大白菜倍性变异引起花发育变化的microRNA调控机制研究
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摘要
为探索microRNAs(miRNAs)在大白菜花发育中的调控机制,以大白菜DH系FT为试材,通过游离小孢子培养创制出同一基因组的二倍体和四倍体纯系,并稳定遗传。与二倍体相比,四倍体植株在形态上表现出生长发育缓慢、开花时间延迟、花器官整体变大、花瓣颜色更深等特征。利用sRNA-seq技术,分别构建了上述二倍体和四倍体的small RNA(sRNA)文库。通过与植物数据库miRBase比对,分别获得92个和95个成熟的bra-miRNAs,新预测的分别有70个和74个novel miRNAs。通过差异表达分析,筛选获得了34个差异表达的miRNAs,并预测了其287个靶基因。根据基因功能注释分析,筛选出9个与花发育相关的miRNAs,其靶基因涉及类黄酮合成、激素调控、细胞生长、开花时间调控等多方面。通过对287个靶基因进行GO功能富集和KEGG代谢通路分析,其中179个靶基因GO功能富集到810个条目上。最显著差异的条目有:核酸结合、细胞内组分、细胞器、细胞内的细胞器及DNA结合。KEGG代谢通路分析表明,有126个靶基因参与到39条代谢通路上。富集基因显著且数量最多的通路是代谢途径,其次是氰基氨基酸代谢、木质素生物合成以及淀粉和蔗糖代谢。通过对涉及影响四倍体植株花发育变化的靶基因功能进行分析,讨论其9个miRNAs在花发育过程中可能参与的调控作用,推测出有6个miRNAs(bra-miR156a-5p、bra-miR172b-5p、brami R172c-3p、bra-miR172c-5p、bra-miR172d-5p和novel_87)在花期通过互相调控多个靶基因导致开花时间的延迟;有2个miRNAs(bra-miR395d-3p、novel_122)参与调控生长素和脱落酸的变化,可能导致花瓣变大及生长发育变缓等表型特征;brami R395d-3p调控的靶基因还涉及类胡萝卜素生物合成,同时另一个miRNA (bra-miR9552a-3p)可能参与类黄酮生物合成的调控,它们的调控可能是使花瓣颜色变深的主要原因。本研究为揭示由大白菜倍性变异引起的花发育表型变化的分子机制奠定了基础。
In order to explore the regulation mechanism of microRNAs(miRNAs) in the floral development of Chinese cabbage,the DH line FT of Chinese cabbage was used as a test material to create diploid and tetraploid pure lines of the same genome through isolated microspore culture, and stable inheritance. Compared with diploids, tetraploid plants are characterized by slow growth and development, delayed flowering time, larger flower organ, and deeper petal color. Using the s RNA-seq technique, we constructed the above diploid and tetraploid small RNA(sRNA) libraries, respectively. By comparison with the plant database miRBase, 92 and 95 mature bra-miRNAs were obtained, respectively. Moreover, 70 and 74 novel miRNAswere obtained,respectively. Through the analysis of differentially expressed genes, 34 differentially expressed miRNAs were selected and their287 target genes were predicted. According to the analysis of gene function annotation, nine miRNAs related to floral development were selected, and their target genes involved flavonoid synthesis, hormone regulation, cell growth, flowering time regulation and so on. By performing GO functional enrichment and KEGG pathway analysis on 287 target genes, 179 target genes were enriched into 810 terms. The most notable differences were: nucleic acid binding, intracellular part, organelle,intracellular organelle, and DNA binding. KEGG pathway analysis showed that 126 target genes were involved in 39 metabolic pathways. The most significant and abundant pathways for enriched genes were metabolic pathways, followed by cyanoamino acid metabolism, phenylpropanoid biosynthesis, and starch and sucrose metabolism. By analyzing the function of target genes involved in floral development changes affecting tetraploid plants, the regulation of the possible involvement of nine miRNAs in floral development was discussed. It is speculated that six miRNAs(bra-miR156 a-5 p, bra-miR172 b-5 p, bra-miR172 c-3 p, bra-miR172 c-5 p, bra-miR172 d-5 p, novel_87) delayed in flowering time by intermodulating multiple target genes at flowering stage;Two miRNAs(bra-miR395 d-3 p, novel_122) participate in the regulation of auxin and abscisic acid, which could lead to phenotypic characteristics such as large petals and slow growth and development. The target gene regulated by bra-miR395 d-3 p also involves carotenoid biosynthesis. Another miRNA(bra-miR9552 a-3 p) could be involved in the regulation of flavonoid biosynthesis, and their regulation could be the main reason for the deeper color of the petals. This study laid the foundation for revealing the molecular mechanism of floral development phenotypic changes caused by ploidy variation of Chinese cabbage.
