黄花棘豆响应非生物胁迫转录组分析及干旱胁迫信号通路的研究

发布时间：2018-01-06 08:07

本文关键词：黄花棘豆响应非生物胁迫转录组分析及干旱胁迫信号通路的研究　出处：《西北大学》2016年博士论文　论文类型：学位论文

【摘要】：黄花棘豆(Oxytropis ochrocephala Bunge)是豆科棘豆属多年生草本植物,其植株富含生物碱等有毒成分,动物大量采食后会引起中毒甚至死亡,其分布面积广,蔓延迅速,容易形成优势群落。目前关于黄花棘豆的研究主要集中在成分鉴定、毒理分析以及化感作用等方面,在分子水平上对其环境适应性和抗逆机理的研究十分有限。为了明确黄花棘豆逆境生理特征及抗逆分丫机制,以便制定有效可行的防控措施,本研究以黄花棘豆幼苗为材料,利用高通量测序技术获得黄花棘豆响应干旱、盐、低温胁迫的转录组信息,并利用生物信息学方法筛选一系列逆境响应功能基因,在转录水平上分析功能基因的表达模式；同时重点研究了在干旱胁迫下黄花棘豆内源信号分子NO参与依赖ABA的信号通路,调控植株干旱适应性的过程,为研究黄花棘豆的抗逆机制提供理论依据。主要研究结果如下：(1)黄花棘豆为中等抗旱耐低温植物,能在低盐环境中生长。低温胁迫的植株电导率和丙二醛含量低于干旱和盐处理植株,并积累更多脯氨酸。在干旱和低温胁迫过程中,植株SOD和POD酶活性总体呈上升趋势；在盐胁迫下,SOD和POD活性呈现先增加后降低的趋势。由于植物的生理指标变化通常滞后于基因表达,选择三种胁迫处理12h内的植株,提取RNA进行转录组测序。(2)利用HiSeq2000测序平台对黄花棘豆四个样本进行测序,共获得23.22G总数据并上传至NCBI BioProject(登录号：PRJNA292613)。经Trinity软件拼接组装后获得217,270个transcripts和118,596个unigenes,其中注释到植物物种中的unigenes有88,942个,与黄花棘豆亲缘关系较近的物种有鹰嘴豆、蒺藜苜蓿、大豆等。共10,465(11.76%)个基因注释到267条KEGG代谢通路。经过GO功能注释和KEGG代谢途径富集,三种胁迫下差异表达基因(DEGs)主要参与渗透调节、次生代谢、不饱和脂肪酸合成与代谢,植物激素合成及信号转导,氧化磷酸化、转录调控等途径。(3)在建立起黄花棘豆本地blast数据库的基础上,筛选12个候选内参基因,并检测候选基因在干旱、盐、低温胁迫下的表达模式,利用geNorm、NormFinder和BestKeeper程序对基因的表达稳定性进行评估。经综合分析,在三种胁迫处理的黄花棘豆样本中ACT7、HIS、ACT101基因表达最稳定,18S、TUA、ACT11最不稳定。从DEGs中挑选30条unigenes进行qRT-PCR检测,其中24个unigenes在三种胁迫下的表达模式与转录组测序结果相符。(4)筛选黄花棘豆逆境响应功能基因并鉴定其表达特征,包括逆境蛋白、次生代谢、转录因子、植物激素以及钙信号相关基因。在逆境蛋白中,起分子伴侣作用的OoLEA、 OoDHN在三种胁迫下均显著上调表达；参与细胞壁组分合成的OoPRP、OoGS在干旱和盐胁迫中后期大量表达；参与植物防御反应的OoGLP在干旱胁迫下表达量最高；胁迫应激蛋白OoHSP70仅在盐胁迫下表达量上调。多个参与木质素及类黄酮合成途径的基因OoPAL、OoC4H、Oo4CL、OoCHR、OoADH在三种胁迫过程中上调表达。转录因子相关基因中OoAREB与ABA信号通路有关；OoMYB快速应答低温胁迫；OoNAC早于其他转录因子表达；OoWRKY和OoERF与黄花棘豆的低温响应有关。在三种胁迫下黄花棘豆内源激素含量总体向着气孔关闭,根系伸长,抗性增强的趋势变化,多个抗逆激素ABA、ETH、JA合成及信号转导相关基因上调表达。在低温胁迫。下多个钙信号相关基因表达量上调。(5)克隆黄花棘豆OoNCED和OoAREB基因编码区并进行生物信息学分析。OoNCED编码612个氨基酸,分子量为68.4kDa,等电点为8.18;OoAREB编码426个氨基酸,分子量为46.4 kDa,等电点为9.38。OoNCED和OoAREB编码蛋白整体表现为亲水性,不具有跨膜结构域和信号肽。OoNCED和OoAREB编码蛋白分别定位于叶绿体和细胞核中,二级结构主要是α-螺旋、β-折叠和无规则卷曲。通过序列同源性分析,黄花棘豆OoNCED和OoAREB氨基酸序列分别与柠条NCED1、ACU86971.1)和AREB(ABG90380.1)亲缘关系最近。(6)研究干旱胁迫下黄花棘豆中内源信号分子NO的动态平衡以及与ABA的相互关系。PEG诱导黄花棘豆根尖中NO急剧增加,主要分布于根毛、根系皮层细胞和维管组织。在黄花棘豆植株各部位NO的来源不同,根尖中主要有非酶途径和NR途径,在整株中NOA途径也参与NO的合成。OoNR、OoNOA、OoGSNOR共同调节黄花棘豆中NO的平衡。由PEG诱导的根尖中NO积累是暂时的,H202含量随着NO水平降低而增加,NO和H202作为信号分子在一定范围内保持平衡。干旱胁迫条件下,黄花棘豆ABA含量增加,ABA合成抑制剂FLU加深植株脂质过氧化程度,证明黄花棘豆中依赖ABA的抗旱机制。另外,FLU抑制了干旱胁迫下NO的积累,而内源NO清除剂cPTIO不影响ABA含量变化,证明NO是ABA的下游信号。同时当NO清除后植株MDA含量显著增加,说明干旱胁迫下黄花棘豆内源NO参与依赖ABA的信号通路进而减轻植株氧化损伤和脂质过氧化程度。
[Abstract]:O.ochrocephala (Oxytropis ochrocephala Bunge) is the Oxytropis perennial herbaceous plants, the plants rich in alkaloids and other toxic ingredients, a large number of animal feed will cause poisoning and even death, the distribution area of wide spread quickly, easy to form a dominant community. The current research on O.ochrocephala mainly focus on the identification and analysis of toxicological aspects. Allelopathy, at the molecular level on the study of environmental adaptability and resistance mechanism is very limited. In order to clarify the physiological characteristics and O.ochrocephala adversity resistance cents mechanism, in order to develop effective prevention and control measures, this research takes O.ochrocephala seedlings as materials, obtain O.ochrocephala in response to drought, salt and the use of