大白菜雌不育突变体fsm花蕾激素代谢的转录组分析
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摘要
以大白菜DH系FT为试材,通过游离小孢子培养结合EMS诱变处理,创制出一份雌性不育突变体fsm。与野生型FT相比,fsm花器官瘦小、子房干瘪、胚珠败育、自交和杂交均不结子。雌蕊的石蜡切片显示fsm中央隔膜融合异常,胚珠数量减少。内源激素测定显示,突变体fsm花蕾的生长素和油菜素内酯含量显著增高、细胞分裂素显著降低。根据转录组测序分析,差异表达基因(DEGs)主要在内源性刺激反应、非生物刺激反应、细胞膜组分和细胞外周等GO功能条目富集。KEGG分析将6921个差异表达基因比对到275条代谢途径,其中,显著富集的代谢途径包括新陈代谢、次生代谢物合成、植物-病原体互作和植物激素信号转导。YUCCA基因的表达上调可能是生长素含量增多的关键因素。激素代谢途径中,大量AUX1/IAA、GH3、SAUR和PIN类基因表达下调,可能使生长素转运受到抑制,导致雌蕊生长受阻。细胞分裂素代谢通路中,CUC、STM、WUS和CKX基因表达下调。花蕾中油菜素内酯含量增高,而BRI1类基因表达下调,表明突变体fsm可能是BR不敏感突变体,SEUSS-LIKE3和CYP85A2基因的表达下调可能导致雌蕊输导组织融合缺陷。突变体花蕾中激素间相互作用的紊乱可能是雌蕊发育异常的重要原因。利用qRT-PCR对激素代谢通路的25个差异表达基因进行验证,其表达模式与RNA-seq结果一致。研究结果为深入分析大白菜雌性不育突变体fsm雌蕊败育机理提供了重要参考价值,也为探究突变基因的生物学功能奠定了基础。
The female sterile mutant fsm was derived fr om a Chinese cabbage DH line 'FT', using free microspore culture combined with EMS mutagenesis. Compared with wild-type 'FT', the mutant fsm has small organ organs, shriveled ovary, reduced ovule count and no seeds produced when self-crossing or hybridization. Paraffin sections of fsm pistil showed an abnormal fusion of the central transmitting tract and reduced number of ovules. Flower bud endogenous hormone content test showed that, auxin and brassinolide in fsm were significantly increased, and cytokinin was significantly decreased. According to transcriptome sequencing analysis, differentially expressed genes(DEGs) are mainly enriched in GO functional items such as response to endogenous stimulus,response to abiotic stimulus, membrane and cell periphery. The KEGG metabolic pathway analysis matched 6,921 differentially expressed genes into 275 metabolic pathways. The most abundant pathways were metabolism, biosynthesis of secondary metabolites,plant-pathogen interaction and plant hormone signal transduction. The up-regulation of YUCCA gene may be a key factor for auxin content increase. In the transcriptome-hormone pathway of mutant fsm, the expression levels of AUX1/IAA, GH3, SAUR and PIN genes were down-regulated, which may inhibit the transport of auxin, leading to inhibition of pistil growth. In the cytokinin metabolic pathway, CUC, STM, WUS and CKX genes were down-regulated. The content of brassinolide in the fsm flower buds increased, while the expression of BRI1 genes were down-regulated, indicating that the mutant fsm may be a BR-insensitive mutant.Down-regulation of the expression of the SEUSS-LIKE3 and CYP85 A2 genes may result in defects in pistil transport tissue fusion.The disorder of hormone interaction in mutant flower buds was an important cause of pistil dysplasia. The 25 differentially expressed genes involved in the hormone metabolism pathway were verified by qRT-PCR, and the expression pattern was consistent with the transcriptome sequencing results. These provide important reference value for further study on the mechanism of female sterile mutant in Chinese cabbage, also laid the foundation for exploring the biological functions of mutant genes.
