利用RNA-Seq发掘玉米叶片形态建成相关的调控基因
利用RNA-Seq发掘玉米叶片形态建成相关的调控基因利用RNA-Seq发掘玉米叶片形态建成相关的调控基因郭书磊1,2，鲁晓民1，齐建双1，魏良明1，张新1，韩小花1，岳润清1，王振华1，铁双贵1，陈彦惠2（1河南省农业科学院粮食作物研究所/河南省玉米生物学重点实验室，郑州 450002；2河南农业大学农学院，郑州 450046）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。关键词：玉米；叶宽；叶长；RNA-Seq；形态建成；调控基因0 引言【研究意义】玉米作为增产潜力巨大的粮食作物，对于保障粮食安全，促进农业发展具有重要作用。近年来，中国玉米的种植密度维持在4.8—6.77万株/hm2，与美国8.5—10.95万株/hm2的种植密度存在显著差距，提高玉米的耐密植特性，成为实现中国玉米高产突破的重要措施。叶片是玉米进行光合作用和抗逆作用的主要营养器官，是耐密植理想株型的关键组成因素，对于植株干物质的积累及抗逆性有非常重要的影响。对于大面积密植的农作物，高密度条件下容易出现避荫综合症（shade avoidance syndrome，SAS），迫使群体内个体将能量重新分配，用于叶片和植株伸长生长，从而获得更多的阳光，尽管避荫反应对单个植株的适应与生存是有利的，但是由于避荫反应会削弱植株叶片和储藏器官的发育，对提高大面积种植的作物产量是不利的。高密度条件下，叶片宽大的群体冠层间通风透气性差，降低植株的抗病性，加剧病虫害的发生，导致生物量和籽粒产量降低。玉米耐密植株型的合理叶宽、叶长有利于改良理想株型，减少植株的避荫反应，使个体间竞争最小化，对提高种植密度具有重要的促进作用。叶宽、叶长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，因此，选育适宜高密度种植的合理叶宽、叶长玉米，是提高玉米产量的重要技术手段之一。虽然有大量研究揭示了玉米叶发育及形态建成的遗传基础，但是鲜有关于叶宽、叶长QTL克隆及调控网络的研究结果，利用转录组（RNA-seq）筛选与叶宽、叶长发育相关的调控基因，并发掘其可能的调控路径，对叶宽、叶长主效基因研究及耐密株型设计育种具有重要意义。【前人研究进展】叶片的发育起始于茎顶端分生组织（shoot apical meristem，SAM）周边的初始细胞，主要包括叶原基的形成和叶极性轴向的建立。叶原基是由顶端分生组织中心“干细胞”在生长素诱导下形成成簇分布的外缘叶原初生细胞；叶原基形成后在自身遗传机制和环境因子作用下，建立基-顶轴（叶的基部-顶端）、腹-背轴（茎的近轴面-远轴面）、中-边轴（叶的主脉-边缘）3个极性轴向，这些过程中的发育异常将直接或间接影响叶片的发育和形态建成。玉米叶片的发育和形态建成是由多基因调控的复杂过程，主要由遗传因素控制，同时受环境影响，叶宽的遗传相比其他叶形性状受环境影响较小，而且其具有易观测的线性形态，更有利于研究叶片发育过程的形态变化和细胞学特征，是研究叶宽最为理想的模式植物。关于拟南芥、水稻、金鱼草等植物叶片发育的研究，取得了一系列重要进展，深入解析了叶片发育的调控机理，而玉米叶宽、叶长的研究多为遗传分析及QTL定位的报道，只有少数文献关于叶发育基因lg1、lg2、ns1、ns2、knotted1、rgd1/lbl1、rgd2、mwp1、AN3/GIF1和ZmGRF10遗传机理的分析，揭示了叶宽、叶长发育形成的部分分子机理。高通量转录组测序技术，不仅能够检测全基因组所有基因的转录信息，还可以获得特定时期某一生物学过程显著富集的基因，以及特定组织或细胞中的多种转录本和优势表达基因，对研究特定组织的生物学功能具有重要的指导意义，利用转录组发掘玉米叶片发育相关基因，为叶片遗传机理的解析提供了新途径。【本研究切入点】关于叶宽、叶长等叶形主效基因克隆的研究相对有限，已有研究仅揭示了叶形发育的部分分子机理，叶宽、叶长候选基因及其调控路径有待进一步解析。【拟解决的关键问题】本研究通过RNA-seq测序探讨叶宽差异极显著自交系的转录表达特性，以期获得与叶形发育相关的关键基因及转录调控网络，更好地理解玉米叶片发育的分子机理，增加对窄叶形成机制的认识，进而为叶形候选基因的克隆及耐密分子育种提供参考。(1)通过热力学分析发现,N元素可以显著影响Fe-21Cr-3Ni-1Mo-N双相不锈钢两相比例.当N含量从0.700%增加至2.200%时,奥氏体含量从23.2%增加至62.7%. 1 材料与方法1.1 植物材料2017年夏极端窄叶自交系NL409（样品）和宽叶自交系WB665（对照）种植于河南省农业科学院现代农业科技示范基地，在田间正常生长至7叶期时（对生的叶耳包裹着新鲜的幼茎即将打开），取第7片叶近基部叶片（近基部1/3叶长处，切取2.0 cm），为了消除个体间差异，将相同基因型的材料3株混合取样，每个材料取3次生物学重复，样品经液氮速冻后-80℃保存，用于RNA提取。极端窄叶自交系NL409是欧洲杂交种连续自交选育而来，宽叶自交系WB665是昌7-2改良系，两者亲缘关系较远。1.2 RNA样品的提取及转录组测序委托北京安诺优达基因科技有限公司完成RNA提取、文库构建和转录组测序。首先，采用酚/氯仿法提取Total RNA，通过NanoDrop 2000微量分光光度计、Agilent 2100 Bioanalyzer和Agilent RNA 6000 Nano Kit检测RNA浓度、纯度和完整性；其次，检测合格样品利用Oligo（dT）富集mRNA，并通过Fragmentation Buffer使其短片段化，以短片段mRNA合成双链cDNA，纯化后进行末端修复、加碱基A、加测序接头处理；最后进行PCR扩增完成测序文库制备。采用Illumina NextSeq 550AR平台进行双末端测序，并对获得的原始序列（raw reads）进行过滤，去除含接头、低质量（Q＜30）、含N比例大于5%的序列，得到高质量的Clean Reads用于后续分析。1.3 基因表达数据分析利用TopHat（v2.0.12）软件，将6个样本过滤后的Clean Reads比对到B73基因组（AGPv3 版本），通过FPKM（Fragments per Kilobase per Millon Mapped Fragments）对基因表达量进行标准化，以FPKM>1为表达标准，随后根据极端窄叶与宽叶的比较，用DEseq（|log2FC（fold change）|≥1和P-value/FDR＜0.05）确定差异表达基因（differentially expressed genes，DEGs）。利用Uniprot、Swissprot、COG、NR、GO和KEGG等数据库对DEGs进行功能注释，通过GO富集分析显示DEGs显著富集的功能分类，用KEGG分析DEGs主要参与的代谢途径和信号通路（kyoto encyclopedia of genes and genomes，KEGG）。1.4 实时荧光定量PCR（qRT-PCR）分析选择与叶片发育相关的18个差异表达基因进行qRT-PCR验证。将转录组测序用到的RNA，通过反转录试剂盒（GoScript Reverse Transcription Kit购于promega生物技术有限公司）合成cDNA第一链，以其为模板，利用GoTaq qPCR Master Mix Kit（购于promega生物技术有限公司）在Bio-Rad CFX96实时荧光定量仪上进行qRT-PCR。以玉米中的18SrRNA作为内参基因，每个基因的定量分析设置3次生物学重复，3次技术重复，采用相对定量2-ΔΔCt法分析结果。有一天，我终于又看到了白丽筠，或者说我自认为是白丽筠的女子。我高喊了一声：“白丽筠！”