【摘要】 【目的】叶片宽度和长度等叶形特性是决定植株形态,进而影响种植密度的重要农艺性状,通过转录组测序技术筛选并挖掘玉米叶片形态建成相关的代谢路径及调控基因,为深入认识叶片发育的分子机理和鉴定叶宽、叶长候选基因奠定基础。【方法】以极端窄叶自交系NL409和宽叶自交系WB665为材料,利用RNA-Seq技术鉴定7叶期第七片叶近基部的差异表达基因(DEGs),通过生物信息学分析,筛选与叶片发育密切相关的代谢通路,利用qRT-PCR验证不同激素路径叶形相关基因的表达结果,并结合启动子区域的序列差异挖掘叶形功能基因。【结果】分析对照(WB665)和样品(NL409)高通量测序结果,在叶宽形成关键部位共筛选出5 199个DEGs,其中,2 264(43.55%)个基因表达上调,2 935(56.45%)个基因下调表达,下调基因明显多于上调基因;GO功能富集分析表明,差异基因主要富集在细胞膜相关的细胞组分中,涉及代谢过程和细胞响应刺激;KEGG富集分析表明,差异基因主要参与到核糖体、植物激素信号转导、苯丙烷类代谢、乙醛酸和二羧酸代谢等过程,其中核糖体、植物激素信号转导、鞘脂类代谢下调表达基因较多的路径与叶片发育密切相关。核糖体路径富集到多个PRS(PRESSED FLOWER)基因,分析发现PRS13(PFL2)可能在调控窄叶发育过程中发挥重要作用。鞘脂代谢路径富集的基因几乎全部下调表达,引起抑制叶片发育的AP1(APETALA1)类和MAPK(Mitogen-Activated Protein Kinase)类基因上调,以及促进叶片发育的LFY(LEAFY)下调,与窄叶发育受抑制的表型一致。植物激素信号转导路径富集到的油菜素内酯(BR)响应基因和赤霉素(GA)代谢基因下调,细胞分裂素(CTK)和大部分生长素(Auxin)响应基因上调,与窄叶中DELLA蛋白基因上调表达,抑制GA并促进CTK基因表达的作用模式一致。通过qRT-PCR对18个叶片发育相关基因进行分析,结果表明,其表达趋势与转录组结果一致,分析发现BR相关的ROT3、Auxin相关的NAL7-like、AGO7-like以及TCP类转录因子CYC/TB1等基因与窄叶的形成密切相关。【结论】明确了一些与玉米叶片发育密切相关的代谢路径,还发现植物激素间的动态平衡对叶片发育有着重要影响,尤其是生长素与油菜素内酯、细胞分裂素与赤霉素之间的相互作用对调控叶片形态可能发挥重要作用。更多还原

【Abstract】 【Objective】Leaf shape characteristics are one of important agronomic traits that determine plant morphology andaffect planting density. However, the molecular mechanism related to leaf shape remain unknown in maize. Here, transcriptome sequencing technology was used to screen and explore genome-wide analysis of regulatory genes and metabolic pathways involved in leaf morphogenesis. This study will lay the foundation for further understanding the regulator mechanism of leaf development in plant and identifying candidate genes of leaf shapes, such as leaf width and leaf length.【Method】 Extreme narrow-leaf inbred line NL409 and wide-leaf line WB665 were selected as the experimental materials. By RNA-Seq technology, the differentially expressed genes(DEGs) of the seventh leaf base between these two lines were identified during the 7 th leaf stage. Furthermore, metabolic pathways closely related to leaf development were also analyzed using a series of bioinformatics analysis. qRT-PCR was used to validate the expression level of DEGs in different hormone pathways, and the further promoter analysis were performed to explore leaf-shape functional genes.【Result】By analyzing the high-throughput sequencing in WB665 and NL409, a total of 5 199 DEGs were obtained at the primary section of leaf width formation. Of which, 2 264(43.55%) genes were up-regulated, whereas down-regulated genes were significantly more than up-regulated genes with 2 935(56.45%) decreased genes. GO enrichment analysis showed that these DEGs were mainly enriched in cell membrane-associated function terms of cellular components, including metabolic process and cell stimulus response. KEGG enrichment analysis showed that these DEGs were mainly involved in ribosome, plant hormone signal transduction, sphingolipid metabolism pathways, phenylpropanoid biosynthesis, glyoxylate and dicarboxylate metabolism and other processes. among which ribosome, plant hormone signal transduction, sphingolipid metabolism pathways with more down-regulated genes were closely related to leaf development. One of PRS(PRESSED FLOWER) family genes, which were enriched in the ribosomal pathway in this study, PRS13(PFL2) was identified to participate in regulating the development of narrow leaves. The expression pattern of genes enriched in sphingolipid metabolism pathway and its related MAP kinase, AP1-like, and LFY-like were consistent with the result of the inhibited development of narrow leaves. Notably, all of BR(Brassinosteroid) response genes and most of GA(Gibberellin) metabolic genes were down-regulated in plant hormone signal transduction pathway, while the expression level of all the CTK(Cytokinine) response genes and Auxin genes are mostly increased. The action of up-regulated expression of DELLA protein gene affecting the GA and CTK pathways was consistent with the phenotypic result of narrow leaves. Eighteen genes were validated by qRT-PCR. The result showed that the expression trend was consistent with the transcriptome data. Moreover, the BR-related ROT3, auxin-related NAL7-like, AGO7-like and TCP-like transcription factors CYC/TB1 were identified to be closely associated with the formation of narrow leaves.【Conclusion】Summarily, this study unveils several metabolic pathways closely related to leaf development in maize, and find the dynamic balance between plant hormones plays an important role in leaf development, especially the interaction between Auxin and BR as well as CTK and GA.更多还原