high-throughput sequencing technology the transcriptome information, low temperature stress, and the use of bioinformatic methods a series of stress responsive genes, analysis of functional genes at the transcriptional level The expression pattern at the same time; studied under drought stress in O.ochrocephala endogenous signal molecules NO stress signaling pathways involved in ABA dependent process, regulation of Plant Drought Adaptability, to provide a theoretical basis for the research on the mechanism of anti inverse O.ochrocephala. The main results are as follows: (1) yellow Oxytropis was a moderate drought resistant low temperature resistant plants can grow in the low salt environment. Plant conductivity and MDA content of low temperature stress under drought and salt processing plants, and accumulated more proline. In the process of drought and cold stress in plants SOD and POD activity showed an increasing trend; under salt stress, SOD and POD activity increased first and then decreased as. The changes of physiological index of plant usually lags behind the expression of three kinds of stress in 12h plants, RNA was extracted for transcriptome sequencing. (2) using HiSeq2000 sequencing platform of yellow bean spine four Sequencing samples were obtained 23.22G data and uploaded to the NCBI of BioProject (accession number: PRJNA292613). The Trinity software assembly obtained after 217270 transcripts and 118596 unigenes, which notes unigenes to plant species in 88942, and a close relationship between the pro O.ochrocephala species chickpea, Medicago truncatula a total of 10465 soybean, et al. (11.76%) 267 KEGG pathway gene annotations to GO. After the functional annotation and metabolic pathways of KEGG enrichment, under the stress of three differentially expressed genes (DEGs) mainly involved in osmoregulation, secondary metabolism, unsaturated fatty acid synthesis and metabolism, plant hormone synthesis and signal transduction, oxidative phosphorylation, transcriptional regulation and other ways. (3) on the basis of O.ochrocephala local blast database on the screening of 12 candidate reference genes, and the detection of candidate genes in drought, salt, low temperature stress under the mode of expression Type, using geNorm, NormFinder and BestKeeper expression stability of genes program for evaluation. Through comprehensive analysis, in the three ACT7 stress treatment O.ochrocephala samples HIS, ACT101 gene expression is the most stable, 18S, TUA, ACT11 of the most unstable. From the DEGs to select 30 unigenes and 24 of them were detected by qRT-PCR. Unigenes in three under the stress of the expression pattern and transcriptome sequencing results. (4) screening O.ochrocephala stress response genes and identify their expression characteristics, including stress protein, secondary metabolism, transcription factors, hormones and calcium signaling related genes. The stress protein, molecular chaperone OoLEA, OoDHN three kinds of stress were significantly up-regulated; participate in cell wall components in the synthesis of OoPRP, OoGS in drought and salt stress in the expression; involved in plant defense