那个在我前面走过去的女子回过头来，却是一副不曾相识的表情，马上回过头去，向大堂深处走去。我急忙冲上前去，想要拉她的手，这时不知从哪里冒出来一个保安，照我的下巴狠狠地打了一记钩拳，我一个趔趄，栽倒在地上。 2 结果2.1 叶宽表型和RNA-Seq测序质量分析极端窄叶自交系NL409和宽叶自交系WB665叶宽形态稳定时期（散粉后一周）的叶宽表型（图1），以及第7叶期的叶宽达到极显著差异（表1），在这两个时期，NL409的叶宽、叶长、叶面积均显著小于WB665。6个RNA-Seq文库原始测序过滤后分析结果（表2），各样本生物学重复间的相关系数为0.984—0.995，其中约88%高质量序列被比对到外显子区域，表明测序结果良好，数据可用于进一步分析。植物器官发育过程中极性建立是器官形态建成的核心。叶片的发育起始于叶原基细胞的分裂增殖，伴随着一系列特定基因的精确调控，叶原基建立多维空间的极性轴向，促使叶原基细胞朝特定的方向分化增殖，并最终影响叶片的形态和大小。The traditional OTA-based and inverter-based integrator are shown in Figs. 1(a) and 1(b) respectively. 表1 自交系NL409和WB665第7叶期以及稳定时期的叶宽、叶长、叶面积Table 1 Leaf width, leaf length and leaf area of inbred lines NL409 and WB665 at the 7th leaf stage and in the stable periodhttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/c044502b7fe1843e47fce896af3cc8de.jpg&q=30 材料1)Material叶宽leaf width (cm)叶长leaf length (cm)叶面积leaf area (cm2) NL409-72.49±0.13A36.44±2.92A66.12±8.59A WB665-74.26±0.34B29.67±1.67B89.44±12.45B NL409-S4.67±0.27A65.47±2.47A223.31±13.11A WB665-S10.88±0.58B59.89±2.57B475.60±24.80B1) 7表示第7叶期，S表示稳定时期。不同大写字母表示在P＜0.01水平差异显著1) 7 indicate the 7th leaf period, S indicate the stable period. different uppercase letters indicate significant differences at P＜0.01 levelshttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/a9fc9fb9b65a48a7089ec73b9af13082.jpg 图1 自交系NL409（左）和WB665（右）稳定时期的叶片Fig. 1 Leaves of inbred lines NL409 (left) and WB665 (right) in the stable periods2.2 基因表达数据分析选取第7叶期窄叶自交系NL409（T7，样品）和宽叶自交系WB665（C7，对照）叶片发育最宽处进行转录组差异比较分析，发现5 199个差异表达基因，其中2 264（43.55%）个基因上调表达，2 935（56.45%）个基因下调表达。上（下）调1—3倍（fold change）的基因分别占差异表达上（下）调基因总数的29.37%（665）和21.69%（756）；上（下）调3—10倍的基因分别占43.86%（993）和29.24%（1 019）；上（下）调10—60倍的基因分别占20.76%（470）和12.82%（447）；上（下）调60倍以上的基因分别占6.01%（136）和24.29%（713）。从火山图中可以直观地看出对照组与比较样本组之间的表达水平具有显著差异，而且差异60倍以上的下调基因明显多于上调基因（图2）。表2 样品reads分布情况Table 2 Overview of the sequencing readshttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/cb2eb05cdf06a48afa2c92a97e7121c5.jpg&q=30 样品1)Sample 原始序列raw reads过滤后序列clean reads高质量序列clean reads (%)比对到基因组序列mapped reads锚定比例mapped (%)单一位置序列unique mapped reads (%)过滤后Q30比例Q30 after filter (%) NL409-71485701844780571498.433824030679.9977.1293.19 NL409-72452150544423690697.843491800278.9376.2392.06 NL409-73468223404615671698.583721174980.6277.7995.02 WB665-71456257224479202898.173416604577.9673.3492.53 WB665-72438488944312027898.343361820678.8075.2194.68 WB665-73478388744720295298.673719386878.0276.0095.38
1) 71、72、73分别代表7叶期的第1、2、3个样品1) 71, 72 and 73 indicate the first, second and third samples of the 7th leaf period respectivelyGO功能分析表明，差异表达基因被注释到24个生物过程、22个细胞组分、19个分子功能类别中，差异基因主要富集在细胞膜相关的细胞组分中。其中生物过程显著富集在次生代谢、花粉雌蕊相互作用、花粉识别、防卫反应和细胞识别5个过程（图3）；细胞组分分析中差异基因显著富集在细胞外缘、质膜、细胞膜固有成分、细胞膜基本成分、膜部分、共质体、胞间接合和胞间连丝等10个组分类别（图4）；分子功能显著富集在单加氧酶活性、氧化还原活性、催化活性、纤维素合成酶活性、ADP结合、碳水化合物结合、铁离子结合、葡糖基转移酶活性等19个类别（图5）。http://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/fd898482d3c7bc400f5a9923ca5d66f0.png 横坐标为差异表达基因表达倍数变化，纵坐标为表达量变化的统计学显著程度The abscissa is the change in the expression fold of DGEs, the ordinate is the statistical significance of the change of expression level图2 差异表达基因火山图Fig. 2 Volcano Plot of DEGs利用MapMan工具对差异表达基因进行pathway富集分析，差异基因被注释到125条代谢通路中，主要涉及生物合成、能量代谢、信号转导及次生代谢。