The female sterile mutant fsm was derived fr om a Chinese cabbage DH line 'FT', using free microspore culture combined with EMS mutagenesis. Compared with wild-type 'FT', the mutant fsm has small organ organs, shriveled ovary, reduced ovule count and no seeds produced when self-crossing or hybridization. Paraffin sections of fsm pistil showed an abnormal fusion of the central transmitting tract and reduced number of ovules. Flower bud endogenous hormone content test showed that, auxin and brassinolide in fsm were significantly increased, and cytokinin was significantly decreased. According to transcriptome sequencing analysis, differentially expressed genes(DEGs) are mainly enriched in GO functional items such as response to endogenous stimulus,response to abiotic stimulus, membrane and cell periphery. The KEGG metabolic pathway analysis matched 6,921 differentially expressed genes into 275 metabolic pathways. The most abundant pathways were metabolism, biosynthesis of secondary metabolites,plant-pathogen interaction and plant hormone signal transduction. The up-regulation of YUCCA gene may be a key factor for auxin content increase. In the transcriptome-hormone pathway of mutant fsm, the expression levels of AUX1/IAA, GH3, SAUR and PIN genes were down-regulated, which may inhibit the transport of auxin, leading to inhibition of pistil growth. In the cytokinin metabolic pathway, CUC, STM, WUS and CKX genes were down-regulated. The content of brassinolide in the fsm flower buds increased, while the expression of BRI1 genes were down-regulated, indicating that the mutant fsm may be a BR-insensitive mutant.Down-regulation of the expression of the SEUSS-LIKE3 and CYP85 A2 genes may result in defects in pistil transport tissue fusion.The disorder of hormone interaction in mutant flower buds was an important cause of pistil dysplasia. The 25 differentially expressed genes involved in the hormone metabolism pathway were verified by qRT-PCR, and the expression pattern was consistent with the transcriptome sequencing results. These provide important reference value for further study on the mechanism of female sterile mutant in Chinese cabbage, also laid the foundation for exploring the biological functions of mutant genes.
引文
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[23] MARTINEZ I,SANCHIS S,MARINI N,et al.The effect of NGATHA altered activity on auxin signaling pathways within the Arabidopsis gynoecium[J].Front Plant Sci,2014,5:210.
[24] EL-SHOWK S,RUONALA R,HELARIUTTA Y.Crossing paths:cytokinin signalling and crosstalk[J].Development,2013,140(7):1373-1383.
[25] KAMIUCHI Y,YAMAMOTO K,FURUTANI M,et al.The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development[J].Front Plant Sci,2014,5:165.
[26] CLOUSE S D,LANGFORD M,MCMORRIS T C.A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development[J].Plant Physiol,1996,111(3):671-678.
[27] NOMURA T,NAKAYAMA M,REID J B,et al.Blockage of brassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea[J].Plant Physiol,1997,113(1):31-37.
[28] BISHOP G J,HARRISON K,JONES J D.The tomato dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family[J].Plant Cell,1996,8(6):959-969.
[2] YANG L,WU Y,YU M,et al.Genome-wide transcriptome analysis of female-sterile rice ovule shed light on its abortive mechanism[J].Planta,2016,244(5):1011-1028.
[3] YAO Y,HAN R,GONG Z,et al.RNA-seq analysis reveals gene expression profiling of female fertile and fterile ovules of PinusTabulaeformis Carr.during free nuclear mitosis of the female gametophyte[J].Int J Mol Sci,2018,19(8):2246.
[4] CHEN L,ZHANG J,LI H,et al.Transcriptomic analysis reveals candidate genes for female sterility in Pomegranate flowers[J].Front Plant Sci,2017,8:1430.
[5] NEMHAUSER J L,FELDMAN L J,ZAMBRYSKI P C.Auxin and ETTIN in Arabidopsis gynoecium morphogenesis[J].Development,2000,127(18):3877-3888.
[6] SCHUSTER C,GAULLOCHET C,LOHMANN JU.Arabidopsis HECATE genes function in phytohormone control during gynoecium development[J].Development,2015,142(19):3343-3350.