responses to OoGLP in the highest expression level under drought stress; Stress protein OoHSP70 only under salt stress. The expression of OoPAL gene, more involved in lignin and flavonoid synthesis pathway of OoC4H, Oo4CL, OoCHR, OoADH rise in the three kinds of stress in the process of expression of transcription factor related genes OoAREB and ABA signaling pathway; OoMYB fast response to low temperature stress; the expression of OoNAC in early in other transcription factors; OoWRKY and OoERF and O.ochrocephala temperature response. In three under the stress of overall endogenous hormone content of Oxytropis ochrocephala to stomatal closure, root elongation, trend enhanced resistance, multiple resistant hormone ABA, ETH, JA expression synthesis and signal transduction related genes up-regulated. Under low temperature stress. Calcium signal of multiple gene expression. (5) the bioinformatics analysis of.OoNCED encoding 612 amino acids was cloned O.ochrocephala OoNCED and OoAREB gene encoding region and molecular weight of 68.4kDa and isoelectric point 8.18; OoAREB encoding 426 amino acids. The molecular weight of 46.4 kDa and isoelectric point of 9.38.OoNCED protein and OoAREB encoding for the overall performance of hydrophilic, has no transmembrane domain and signal peptide encoding.OoNCED and OoAREB proteins were localized in the chloroplast and nucleus, two level structure mainly alpha helices, beta folding and random coil. Through sequence analysis, OoNCED and OoAREB amino acid sequences of O.ochrocephala respectively with NCED1 ACU86971.1 and AREB) of Caragana, (ABG90380.1) the closest genetic relationship. (6) study on the dynamic balance of endogenous signal under drought stress in Oxytropis ochrocephalabunge molecules NO and.PEG and the relationship between ABA induced yellow spines root tip NO increased sharply, mainly distributed in the root tube root cortical cells and vascular tissue. In the different sources of O.ochrocephala plants of each part of NO, a non enzymatic pathway and NR pathway mainly in root, in the whole plant NOA pathways are also involved OoNOA and synthesis of.OoNR, NO, OoGSNOR in NO regulated O.ochrocephala balance. Root tip induced by PEG in NO accumulation is temporary, the content of H202 increased with the decrease of NO level, NO and H202 as signal molecules to maintain balance in a certain range. Under drought stress, increase O.ochrocephala content of ABA, ABA the synthesis of inhibitors of FLU plants enhance the degree of lipid peroxidation, demonstrated ABA dependent mechanism of drought resistance in Oxytropis ochrocephala. In addition, FLU inhibited NO accumulation under drought stress, and endogenous NO scavenger cPTIO does not affect the change of the content of ABA, that NO is downstream of ABA signal. At the same time when the content of MDA was significantly increased after the removal of NO, explained under drought stress O.ochrocephala endogenous NO signaling pathways involved in ABA dependent and thus reduce oxidative damage and lipid peroxidation of plant.

【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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