以P-value≤0.05为筛选标准，发现这些基因显著富集到11条代谢路径（表3），其中核糖体路径富集差异基因数目最多达到83个，37个基因表达上调，46个基因下调表达；其次，植物激素信号转导通路富集57个差异基因，24个基因上调，33个基因下调；苯丙烷类代谢路径18个基因上调，15个基因下调；乙醛酸和二羧酸代谢通路15个基因上调，10个基因下调；谷胱甘肽代谢路径13个基因上调，10个基因下调；鞘脂类代谢路径2个基因上调，11个基因下调（表4）；油菜素内酯生物合成路径4个基因上调，3个基因下调（表5）；类黄酮生物合成路径6个基因上调，2个基因下调；苯并恶唑嗪类生物合成和苯乙烯类、二芳庚酸类、姜酚类生物合成通路也富集到较多差异基因，分别有7个和5个基因下调表达。基于不同代谢通路中基因上下调比例，推断核糖体、植物激素信号转导、鞘脂类代谢这些基因下调表达较多的路径可能与叶片发育，尤其是窄叶的形成密切相关。在高中数学教学过程中，教师应当引导学生进行知识探究，营造轻松愉悦的课堂氛围，有助于加强课堂互动交流，提高学生的数学学习兴趣。本文从以下几方面简要阐述如何在高中数学教学中巧妙设置疑问，希望对教育教学工作有所借鉴和帮助。 http://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/293291b6a737d558b71c9b4b236302b2.png Size代表圆点的大小，表示富集到该GO条目基因的数量，q-value表示该GO条目的富集程度，颜色越趋近于红色表示富集程度越高。下同Size represents the size of the dot, indicating the number of genes enriched to the GO item, q-value indicates the enrichment degree of the GO item, and the closer the color is to red, the higher the enrichment. The same as below图3 差异表达基因显著富集的生物过程Fig. 3 Biological process enriched analysis of DEGshttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/0c2b2db5ede962c81eb5a9cd2cb7fe01.png 图4 差异表达基因显著富集的细胞组分Fig. 4 Cellular component enriched analysis of DEGs表3 差异表达基因显著富集的代谢通路Table 3 Pathway enriched analysis of DEGshttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/9789b7f0ad1d6bdca8fb36805000fffb.jpg&q=30 代谢通路Pathway代谢通路IDPathway ID差异基因数目Counts of DEGsP值P-value 油菜素内酯生物合成Brassinosteroid biosynthesismap00905 70.00088 乙醛酸和二羧酸代谢Glyoxylate and dicarboxylate metabolismmap00630250.00108 核糖体Ribosomemap03010830.00464 苯丙烷类代谢Phenylpropanoid biosynthesismap00940330.00854 植物激素信号转导Plant hormone signal transductionmap04075570.01099 苯并恶嗪类生物合成Benzoxazinoid biosynthesismap00402 70.02726 谷胱甘肽代谢Glutathione metabolismmap00480230.02847 鞘脂类代谢Sphingolipid metabolismmap00600130.03916 二萜类生物合成Diterpenoid biosynthesis map00904 60.04792 类黄酮生物合成Flavonoid biosynthesismap00941 80.04716 苯乙烯类、二芳庚酸类和姜酚类生物合成Stilbenoid, diarylheptanoid and gingerol biosynthesismap00945 70.04825
植物内源激素广泛参与到叶片发育过程，对叶片形态建成起到一定调节作用。进一步分析植物激素信号转导路径发现，在叶宽形成的关键部位检测到大量参与激素信号响应和转导的差异基因，涉及生长素、油菜素内酯、细胞分裂素、赤霉素、脱落酸、乙烯、茉莉酸等多种激素（图6）。分析不同响应路径（表5和图6）发现，生长素信号转导通路富集到18个差异基因，有12个响应基因表达上调；油菜素内酯信号响应路径富集到6个差异基因，全部下调表达；细胞分裂素响应路径富集的3个基因，表达均上调；赤霉素和水杨酸响应通路分别富集到1个差异基因，表达均上调；脱落酸路径富集到8个基因下调表达，4个上调表达基因；乙烯路径中4个基因上调表达，4个基因下调表达；茉莉酸路径中1个基因上调表达，7个基因下调表达。表4 鞘脂类代谢路径及其相关基因Table 4 Sphingolipid metabolism pathway and related geneshttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/61510f7327ae2158bd745e74a1637a04.jpg&q=30 路径pathway基因编号Gene ID差异倍数FC基因描述/注释Gene description/annotation 鞘脂类代谢Sphingolipid metabolismGRMZM2G1656130.00下调Down丝氨酸软脂酰转移酶，长链碱基生物合成蛋白1bSerine palmitoyltransferase, Long chain base biosynthesis protein 1b GRMZM2G4443780.00下调Down丝氨酸软脂酰转移酶，PKC激活蛋白磷酸酶1型抑制剂Serine palmitoyltransferase, PKC-activated protein phosphatase-1 inhibitor GRMZM2G4497630.00下调Down丝氨酸软脂酰转移酶，胺核黄素氧化还原酶Serine palmitoyltransferase, Flavin containing amine oxidoreductase GRMZM2G0763980.00下调Down丝氨酸软脂酰转移酶，长链碱基生物合成蛋白1bSerine palmitoyltransferase, Long chain base biosynthesis protein 1b GRMZM2G1342480.24下调Down丝氨酸软脂酰转移酶，长链碱基生物合成蛋白1bSerine palmitoyltransferase, Long chain base biosynthesis protein 1b GRMZM2G1449850.33下调Down丝氨酸软脂酰转移酶，长链碱基生物合成蛋白1bSerine palmitoyltransferase, Long chain base biosynthesis protein 1b GRMZM2G1528880.30下调Down丝氨酸软脂酰转移酶，长链碱基生物合成蛋白2aSerine palmitoyltransferase, Long chain base biosynthesis protein 2a GRMZM2G3577340.02下调Down丝氨酸软脂酰转移酶，胺核黄素氧化还原酶Serine palmitoyltransferase, Flavin