不同四倍体泡桐干旱胁迫应答基因的鉴定与分析

发布时间：2018-03-10 03:01

  本文选题：同源四倍体泡桐　切入点：转录组　出处：《河南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：泡桐是我国速生用材和绿化树种,用途多,适应性强,分布广,具有重要的经济和生态价值。为满足人们的生产需要,我们利用秋水仙素成功诱导出了同源四倍体泡桐。之前研究显示同源四倍体泡桐具有很多优良特性比如抗性。干旱是影响泡桐的生物产量的非生物因素之一。泡桐的干旱应答是多水平的复杂调控,但分子机制还不清楚。本研究以白花泡桐二四倍体、南方泡桐二四倍体、毛泡桐二四倍体和豫杂一号泡桐二四倍体盆栽苗为试验材料,对其分别进行15天的25%和75%土壤含水量干旱处理。通过高通量转录组测序、s RNA测序和降解组测序,以白花泡桐基因组为参考序列,基于生物信息学分析mRNA和miRNA的表达变化,鉴定出差异表达的转录本和miRNA,同时利用降解组预测差异表达miRNA的靶基因。本研究主要结果如下:1、四种泡桐二四倍体构建的16个文库都具有较高的测序质量。clean read的GC含量都大于45.68%,Q20都大于94.94%,Q30都大于86.65%,N比例都为0%;clean read与泡桐基因组的比对率都大于79.83%,与泡桐基因比对率都高于44.89%。26个基因的实时荧光定量PCR表达趋势和高通量测序获得的表达趋势大致一致,说明测序的可靠性高。2、从四倍体白花泡桐、毛泡桐、南方泡桐和豫杂一号泡桐中鉴定出了302、453、441和372个与其二倍体的干旱应答差异基因。这些基因的Pathway富集在植物信号转导通路、淀粉和糖的代谢通路、苯丙烷的生物合成通路、谷胱甘肽的代谢过程、次生代谢物的合成代谢、过氧化物酶代谢途径和乙醇酸的代谢。两种水通道蛋白(PIP和TIP)和脱水蛋白在干旱胁迫后上调表达有利于泡桐根部对于水分的吸收。GO富集分析显示下调的转录本作用的细胞组分主要集中在光合单位的膜系统,发挥的分子功能富集在结合作用和催化功能,参与的生物过程多富集在了光合作用的光反应和暗反应过程。它们保护了泡桐的光合体系免受来自还原力和氧化电子的破坏,但减少了泡桐生物量的积累,其中尤为显著的就是碳水化合物的合成。但与二倍体相比较,这些基因在同源四倍体泡桐中下调的幅度较小,能够在干旱胁迫条件下积累较多的光合产物。3、通过sRNA测序,从不同倍性不同干旱处理的白花泡桐、毛泡桐、南方泡桐和豫杂一号泡桐分别鉴定出miRNA有180、169、178和246条,四个库中19、27、11、25个mi RNA预测到了28、23、16和46靶基因。s RNA测序水平上,MIR398、MIR171、MIR172、MIR159、Pau-mir-10、Ptf-mir68和Ptf-mir116差异表达,它们靶定的基因包括钾离子通道KAT1、Ca2+结合蛋白、脯氨酸-t RNA连接酶、葡聚糖合成酶、氨基酸透性酶、ABC转运家族、ABA受体和脯氨酸氨肽酶,参与逆境激素ABA的信号转导,铁氧合酶的活性调节,脯氨酸的合成代谢以及细胞内可溶性糖、可溶性蛋白的合成、运输和积累过程。维持泡桐体内渗透平衡和氧化平衡,最终提高泡桐在干旱逆境下根部的吸水能力,减少地上部分的失水量。综上所述,以泡桐基因组为背景,通过高通量测序和生物信息学鉴定和分析大量干旱应答相关的基因。植物激素ABA上调与干旱耐性相关,可溶性蛋白和糖类上调有利于维持细胞膨压和渗透压平衡与干旱耐性相关,抗氧化酶和非抗氧化酶(抗坏血酸酶和谷胱甘肽酶)上调维持细胞的氧化平衡和离子平衡相关。从不同层次、不同角度,研究和探讨了泡桐的干旱耐性相关的分子机制,为将来泡桐的分子育种奠定理论基础。
[Abstract]:Paulownia is a fast-growing timber tree species in China and, multiple purposes, strong adaptability, wide distribution, has important economic and ecological value. In order to meet the production needs of people, we successfully induced by colchicine in Autotetraploid Paulownia. Previous studies indicated that tetraploid Paulownia foam has many good characteristics such as resistance. The drought is one of the the non biological factors of Paulownia biomass. The drought response of Paulownia is a complex multi level regulation, but the molecular mechanism is not clear. In this study, p.fortunei 24 times, 24 times south of Paulownia, Paulownia 24 times and 24 times of Paulownia tomentosa * p.fortune body potted seedlings as the experimental materials, the were 15 days of drought treatment of water containing 25% and 75% soil. Through high-throughput transcriptome sequencing, s sequencing and RNA sequencing to Degradome, Paulownia genome reference sequence, based on Bioinformatics Analysis of the changes of the expression of mRNA and miRNA, identified differentially expressed transcripts and miRNA, at the same time the degradation group predicted the differentially expressed target genes of miRNA. The main results of this study are as follows: 1. The GC content of four species of Paulownia was 24 times as much as the construction of the 16 libraries have high quality.Clean read sequencing is higher than 45.68%. Q20 is greater than 94.94%, Q30 greater than 86.65%, the proportion of N is 0%; clean and read ratio of Paulownia genome rate is greater than 79.83%, and the gene expression trend of Paulownia comparison rate is higher than the real time fluorescence quantitative PCR 44.89%.26 gene expression