玉米BRI1基因的功能分析及其响应ABA调控的蛋白组学研究

发布时间：2018-02-12 17:36

本文关键词： 玉米油菜素内酯 ABA 丙环唑 ZmBRI1　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：油菜素内酯(BRs)是一种重要的甾族类激素,在植物生长发育过程中起着重要的调控作用,同时也参与植物对逆境响应调控。论文针对玉米生产中增密或肥料管理不当等导致群体质量下降以及非生物逆境如干旱等影响植株生长等生产问题,利用BRs调节株型与响应逆境调控效应,开展BR抑制剂丙环唑(Pcz)调节玉米生长效应及其调控机制,为Pcz在玉米株型塑造上应用提供依据;同时,克隆玉米的BR受体基因ZmBRI1,分析其功能,研究了 BR信号表达响应ABA的蛋白质调控机制,为揭示BR与ABA互作响应逆境提供理论依据。主要研究结果如下:1)Pcz处理显著抑制玉米中胚轴与胚芽鞘的生长,降低了株高,缩短了叶片和叶鞘长度,减小了叶夹角,同时显著抑制了叶片和叶鞘细胞的纵向伸长,促使叶枕细胞排列由疏松变为紧密;降低了玉米中赤霉素(GA)的含量,下调了GA3ox 表达,上调了 GA失活的酶GA2ox5和GA2ox8表达,而GA合成酶GA20ox1的表达呈现先上调后下调模式;降低了油菜素内酯(BR)含量,但BR合成基因CPD和DWF4的表达上调,可能是由于反馈调节;下调了扩张蛋白EXZR4、EXPA5和木葡聚糖内糖基转移酶/水解酶基因XTH1、XET1的表达。2)利用同源克隆的方法,从玉米B73自交系中获得了油菜素内酯受体蛋白编码基因ZmBRI1,全长为3369bp,编码1122个氨基酸;亚细胞定位分析表明ZmBRI1蛋白定位于细胞膜上,且在玉米各个组织器官中都有表达;利用转基因技术将ZmBRI1导入拟南芥BR不敏感突变体bri1-5中,修复了突变体bri1-5的表型,如植株高度、叶片形态和果荚大小;修复了突变体bri1-5的BR信号,如根长、下胚轴和BR合成酶基因CPD、DWF4的表达;在ABA处理条件下,ZmBRI1超表达株系line2和line5显著提高了种子的萌发率和根长,且ABA响应基因RS29A、RD29B、ABI5和RAB18表达量降低,对ABA敏感性减弱。3)利用iTRAQ技术对野生型Ws、突变体bri1-5和ZmBRI1超表达株系line5在幼苗生长过程中响应ABA的蛋白质组进行了分析,共有5479个蛋白被鉴定,其中差异蛋白263个;鉴定出的差异蛋白涉及了初生代谢和次生代谢、能量和运输、细胞生长和分裂、蛋白合成和降解、转录和信号传导、胁迫响应和细胞结构等多种生物学途径;突变体bri1-5对ABA最为敏感,物质代谢、能量转化和蛋白合成平衡被打破,胁迫响应基因的表达上调;ZmBRI1超表达株系line5对ABA的敏感性最低,物质代谢相对稳定,胁迫蛋白上调的倍数较小,缓解了 ABA对种子萌发和幼苗生长的抑制作用。互作网络分析表明,BR信号主要是通过BIN2与ABI5进行互作,影响ABA信号途径的抗氧化酶系统、钙离子信号、MAPK激酶途径、以及ZIP转录因子等,从而抑制ABA信号下游基因的表达,促进种子萌发和幼苗生长。
[Abstract]:Brassinolide (BRs) is an important steroid hormone, which plays an important regulatory role in plant growth and development. At the same time, it is also involved in the regulation of plant response to stress. In this paper, the effects of abiotic stress, such as drought, on plant growth and other problems, such as increasing density or improper management of fertilizer in maize production, have led to the decline of population quality. Using BRs to regulate plant type and response to stress, the growth effect and regulation mechanism of maize were regulated by Br inhibitor Pcz, which provided the basis for the application of Pcz in maize plant shape. The Br receptor gene ZmBRI1 of maize was cloned and its function was analyzed. The protein regulation mechanism of Br signal expression in response to ABA was studied. In order to reveal the theoretical basis of the interaction between Br and ABA in response to stress, the main results were as follows: 1. Pcz treatment significantly inhibited the growth of maize mesocotyl and coleoptile, decreased plant height, shortened the length of leaf and leaf sheath, and reduced the angle between leaves. At the same time, the longitudinal elongation of leaf and leaf sheath cells was significantly inhibited, the arrangement of leaf occipital cells was changed from loose to tight, the content of gibberellin (GA) in maize was decreased, the expression of GA3ox was down-regulated, and the expression of GA2ox5 and GA2ox8, which was inactivated by GA, was upregulated. The expression of GA synthase GA20ox1 was up-regulated and then down-regulated, and the content of Br-R was decreased, but the expression of CPD and DWF4 was up-regulated by feedback regulation. The down-regulated expression of exzr4xyltransferase / hydrolase gene XTH1 XET1 and xylglucan intra glycosyltransferase / hydrolase gene XTH1 XET1 was down-regulated. By using homologous cloning method, a rapesinolactone receptor protein encoding gene ZmBRI1 was obtained from maize inbred line B73. The length of ZmBRI1 was 3369 BP, encoding 1122 amino acids. Subcellular localization analysis showed that ZmBRI1 protein was located on the cell membrane and expressed in all tissues and organs of maize, and ZmBRI1 was introduced into Arabidopsis thaliana Br insensitive mutant bri1-5 by transgenic technique to repair the phenotype of the mutant bri1-5, such as plant height. Leaf morphology and pod size, Br signal of mutant bri1-5, such as root length, Hypocotyl and the expression of Br synthase gene CPDN DWF4, were repaired. Under ABA treatment, line2 and line5 increased seed germination rate and root length significantly. The expression of ABA response gene RS29Agna RD29BHABI5 and RAB18 was decreased, and the sensitivity to ABA was weakened. 3) the proteome of wild type Ws, mutant bri1-5 and ZmBRI1 superexpression line line5 were analyzed during seedling growth, and a total of 5479 proteins were identified. The identified differential proteins are related to primary and secondary metabolism, energy and transport, cell growth and division, protein synthesis and degradation, transcription and signal transduction, The mutant bri1-5 was most sensitive to ABA, the balance of substance metabolism, energy transformation and protein synthesis was broken, and the expression of stress response gene up-regulated ZmBRI1 strain line5 had the lowest sensitivity to ABA. Substance metabolism was relatively stable, stress protein upregulation was small, which alleviated the inhibitory effect of ABA on seed germination and seedling growth. The interaction network analysis showed that the signal of Br was mainly interacted by BIN2 and ABI5. The antioxidant enzyme system, Ca ~ (2 +) signaling MAPK kinase pathway and ZIP transcription factors, which affect the ABA signaling pathway, can inhibit the expression of downstream genes of ABA signal and promote seed germination and seedling growth.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S513

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