containing amine oxidoreductase GRMZM2G4023680.01下调DownSUR2，鞘氨醇C4型单加氧酶2SUR2, sphinganine C4-monooxygenase 2 GRMZM2G0301180.11下调DownSUR2，鞘氨醇C4型单加氧酶2SUR2, sphinganine C4-monooxygenase 2 GRMZM2G3464550.46下调Downα-半乳糖苷酶，参与植物细胞壁Alpha-galactosidase 1, involved in plant-type cell wall GRMZM2G0951262.66上调Upα-半乳糖苷酶，参与植物细胞壁Alpha-galactosidase 1, involved in plant-type cell wall GRMZM2G1059222.27上调UpGBA2，非溶酶体葡糖神经酰胺酶GBA2, non-lysosomal glucosylceramidase MAP激酶MAP kinaseGRMZM2G13590413.57上调UpMAPK12，丝裂原激活蛋白激酶家族蛋白MAPK12, MAP kinase family protein GRMZM2G4598248.117上调UpMAPKKK2，丝裂原激活蛋白激酶激酶激酶ANP1MAPKKK2, MAP kinase kinase kinase ANP1-like GRMZM5G8783793.00上调UpMAPKK4，丝裂原激活蛋白激酶家族蛋白MAPKK4, MAP kinase family protein GRMZM2G0646012.49上调UpMAPKKK，丝裂原激活蛋白激酶激酶激酶YODAMAPKKK, MAP kinase kinase kinase YODA GRMZM2G0627612.04上调UpMAPK17，丝裂原激活蛋白激酶家族蛋白MAPK17, MAP kinase family protein AP1类AP1-likeGRMZM2G05578217.58上调UpMADS-box型转录因子27MADS-box transcription factor 27-like GRMZM2G1477163.78上调UpAGL8/AP1，K-box和MADS-box型转录因子家族AGL8/AP1, K-box region and MADS-box transcription factor family GRMZM2G0700342.88上调UpMADS-box型转录因子56MADS-box transcription factor 56-like LFY 类LFY-likeGRMZM2G1628140.25下调Down多叶，花分生组织蛋白，影响花和叶片的发育Leafy, floral meristem identity protein, affect flower and leaf development
FC：表达量差异倍数（NL409/WB665）FC：Differential multiple of expression (NL409/WB665)http://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/3aeb498fae92f7ec7462eaf3bc509cd4.png 图5 差异表达基因显著富集的分子功能Fig. 5 Molecular function enriched analysis of DEGs2.3 荧光定量PCR验证RNA-Seq结果研究表明多种转录因子参与叶细胞分化及叶片形态建成，如YABBY、MADS类转录因子决定叶原细胞命运向不同方向分化形成不同组织，GRF类调控特定部位细胞的增殖和生长；ARF、SBP、TCP、Myb、HB类调控叶细胞的分化和维管的形成；同时ARF、ARR、GRAS类在叶片发育过程分别参与生长素、细胞分裂素、赤霉素信号的响应和生物合成，影响叶片形态建成。为了进一步验证RNA-Seq结果的准确性，选取与叶片发育密切相关的18个不同的基因进行了qRT-PCR定量分析。对qRT-PCR与RNA-Seq结果进行相关性分析，发现两者的相关系数R2=0.962，说明RNA-Seq结果良好。3 讨论3.1 叶片发育密切相关的代谢通路多条路径显著富集到大量与玉米叶片发育相关的基因。拟南芥PFL（POINTED FIRST LEAVES）和AE（Asymmetric Leaves Enhancer）分别编码不同亚族的核糖体蛋白（PRS），与rRNA形成蛋白复合物促进叶片近轴组织的建立。本文核糖体路径显著富集到多个PFL和AE（电子附表1），并鉴定出玉米PRS13（PFL2）启动子区域存在显著差异（电子附图1），其在窄叶中下调表达的作用模式（图7）与拟南芥ae5、ae6缺失突变导致叶柄变长、叶片变窄的作用机理相似。鞘脂类（Sphingolipid）是细胞膜骨架及脂蛋白的重要组成成分，鞘脂代谢在细胞生长、不同功能细胞分化、细胞内以及细胞间信号传递发挥重要作用，鞘脂合成紊乱含量较低时，能够激活MAPK（mitogen-activated protein kinase）路径和转录因子AP1（APETALA1），进而调控细胞增殖、分化。AP1抑制拟南芥花萼叶和茎叶形成，LFY（LEAFY）能够解除AP1的抑制作用，促进叶片发育。本文鞘脂代谢路径富集到的基因大部分下调表达（表4），AP1类和MAPK类基因表达上调（表4），LFY表达下调（表4），与窄叶发育受抑制的表型一致。研究人员分析叶片发育的动态转录组学，不仅发现大量激素信号基因，细胞壁合成前体、纤维素合成酶、蛋白酶体、翻译后修饰基因也被大量检测到。Bolduc等解析KN1调控网络，不仅在叶片和分生组织中发现大量激素合成和响应基因，而且富集到脂类代谢、次生代谢、细胞壁合成和调控RNA转录相关的基因。叶片发育过程不同部位、不同时期的蛋白质组学分析，也富集到大量的尿苷二磷酸-葡糖基/葡聚糖转移酶、细胞壁前体、细胞壁受体激酶、纤维素合成酶和RNA转录相关的基因，本文显著富集的路径与前人的研究结果一致。由此可知，上述显著富集的代谢通路（表3）与叶片发育密切相关，这些代谢路径中的基因可能参与叶宽、叶长的形态建成。http://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/a86d7f0e2d7b35bc2f65bff4cfd8ebb5.jpg Tryptophan metabolism：色氨酸代谢；Auxin：生长素；Ubiquitin mediated proteolysis：泛素介导的蛋白水解；Cell enlargement：细胞增大；Plant growth：植物生长；Zeatin biosynthesis：玉米素的生物合成；Cytokinine：细胞分裂素；Cell division：细胞分裂；Shoot initiation：根的起始；Diterpenoid biosynthesis：二萜类化合物的生物合成；Gibberellin：赤霉素；Stem growth：茎的生长；Induced germination：诱导萌发；Carotenoid biosynthesis：类胡萝卜素的生物合成；Abscisic acid：脱落酸；Stomatal closure：气孔闭合；Seed dormancy：种子休眠；Cysteine and methionine metabolism：半胱氨酸和蛋氨酸代谢；Ethylene：乙烯；Endoplasmic reticulum(ER)：内质网；Fruit ripening：果实成熟；Senescence：衰老；Brassinosteroid biosynthesis：油菜素内酯生物合成；Brassinosteroid：油菜素甾醇；Proteasomal degradation：蛋白酶体降解；Cell elongation：细胞伸长；Alpha-Linolenic acid metabolism：α-亚麻酸代谢；Jasmonic acid：茉莉酸；Monoterpenoid biosynthesis类单萜生物合成；Indole alkaloid biosynthesis：吲哚生物碱生物合成；Stress response：应激反应；Phenylalanine metabolism：苯丙氨酸代谢；Salicylic acid：水杨酸；Disease resistance：抗病性。