downstream genes of phenylpropanoid biosynthesis, such as 4-coumarate-CoA ligase 3 and peroxidase, played regulation roles in response to water during germination of foxtail millet. Sinapic acid participated in the dormancy and germination of millet seeds, which were the medium product of phenylpropanoid biosynthesis pathway.

Key words: foxtail millet, water uptake phase of seed germination, transcriptome analysis, phenylpropanoid biosynthesis, phenylalanine metabolism

YU AiLi,ZHAO JinFeng,CHENG Kai,WANG ZhenHua,ZHANG Peng,LIU Xin,TIAN Gang,ZHAO TaiCun,WANG YuWen. Screening and Analysis of Key Metabolic Pathways in Foxtail Millet During Different Water Uptake Phases of Germination[J].Scientia Agricultura Sinica, 2020, 53(15): 3005-3019.

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基因名称 Gene name	基因ID Gene ID	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')
β-Actin	Seita.7G294000	TGTGCCGGCCATGTATGT	CACACCATCACCAGAGTCCAA
Cationic peroxidase SPC4 precursor	Seita.3G004800	CGGCATCGTCAGGGATTT	AGGCGGGCTTTGTTGGT
4-coumarate--CoA ligase 3	Seita.1G065800	TGGAGTTCGCCGAGGTGAT	CGAGTTGAGCGAGTAGATGTGG
Beta-glucosidase 6 precursor	Seita.9G492600	CCGGCGTGTCACTAATGCT	GTCTTCCCTCTCCCATCTTCCT
Peroxidase 4 precursor	Seita.5G145500	TCTCGACGCTCCTGTCCAT	TTGGTGGCGGTGTCGTT

分类 Classification	开始快速吸水期PhaseⅠ		滞缓吸水期PhaseⅡ		重新大量吸水期PhaseⅢ
分类 Classification	CK2	CK2R	CK8	CK8R	CK14	CK14R
总reads Clean reads	38845760	39679812	56164700	43525742	46838670	49151796
总核苷酸 Clean bases	4894565760	4986871932	7076752200	5472201146	5901672420	6178141042
GC含量 GC content (%)	58.72	57.83	57.53	56.81	57.21	56.36
碱基质量值 Q30 (%)	89.74	85.29	90.33	85.17	91.62	85.13
匹配的reads Mapped reads	31007621 (79.82%)	31533133 (79.47%)	45812237 (81.57%)	35213766 (80.90%)	38024006 (81.18%)	39595878 (80.56%)
唯一位点匹配reads Unique mapped reads	26519605 (68.27%)	27126368 (68.36%)	42124995 (75.00%)	31824703 (73.12%)	35017461 (74.76%)	35922812 (73.09%)
多位点匹配reads Multiple mapped reads	4488016 (11.55%)	4406765 (11.11%)	3687242 (6.57%)	3389063 (7.79%)	3006545 (6.42%)	3673066 (7.47%)

Screening and Analysis of Key Metabolic Pathways in Foxtail Millet During Different Water Uptake Phases of Germination

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[9]	LI HuiXia,TIAN Gang,WANG YuWen,LIU Xin,LIU Hong. Genetic Correlation Coefficients of Foxtail Millet Traits Between Parents and Hybrids [J]. Scientia Agricultura Sinica, 2020, 53(2): 239-246.
[10]	CHANG GuoRong,LI RenJian,ZHANG Qi,ZHANG YuMing,HAN YuanHuai,ZHANG BaoJun. Identification of Co-Expression Genes Related to Endogenous Abscisic Acid in Response to the Stress of Sclerospora graminicola by WGCNA in Foxtail Millet [J]. Scientia Agricultura Sinica, 2020, 53(16): 3280-3293.
[11]	ZHANG Bin,LI Meng,LIU Jing,WANG JunJie,HOU SiYu,LI HongYing,HAN YuanHuai. Expression Analysis of the Chlorophyll Biosynthesis Structural Genes in Green and White Foxtail Millet [Setaria italica (L.) Beauv] [J]. Scientia Agricultura Sinica, 2020, 53(12): 2331-2339.
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[14]	JianFeng LI,Bo ZHANG,JianZhang QUAN,YongFang WANG,XiaoMei ZHANG,Yuan ZHAO,XiLei YUAN,XiaoPing JIA,ZhiPing DONG. Associated Loci Detection and Elite Allelic Variations Analysis of Main Agronomic Traits in Foxtail Millet (Setaria italica L.) Based on SSR Markers [J]. Scientia Agricultura Sinica, 2019, 52(24): 4453-4469.
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