trends and high-throughput sequencing was roughly consistent, indicating a high reliability.2 sequencing. From the tetraploid Paulownia, Paulownia, Paulownia australis and Paulownia tomentosa * p.fortune identified genetic differences between the 302453441 and 372 drought response and its diploid. These genes Pathway enrichment in plant signal transduction Pathway, metabolic pathway of starch and sugar, phenylpropanoid biosynthesis pathway, metabolism of glutathione, biosynthesis of secondary metabolites, the metabolic pathway of peroxidase and glycolate metabolism. Two kinds of water channel proteins (PIP and TIP) and dehydration protein after drought stress expression is conducive to root for the absorption of.GO enrichment analysis of Paulownia display cell group down transcripts function focused on membrane system of photosynthetic unit water, molecular function enrichment in binding and catalytic function, biological process in the enrichment in the light reaction of photosynthesis and dark reaction process. They protect the photosynthetic system from Paulownia reduction stress and oxidative damage of electronic, but reduced the biomass of Paulownia accumulation, which is particularly significant is the synthesis of carbohydrates. But compared with the diploid, the homologous gene in Small scale down of tetraploid Paulownia, will accumulate more in drought stress conditions of photosynthetic products in.3, through sRNA sequencing, from different times of different drought treatment of Paulownia, Paulownia, Paulownia australis and Paulownia tomentosa * p.fortune were identified with 180169178 miRNA and 246 19,27,11,25, MI RNA four a library to 28,23,16 46 and.S RNA sequencing of target gene level, MIR398, MIR171, MIR172, MIR159, Pau-mir-10, Ptf-mir68 and Ptf-mir116 expression differences, their target genes including potassium channel KAT1, Ca2+ binding protein, proline -t RNA ligase, glucan synthase, amino acid permease, ABC transporter family. ABA receptor and proline aminopeptidase, signal transduction involved in the stress hormone ABA, regulation of iron oxide synthase activity, proline metabolism and intracellular synthesis of soluble sugar, soluble protein, transport and accumulation Cheng. Maintain the osmotic balance of Paulownia and oxidative balance, and ultimately improve the water absorbing ability of Paulownia in roots under drought stress, reduce the amount of water on the ground part. To sum up, in the background of Paulownia genome, by high-throughput sequencing and bioinformatics identification and analysis of a large number of drought responsive genes related to plant hormone. ABA increased with drought tolerance correlation of soluble protein and carbohydrate increase helps to maintain cell turgor and osmotic balance and drought tolerance, antioxidant enzymes and non antioxidant enzymes (ascorbate peroxidase and glutathione peroxidase) up-regulated maintain oxidative balance and ion balance in cells. Different from the different levels and angles, studied and discussed the drought tolerance of Paulownia related molecular mechanism and lay the theoretical foundation for the molecular breeding of Paulownia in the future.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.43
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本文编号：1591448