红色表示富集到的差异基因表达上调，绿色表示基因下调Red indicates that DEGs are up-regulated, and green indicates down-regulation of DEGs图6 植物激素信号转导Fig. 6 Plant hormone signal transduction under leaf development for 7 leaf stage between narrow leaf sample and wide blade sample表5 植物激素信号转导路径相关基因Table 5 Some genes related to plant hormone signal transductionhttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/0666c16ccfbee3abf94bf44682e90a72.jpg&q=30
http://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/2a96366585a31cbc100f8c62cc4af660.jpg&q=30 激素Hormones基因编号Gene ID差异倍数FC（T7/C7）基因描述/注释Gene description/annotation 生长素信号Auxin signaling GRMZM2G0307103.38上调UpARF，类生长素响应因子15，调控转录ARF, Auxin response factor 15-like, regulation of transcription GRMZM2G0782742.54上调UpARF，类生长素响应因子3，调控转录ARF, Auxin response factor 3-like, regulation of transcription GRMZM5G8741632.07上调UpARF，类生长素响应因子3，激活生长素信号通路ARF, Auxin response factor 3-like, auxin-activated signaling pathway GRMZM2G3613760.00下调DownARF，类生长素响应因子15，细胞响应生长素刺激ARF, Auxin response factor 15-like, cellular response to auxin stimulus GRMZM5G85347970.68上调Up生长素响应蛋白IAA4，细胞响应生长素刺激Auxin-responsive protein IAA4, cellular response to auxin stimulus GRMZM2G1041767.53上调Up生长素响应蛋白IAA2，激活生长素信号通路Auxin-responsive protein IAA2, auxin-activated signaling pathway GRMZM2G0773564.12上调Up生长素响应蛋白IAA13，激活生长素信号通路Auxin-responsive protein IAA13, auxin-activated signaling pathwa GRMZM2G0799573.86上调Up生长素响应蛋白IAA31，激活生长素信号通路Auxin-responsive protein IAA31, auxin-activated signaling pathway GRMZM2G0744270.12下调Down生长素响应蛋白IAA18，细胞响应生长素刺激Auxin-responsive protein IAA18, cellular response to auxin stimulus GRMZM2G0792000.01下调Down生长素响应蛋白IAA23，激活生长素信号通路Auxin-responsive protein IAA23, auxin-activated signaling pathway GRMZM2G0595440.02下调Down生长素响应蛋白IAA25，细胞响应生长素刺激Auxin-responsive protein IAA25, cellular response to auxin stimulus GRMZM2G4292544.12上调UpSAUR家族生长素响应蛋白SAUR32，生长素代谢SAUR family auxin-responsive protein SAUR32, auxin metabolism GRMZM2G3915963.46上调UpSAUR家族生长素响应蛋白SAUR36，生长素代谢SAUR family auxin-responsive protein SAUR36, auxin metabolism GRMZM2G3619930.34下调DownSAUR家族生长素响应蛋白SAUR32，生长素代谢SAUR family auxin-responsive protein SAUR32, auxin metabolism GRMZM2G4756830.24下调DownSAUR家族生长素响应蛋白SAUR12，生长素代谢SAUR family auxin-responsive protein SAUR12, auxin metabolism GRMZM2G4105672.35上调Up生长素响应GH3基因家族，响应生长素刺激Auxin-responsive GH3 gene family, response to auxin stimulus GRMZM2G0670224.17上调UpAUX1，类生长素转运蛋白1，跨膜转运体AUX1, Auxin transporter-like protein 1, transmembrane transporter GRMZM2G1279492.00上调UpAUX1，类生长素转运蛋白1，跨膜转运体AUX1, Auxin transporter-like protein 1, transmembrane transporter 油菜素内酯信号BR signaling GRMZM2G1587750.39下调DownBRI1，油菜素内酯不敏感1，膜受体，激活BZR1BRI1, brassinosteroid insensitive 1, membrane receptor, activation of BZR1 AC194970.5_FG0020.27下调DownBZR1，芸苔素唑拮抗1，油菜素内酯信号转导BZR1, brassinazole-resistant 1, brassinosteroid signal transduction GRMZM2G1270500.49下调DownBSK，油菜素内酯信号激酶，膜受体，BRI1激酶底物BSK, BR-signaling kinase, membrane receptor, substrates of BRI1 kinase