In order to explore the regulation mechanism of microRNAs(miRNAs) in the floral development of Chinese cabbage,the DH line FT of Chinese cabbage was used as a test material to create diploid and tetraploid pure lines of the same genome through isolated microspore culture, and stable inheritance. Compared with diploids, tetraploid plants are characterized by slow growth and development, delayed flowering time, larger flower organ, and deeper petal color. Using the s RNA-seq technique, we constructed the above diploid and tetraploid small RNA(sRNA) libraries, respectively. By comparison with the plant database miRBase, 92 and 95 mature bra-miRNAs were obtained, respectively. Moreover, 70 and 74 novel miRNAswere obtained,respectively. Through the analysis of differentially expressed genes, 34 differentially expressed miRNAs were selected and their287 target genes were predicted. According to the analysis of gene function annotation, nine miRNAs related to floral development were selected, and their target genes involved flavonoid synthesis, hormone regulation, cell growth, flowering time regulation and so on. By performing GO functional enrichment and KEGG pathway analysis on 287 target genes, 179 target genes were enriched into 810 terms. The most notable differences were: nucleic acid binding, intracellular part, organelle,intracellular organelle, and DNA binding. KEGG pathway analysis showed that 126 target genes were involved in 39 metabolic pathways. The most significant and abundant pathways for enriched genes were metabolic pathways, followed by cyanoamino acid metabolism, phenylpropanoid biosynthesis, and starch and sucrose metabolism. By analyzing the function of target genes involved in floral development changes affecting tetraploid plants, the regulation of the possible involvement of nine miRNAs in floral development was discussed. It is speculated that six miRNAs(bra-miR156 a-5 p, bra-miR172 b-5 p, bra-miR172 c-3 p, bra-miR172 c-5 p, bra-miR172 d-5 p, novel_87) delayed in flowering time by intermodulating multiple target genes at flowering stage;Two miRNAs(bra-miR395 d-3 p, novel_122) participate in the regulation of auxin and abscisic acid, which could lead to phenotypic characteristics such as large petals and slow growth and development. The target gene regulated by bra-miR395 d-3 p also involves carotenoid biosynthesis. Another miRNA(bra-miR9552 a-3 p) could be involved in the regulation of flavonoid biosynthesis, and their regulation could be the main reason for the deeper color of the petals. This study laid the foundation for revealing the molecular mechanism of floral development phenotypic changes caused by ploidy variation of Chinese cabbage.
引文
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[24]赵佳媛,陈婧,钟扬,等.植物microRNA的研究进展[J].植物生理学报,2013(9):847-854.
[25]徐妙云,王磊.microRNA与植物花发育调控的研究进展[J].中国农业科技导报,2011,13(2):9-16.
[26] CHUCK G,CIGAN AM,SAETEURN K,et al.The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA[J].Nat Genet,2007,39:544-549.
[27] SCHMID M,UHLENHAUT NH,GODARD F,et al.Dissection of floral induction pathways using global expression analysis[J].Development,2003,130:6001-6012.
[28]黄赫,徐启江.MicroRNA调控被子植物花发育的研究进展[J].植物生理学报,2012,48:929-940.
[29] NAGPAL P,ELLIS CM,WEBER H,et al.Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation[J].Development,2005,132(18):4107-4118.
[30]刘振华,于延冲,向凤宁.生长素响应因子与植物的生长发育[J].遗传,2011,33(12):1335-1346.
[31]关颖谦,方建雄,张映璜,等.脱落酸对离体水稻叶片衰老的效应[J].植物生理学报,1981(3):52-58.
[2] MALLORY AC,VAUCHERET H.Functions of microRNAs and related small RNAs in plants[J].Nat Genet,2006,38:S31-S36.
[3] LI PP,MONTGOMERYTA,FAHLGREN N,et al.Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages[J].Plant J,2007,52(1):133-146.
[4] MOXON S,JING R,SZITTYA G,et al.Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening[J].Genome Res,2008,18(10):1602-1609.
[5] WU L,LIU D,WU J,et al.Regulation of FLOWERING LOCUS T by a microRNA in Brachypodium distachyon[J].Plant Cell,2013,25(11):4363-4377.