[7] WU Y F,REED G W,TIAN C Q.Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression,and regulates both female and male reproduction[J].Development,2006,133(21):4211-4218.
[8] NOLE-WILSON S,RUESCHHOFF E E,BHATTI H,et al.Synergistic disruptions in seuss cyp85A2 double mutants reveal a role for brassinolide synthesis during gynoecium and ovule development[J].BMC Plant Biol,2010,10(1):198.
[9] MARSCH-MARTINEZ N,DE F.Hormonal control of the development of the gynoecium[J].Curr Opin Plant Biol,2016,29:104-114.
[10] EL-SHOWK S,RUONALA R,HELARIUTTA Y.Crossing paths:cytokinin signalling and crosstalk[J].Development,2013,140(7):1373-1383.
[11] SCHMULLING T,WENER T,RIEFLER M,et al.Structure and function of cytokinin oxidase/dehydrogenase genes of maize,rice,Arabidopsis and other species[J].J Plant Res,2003,116(3):241-252.
[12] CHEN M L,FU X M,LIU J Q,et al.Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC–ESI-Q-TOF-MS analysis[J].Journal of Chromatography B,2012,905:67-74.
[13] LI R,YU C,LI Y,et al.Soap2:an improved ultrafast tool for short read alignment[J].Bioinformatics,2009,25(15):1966-1967.
[14] ASHBURNER M,BALL C A,BLAKE J A,et al.Gene ontology:tool for the unification of biology[J].Nat Genet,2000,25(1):25-29.
[15] KANEHISA M,ARAKI M,GOTO S,et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Res,2007,36(Database issue):D480-D484.
[16] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,2001,25(4):402-408.
[17] PANOLI A,MARTIN M V,ALANDETE-SAEZ M,et al.Auxin import and local auxin biosynthesis are required for mitotic divisions,cell expansion and cell specification during female gametophyte development in Arabidopsis thaliana[J].PLoS One,2015,10(5):e0126164.
[18] PAGNUSSAT G C,ALANDETE-SAEZ M,BOWMAN J L,et al.Auxin-dependent patterning and gamete specification in the Arabidopsis female gametophyte[J].Science,2009,324(5935):1684-1689.
[19] FURUTANI M,VERNOUX T,TRAAS J,et al.PIN-FORMED1 and PINOID regulate boundary formation and cotyledon development in Arabidopsis embryogenesis.Development,2004,131(20):5021-5030.
[20] WU Y F,REED G W,TIAN C Q.Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression,and regulates both female and male reproduction[J].Development,2006,133(21):4211-4218.
[21] GALBIATI F,SINHA R D,SIMONINI S,et al.An integrative model of the control of ovule primordia formation[J].Plant J,2013,76:446-455.
[22] ZHOU Y,LU D,LI C,et al.Genetic control of seed shattering in rice by the APETALA2 transcription factor SHATTERING ABORTION1[J].Plant Cell,2012,24(3):1034-1048.
[23] MARTINEZ I,SANCHIS S,MARINI N,et al.The effect of NGATHA altered activity on auxin signaling pathways within the Arabidopsis gynoecium[J].Front Plant Sci,2014,5:210.
[24] EL-SHOWK S,RUONALA R,HELARIUTTA Y.Crossing paths:cytokinin signalling and crosstalk[J].Development,2013,140(7):1373-1383.
[25] KAMIUCHI Y,YAMAMOTO K,FURUTANI M,et al.The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development[J].Front Plant Sci,2014,5:165.
[26] CLOUSE S D,LANGFORD M,MCMORRIS T C.A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development[J].Plant Physiol,1996,111(3):671-678.
[27] NOMURA T,NAKAYAMA M,REID J B,et al.Blockage of brassinosteroid biosynthesis and sensitivity causes dwarfism in garden pea[J].Plant Physiol,1997,113(1):31-37.
[28] BISHOP G J,HARRISON K,JONES J D.The tomato dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family[J].Plant Cell,1996,8(6):959-969.