GRMZM2G1642240.31下调DownBSK，油菜素内酯信号激酶，膜受体，BRI1激酶底物BSK, BR-signaling kinase, membrane receptor, substrates of BRI1 kinase GRMZM2G0546340.17下调DownBSK，油菜素内酯信号激酶，膜受体，BRI1激酶底物BSK, BR-signaling kinase, membrane receptor, substrates of BRI1 kinase AC210669.3_FG0010.05下调Down木葡聚糖转移酶TCH4，细胞伸长和扩展xyloglucosyl transferase TCH4, cell elongation and expansion 续表5 Continued table 5 激素Hormones基因编号Gene ID差异倍数FC（T7/C7）基因描述/注释Gene description/annotation 油菜素内酯合成BR biosynthesisGRMZM2G10377311.89上调Up油菜素内酯-6-氧化酶2，细胞膜固有成分Brassinosteroid-6-oxidase 2, intrinsic component of membrane GRMZM2G1071995.83上调UpD11，细胞色素P450，细胞膜固有成分，氧化还原活性D11, cytochrome P450, integral component of membrane, oxidoreductase activity GRMZM2G1432355.57上调UpD2，类固醇3-氧化酶，细胞膜部分，氧化还原活性D2, steroid 3-oxidase, membrane part, oxidoreductase activity GRMZM2G0328962.78上调UpD2，类固醇3-氧化酶，细胞膜固有成分，氧化还原活性D2, steroid 3-oxidase, intrinsic component of membrane, oxidoreductase activity GRMZM2G0656350.35下调DownDWF4，类固醇22-α-羟化酶，细胞膜固有成分DWF4, steroid 22-alpha-hydroxylase, intrinsic component of membrane GRMZM2G1627370.04下调DownCPD，细胞色素P450，细胞膜固有成分，氧化还原活性CPD, cytochrome P450, integral component of membrane, oxidoreductase activity GRMZM5G8723680.03下调DownBR6OX2，油菜素内酯-6-氧化酶2，细胞膜固有成分BR6OX2, brassinosteroid-6-oxidase 2, intrinsic component of membrane 细胞分裂素信号CTK signaling GRMZM5G88491473.11上调Up双组分响应调节器ARR-B家族，响应刺激Two-component response regulator ARR-B family, response to stimulus GRMZM2G4099742.32上调Up双组分响应调节器ARR-B家族，响应刺激Two-component response regulator ARR-B family, response to stimulus GRMZM2G0136122.11上调Up类双组分响应调节器ARR-10，调控转录Two-component response regulator ARR10-like, regulation of transcription 赤霉素信号GAsGRMZM2G0130166.80上调UpGAI1型DELLA蛋白，膜上细胞器，信号转导DELLA protein GAI1-like, membrane-bounded organelle, signal transduction 赤霉素代谢相关基因GAmrgGRMZM2G0737790.38下调DownGRAS家族参与赤霉素代谢，调控植株生长与发育GRAS family, as a player in GA metabolism, regulate plant growth and development GRMZM2G0738050.29下调DownGRAS家族参与赤霉素代谢，调控植株生长与发育GRAS family, as a player in GA metabolism, regulate plant growth and development GRMZM2G0985170.23下调DownGRAS家族参与赤霉素代谢，调控植株生长与发育GRAS family, as a player in GA metabolism, regulate plant growth and development GRMZM2G0182540.04下调DownGRAS家族参与赤霉素代谢，调控植株生长与发育GRAS family, as a player in GA metabolism, regulate plant growth and development GRMZM2G0703710.00下调DownGRAS家族参与赤霉素代谢，调控植株生长与发育GRAS family, as a player in GA metabolism, regulate plant growth and development GRMZM2G1105793.02上调UpGRAS家族参与赤霉素代谢，调控植株生长与发育GRAS family, as a player in GA metabolism, regulate plant growth and development GRMZM2G0363400.42下调DownGA 3氧化酶1，赤霉素3-β-双加氧酶活性GA 3-oxidase 1, gibberellin 3-beta-dioxygenase activity GRMZM2G0069640.3下调DownGA 2氧化酶6，赤霉素2-β-双加氧酶活性GA 2-oxidase 6, gibberellin 2-beta-dioxygenase activity GRMZM2G3235040.37下调DownGA受体GID1L2，羟基亚甲丁酸吡喃葡糖转化酶GA receptor GID1L2, tuliposide A-converting enzyme GRMZM2G0496750.29下调DownGA受体GID1L2，羟基亚甲丁酸吡喃葡糖转化酶GA receptor GID1L2, tuliposide A-converting enzyme GRMZM2G1563100.09下调DownGA受体GID1L2，羟基亚甲丁酸吡喃葡糖转化酶GA receptor GID1L2, tuliposide A-converting enzyme AC203966.5_FG0050.21下调DownGA 20氧化酶1，调控叶形和矮化GA 20 oxidase 1, regulate leaves shape and dwarfism