[6]刘畅,刘志勇,李承彧,等.大白菜核雄性不育相关microRNA的鉴定[J].沈阳农业大学学报,2016,47(5):527-535.
[7] RUIZ-FERRER V,VOINNET O.Roles of plant small RNAs in biotic stress responses[J].Annu Rev Plant Biol,2009,60(1):485-510.
[8] JEONG D H,PARK S,ZHAI J,et al.Massive analysis of rice small RNAs:mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage[J].Plant Cell,2011,23(12):4185-4207.
[9] DOYLE JJ,FLAGEL LE,PATERSON AH,et al.Evolutionary genetics of genome merger and doubling in plants[J].Annu Rev Genet,2008,42(1):443-461.
[10]张云,刘青林.植物花发育的分子机理研究进展[J].植物学报,2003,20(5):589-601.
[11] COMAI L.The advantages and disadvantages of being polyploid[J].Nat Rev Genet,2005,6(11):836-846.
[12] CHAO DY,DILKES B,LUO H,et al.Polyploids exhibit higher potassium uptake and salinity tolerance in Arabidopsis[J].Science,2013,341(6146):658-659.
[13] MA Y,XUE H,ZHANG L,et al.Involvement of auxin and brassinosteroid in dwarfism of Autotetraploid Apple(Malus×domestica)[J].Sci Rep,2016,6:26719.
[14] LI X,SHAHID MQ,WU JW,et al.Comparative small RNA analysis of pollen development in autotetraploid and diploid rice[J].International Journal Molecular Sciences,2016,17:499.
[15] ZHANG FJ,ZHAO JY,XU SJ,et al.MicroRNA and putative target discoveries in chrysanthemum polyploidy breeding[J].International Journal of Genomics,2017:1-13.https://doi.org/10.1155/2017/6790478.
[16] HUANG S,LIU Z,LI D,et al.Screening of Chinese cabbage mutants produced by 60Coγ-ray mutagenesis of isolated microspore cultures[J].Plant Breeding,2014,133:480-488.
[17] ZHOU L,CHEN J,LI Z,et al.Integrated profiling of microRNAs and mRNAs:microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma[J].PLoS One,2010,5:e15224.
[18] WANG L,FENG Z,WANG X,et al.DEGseq:an R package for identifying differentially expressed genes from RNA-seq data[J].Bioinformatics,2010,26:136-138.
[19] WU H J,MA Y K,CHEN T,et al.PsRobot:a web-based plant small RNA meta-analysis toolbox[J].Nucleic Acids Res,2012,40:W22-W28.
[20] SHERLOCK G.Gene Ontology:tool for the unification of biology[J].Canadian Institute of Food Science&Technology Journal,2009,22(4):415.
[21] KANEHISA M,ARAKI M,GOTO S,et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Research,2008,36:D480-D484.
[22] NG D W,LU J,CHEN Z J.Big roles for small RNAs in polyploidy,hybrid vigor,and hybrid incompatibility[J].Curr Opin Plant Biol,2012,15(2):154-161.
[23] GUAN X,SONG Q,CHEN ZJ.Polyploidy and small RNA regulation of cotton fiber development[J].Trends Plant Sci,2014,19(8):516-528.
[24]赵佳媛,陈婧,钟扬,等.植物microRNA的研究进展[J].植物生理学报,2013(9):847-854.
[25]徐妙云,王磊.microRNA与植物花发育调控的研究进展[J].中国农业科技导报,2011,13(2):9-16.
[26] CHUCK G,CIGAN AM,SAETEURN K,et al.The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA[J].Nat Genet,2007,39:544-549.
[27] SCHMID M,UHLENHAUT NH,GODARD F,et al.Dissection of floral induction pathways using global expression analysis[J].Development,2003,130:6001-6012.
[28]黄赫,徐启江.MicroRNA调控被子植物花发育的研究进展[J].植物生理学报,2012,48:929-940.
[29] NAGPAL P,ELLIS CM,WEBER H,et al.Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation[J].Development,2005,132(18):4107-4118.
[30]刘振华,于延冲,向凤宁.生长素响应因子与植物的生长发育[J].遗传,2011,33(12):1335-1346.
[31]关颖谦,方建雄,张映璜,等.脱落酸对离体水稻叶片衰老的效应[J].植物生理学报,1981(3):52-58.