γ-氨基丁酸和2-乙酰-1-吡咯啉代谢通路在水稻籽粒发育过程中的变化分析

Metabolism ofγ-aminobutyrate and 2-acetyl-1-pyrroline Analyses at Various Grain Developmental Stages in Rice(Oryza sativa L.)

【目的】结合代谢组和转录组技术,分析香稻和非香稻不同发育时期籽粒中γ-氨基丁酸(GABA)和2-乙酰-1-吡咯啉(2AP)代谢通路主要化合物和基因的动态变化,为高含量GABA和2AP的水稻育种提供理论依据。【方法】本研究收集优质香籼稻美香占2号和优质籼稻黄华占花后8 d(乳熟期)、花后15 d(蜡熟期)、30 d(完熟期)的脱壳籽粒和花后40 d收获的籽粒的精米,对各样本采用LC-MS/MS和RNA-seq技术进行代谢产物检测和转录表达分析,分析两个水稻品种GABA和2AP代谢通路相关化合物的空间分布以及含量变化特征。【结果】代谢组数据PCA分析表明两个水稻品种乳熟期籽粒和花后40 d收获的籽粒的精米明显分离,蜡熟期和完熟期籽粒分离不明显。代谢组共鉴定出623种代谢物,四个时期差异代谢物合计161个。GABA和2AP通路中9个代谢物被检出,两个水稻品种的相关代谢物变化趋势相似,其中腐胺主要定位于籽粒的糊粉层,而亚精胺在内胚乳特异累积。转录组共检出3.3万个左右基因,其中检出GABA通路18个酶中的14个酶对应的基因有较高的表达水平,除Badh2外其他基因均不是差异表达基因,同一基因在两份水稻材料中的表达水平和变化趋势相似,GAD1、GABA-T1、DAO4和PAO4在相应同源基因中表达水平较高。黄华占籽粒中L-精氨酸和腐胺的代谢在蜡熟期已基本结束,美香占2号在籽粒整个发育期具有持续较强的多胺降解水平。美香占2号籽粒具有合成2AP的物质基础,多胺降解途径和谷氨酸-脯氨酸转化通路同时有助于2AP的积累。【结论】水稻籽粒GABA代谢以降解为主,GABA-T1和GABA-T2共同调控GABA的降解。GABA相关代谢物主要集中在糊粉层,黄华占籽粒中代谢物分布和基因表达特征与美香占2号相似,美香占2号籽粒成熟过程中具有持续较高的线粒体活性。

【Objective】It is important to reveal the dynamic changes of metabolites and genes involved inγ-aminobutyric acid(GABA)and 2-acetyl-1-pyrroline(2AP)metabolic pathways at various grain developmental stages in scented and non-scented rice cultivars by metabolomes and transcriptomes.The purpose of this study is to lay a theoretical basis for high GABA and 2AP rice breeding.【Method】The metabolomics and transcriptomics of grains including milky grains(8D,8 days after flowering),dough grains(15D),mature grains(30D)and milled rice(40D)of scented rice cultivar Meixiangzhan 2(MXZ)along with non-scented rice cultivar Huanghuazhan(HHZ)were performed by LC-MS/MS and RNA-seq,respectively.Finally,the metabolites and transcript profiles of GABA and 2AP metabolic pathways were analyzed.【Result】The LC-MS/MS-based metabolic PCA analysis illustrated that the 8D and 40D grains of both varieties were dispersedly distributed,while 15D and 30D grains were concentrated in a relatively narrow area.In a total of 623 metabolites were detected in all samples,among which 161 were different metabolites.The contents of nine metabolites,detected in the GABA and 2AP pathways,followed a similar changing trend in all samples.We found that putrescine was located in embryo and aleurone layer,while spermidine was specifically accumulated in the endosperm.About 33000 genes were identified through the gene expression analysis,among which 14 genes involved in GABA pathway showed high transcriptional levels.These related genes had similar abundance patterns between two varieties,while the Badh2 was the only differentially expressed gene.GAD1,GABA-T1,DAO4 and PAO4 showed higher expression levels as compared with respective homologous genes.The L-arginine and putrescine profiles minimized in HHZ 5D grains,while the polyamine degradation had strong activity in whole MXZ grain development.There were all metabolites required for the 2AP synthesis in the grains,and both polyamine degradation pathway and the glutamate-proline pathway contributed to the accumulation of 2AP in grains.【Conclusion】The GABA metabolite was mainly degraded,and GABA-T1 and GABA-T2 were responsible for GABA degradation.The metabolites involved in GABA pathway were mainly concentrated in the aleurone layer,and the metabolites and gene expression profiles had similar patterns in both rice varieties.A higher mitochondria activity was found in MXZ grains than HHZ.