FC：表达量差异倍数（NL409/WB665）；BR：油菜素内酯Brassinosteroid；CTK：细胞分裂素Cytokinine；GAs：赤霉素信号Gibberellin signaling；GAmrg：赤霉素代谢相关基因Gibberellin metabolism related geneshttp://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/087ce2a3fe1a5b3c4d326c453d5ac498.png 图7 RNA-Seq与qRT-PCR结果比较Fig. 7 Comparison of results between RNA-Seq and qRT-PCR本研究发现油菜素内酯信号转导路径显著富集的基因全部下调（表5），不仅检测到前人已研究表达量降低的BZR类和BRI类基因，还发现BR路径中BSK类、TCH类基因也下调表达。生长素信号转导路径富集到的基因大部分上调表达（表5），其中ARF（GRMZM2G078274、GRMZM2G030710和GRMZM5G874163）、AUX-IAA（GRMZM2G104176、GRMZM2G077356、GRMZM2G079957、GRMZM2G074427）和Auxin transporter（GRMZM2G127949）在KN1调控叶片发育过程中发挥着重要作用。此外，DELLA高表达时，抑制GA信号及叶基部细胞的生长，本文DELLA家族蛋白（GRMZM2G013016）在近基部窄叶中高表达（表5，图6），且赤霉素代谢相关的基因几乎全部下调表达（表5），这与窄叶发育受抑制的结果一致。由此推断油菜素内酯、生长素、赤霉素在叶片发育过程发挥重要作用。3.2 不同激素途径发掘的叶形重要功能基因VOCs监测预警云计算平台的设计与实现·············张 涛 蒋晓濛 焦 正 (5,791) TCP类转录因子通过AUX、BR、GA、CTK等多种激素调控叶片、花发育以及植株形态建成等生长过程。叶片发育过程TCP1可调控DWARF4影响拟南芥叶片及植株形态，而BRI是tcp1发挥功能所必需的；此外，多个AtTCP负向调控叶片边缘区细胞分化增殖，其中TCP3、TCP15分别负调控AUX1、PIN1和YUCCA1、YUCCA4影响生长素分布及含量。过量表达TCP II型基因LANCEOLATE (LA)使西红柿和拟南芥的叶片变小，或表现卷曲叶，拟南芥TCP4的同源基因CYC/TB1（AC205574.3_FG006）在窄叶中上调表达的作用模式（图7，电子附表1）与前人研究结果一致。因此，推测CYC/TB1可能影响多种激素调控玉米叶片发育。激素BR可影响玉米叶片形态建成。AtROT3编码一种细胞色素P450蛋白，作用于叶片纵向极性细胞的伸长生长，其功能缺失影响拟南芥叶柄变短，叶片变大变宽，拟南芥BR合成相关的P450家族det2和dwarf4突变后影响叶细胞变短变少。VP1（VIVIPAROUS1）、VAL（VP1/ABI3-LIKE）、RAV1（Related to ABI3/VP1）、RAVL1（Related to ABI3/ VP1-Like）分别编码不同的B3结构DNA结合蛋白，拟南芥val缺失突变后影响叶片变小且赤霉素含量降低，RAV1作为BR受体负向调控拟南芥莲座叶的生长，与本文中VAL-like下调和RAV-like表达上调（电子附表2），以及赤霉素路径基因下调表达的结果一致（表5）。RAVL1通过E-box（CACCTG/ CACGTG/CACATG）结合位点激活BR响应和合成基因的表达，维持BR平衡对生长的调节作用，本文中BR响应基因的启动子区域存在大量该元件（电子附图4—9），说明植物激素BR与叶片发育密切相关。此外，ROT3（GRMZM2G143235）和VP1-like（GRMZM2G344521）在叶宽形成的关键部位上调表达（图7），且位于已定位的叶宽区间内，同时ROT3启动子区域的序列差异（电子附图2），表明ROT3和VP1-like可能在叶长和叶宽发育过程中发挥着重要作用，由此推测BR通过某种途径影响玉米叶长、叶宽的形成。生长素合成、分布异常影响叶片形态。玉米NAL7-like与水稻NAL7（OsYUC7）和NAL1（OsYUC1）同源，NAL1、NAL2、NAL3和NAL7编码一种生长素合成酶，调控叶原基的形成，其功能缺失突变影响叶细胞的分裂和维管束的形成，导致水稻叶片变窄，与本文NAL7-like在窄叶中下调表达（图7）的作用模式一致。RGD2编码一种AGO7型蛋白，是合成ta-siARF所必需的，RGD2通过ta-siARF调控ARF的转录，影响玉米叶片腹背面生长素分布，导致rgd2突变体叶片变窄卷曲。此外，NAL7-like位于叶宽研究的热点区域，而且其启动子区域序列存在显著差异（电子附图3）。由此可知，NAL7-like（GRMZM2G480386）、AGO7-like（GRMZM5G892991）可能通过影响生长素调控窄叶的形成。玉米叶片发育过程大量与生长素相关的基因参与其形成。玉米KN1的ChIP分析富集大量生长素AUX-IAA和ARF基因，转录组分析发现生长素基因（ARF12、ARF3）在玉米叶细胞朝不同功能区生长的过渡区上调表达，与本研究中ARF12、ARF3（图7）表达结果一致。拟南芥ARF受叶原基近轴端特异表达tasiR-ARF的抑制，在近轴端不表达，在远轴端KAN1（KANAD1）与ARF3、ARF4相互作用的同时，影响YUC2、YUC5和YUC8表达上调，促进叶原细胞的分化及远轴组织的形成；miR165/166抑制HD-ZIP lll类基因PHB、PHV、REV在叶原基远轴端的表达，促进近轴组织的形成。ARF3、KN1-like和KAN3（图7）位于已定位的叶宽位点内。此外，ARF12、GH5、REV-like（图7）分别受miR167、miR164、miR166调控并且与叶长、叶宽位点显著相关，由此可知KN1-like（GRMZM2G017709）、ARF3（GRMZM5G874163）、KAN3（GRMZM2G175827）、ARF12（GRMZM2G078274）、GH5（GRMZM2G055585）、REV-like（GRMZM2G029692）在叶片发育过程可能发挥特定作用。植物激素间的的相互作用可调节叶片发育。BR信号路径中的BIN2激酶能够磷酸化ARF2，导致其结合Auxin响应元件的功能丧失，影响拟南芥叶片等器官增大，同时BR能够诱导BZR1靶基因ARF2的表达。BZR1与ARF6相互作用促进拟南芥下胚轴变长。Auxin能够诱导拟南芥DWARF4表达，同时DWARF4通过BR路径促进细胞分裂生长。此外，Auxin还可促进OsBRI1表达，加强BR信号转导，而拟南芥BRI1通过tcp影响生长素促进细胞膨大生长，ARF能够特异结合Auxin响应元件（TGTCTC），本文BR响应基因启动子富含该元件（电子附图4—电子附图9），而且BR响应基因全部下调，Auxin响应基因上调（表5，图7），这暗示本文中ARF2-like（GRMZM2G030710）、ARF6-like（GRMZM2G078274）、DWARF4-like（GRMZM2G065635）、BRI1-like（GRMZM2G15877）（表5，电子附表1）可能介导Auxin和BR的平衡来调节植物器官以及叶片的发育。HB类转录因子包含多个KNOX、bHLH和HD-ZIP lll基因。KNOTTED1（KN1）和KNOX7属于KNOX家族，在顶端分生组织特定表达，调控细胞分化向膨大生长的过渡，影响叶片多维空间的发育，尤其在基部-末端轴向的形成过程中，KN1与LG3、LG4、KNOX8相互作用调控叶片发育。KNOX蛋白能够促进细胞分裂素（CTK）合成基因上调，同时抑制GA3和GA20氧化酶合成，促进SAM细胞分裂形成叶原基；提高GA含量可以解除DELLA对叶片发育的抑制作用。与本文中KNOX家族基因（KN1-like和KNOX7）（图7）、CTK路径基因（表5）、DELLA基因上调（表5），GA氧化酶基因下调（表5）影响叶片变窄的结果一致。GA可通过DELLA蛋白负向调控CTK响应基因进而影响CTK变化。拟南芥GA信号的抑制因子SPY（SPINDLY）能够正向调控CTK促进植株及叶片的发育。由此可知KN1-like（GRMZM2G017709）和KNOX7（GRMZM2G433591）可能通过影响细胞分裂素与赤霉素的平衡参与玉米窄叶的形成。生长调节因子GRF的互作因子AN3/GIF1作为叶片发育过程的调节开关，主要影响叶细胞横向分裂，分析其调控机理，在叶片基部分裂区富集到大量上调表达的GRF基因，在叶部伸长区仅发现GRF4和GRF10高量表达，其他GRF基因在叶部伸长区表达量逐渐降低，过量表达ZmGRF1和ZmGRF10使玉米叶片变小，与本文中检测到GRF6（GRMZM2G041223）和GRF13（GRMZM2G018414）在窄叶叶片发育的过渡区域上调表达（图7）的趋势一致，同时GRF6和GRF13位于叶宽研究的热点区域。LG2（GRMZM2G143235）编码一种TGA结合域的碱性亮氨酸拉链蛋白，主要在SAM和叶片与叶鞘连接处表达，作用于叶基部叶耳、叶舌的形成，影响叶片的发育。ZmARF2（GRMZM2G082836）是ADP核糖基化转录因子，能够调控拟南芥叶面积和株高。由此推断GRF6、GRF13、LG2、ZmARF2（图7）在玉米叶片发育以及叶宽形成过程中可能发挥重要作用。柔性直流电网串联直流潮流控制器及其控制策略研究………………………………………………………… 徐烁（12-88） 4 结论明确了一些与玉米叶片发育密切相关的代谢路径，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Auxin与BR、CKT与GA之间的相互作用对调控叶片形态可能发挥重要作用。References 明博, 谢瑞芝, 侯鹏, 李璐璐, 王克如, 李少昆. 2005—2016年中国玉米种植密度变化分析. 中国农业科学, 2017, 50(11): 1960-1972.MING B, XIE R Z, HOU P, LI L L, WANG K R, LI S K. 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XIE Y, STRAUB D, EGUEN T, BRANDT R, STAHL M, Jaime F. MARTINEZ-GAECIA J F, WENKEL S. Meta-analysis of Arabidopsis KANADI1 direct target genes identifies a basic growth-promoting module acting upstream of hormonal signaling pathways. Plant physiology, 2015, 169(2): 1240-1253. EMERY J F, FLOYD S K, AIVAREZ J, ESHED Y, HAWKER N P, IZHAKI A, BAUM S F, BOWMAN J L. Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes. Current Biology, 2003, 13(20): 1768-1774. OKUSHIMA Y, MITINA I, QUACH H L, THEOLOGIS A. AUXIN RESPONSE FACTOR 2 (ARF2): a pleiotropic developmental regulator. The Plant Journal, 2005, 43(1): 29-46. SCHRUFF M C, SPIELMAN M, TIWARI S, ADAMS S, FENBY N, SCOTT R J. The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs. Development, 2006, 133(2): 251-261. TIAN H Y, LV B S, DING T T, BAI M Y, DING Z J. Auxin-BR interaction regulates plant growth and development. 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WANG Q L, XUE X J, LI Y L, DONG Y B, ZHANG L, ZHOU Q, DENG F, MA Z Y, QIAO D H, HU C H, REN Y L. A maize ADP-ribosylation factor ZmArf2 increases organ and seed size by promoting cell expansion in Arabidopsis. Physiologia plantarum, 2016, 156(1): 97-107.Explore Regulatory Genes Related to Maize Leaf Morphogenesis Using RNA-SeqGUO ShuLei1,2, LU XiaoMin1, QI JianShuang1, WEI LiangMing1, ZHANG Xin1, HAN XiaoHua1, YUE RunQing1, WANG ZhenHua1, TIE ShuangGui1, CHEN YanHui2(1 Cereal Crops Institute, Henan Academy of Agricultural Sciences/Henan Provincial Key Lab of Maize Biology, Zhengzhou 450002; 2College of Agronomy, Henan Agricultural University, Zhengzhou 450046)Abstract:【Objective】Leaf shape characteristics are one of important agronomic traits that determine plant morphology and affect 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 7th 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.Key words: maize (Zea mays); leaf width; leaf length; RNA-Seq; morphogenesis; regulatory genedoi: 10.3864/j.issn.0578-1752.2020.01.001开放科学（资源服务）标识码（OSID）：http://rtt.5read.com/pdgpath/format?f=513b69a41df69a545bd2023e1d495fb6/df8fffb7075f935583da74b88191dc5e.jpg收稿日期：2019-05-20；接受日期：2019-07-11基金项目：中国博士后科学基金（2017M612404）、河南省科技攻关计划项目（182102110122）、河南省农业科学院优秀青年科技基金联系方式：郭书磊，E-mail：guosl1309@163.com。通信作者王振华，E-mail：wzh201@126.com。通信作者铁双贵，E-mail：tieshuangg@126.com。通信作者陈彦惠，E-mail：chy9890@163.com（责任编辑 李莉）
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