小麦苗期盐胁迫相关转录组表达谱及蛋白质乙酰化和琥珀酰化修饰研究

发布时间：2018-05-13 10:28

  本文选题：普通小麦 + 盐胁迫　； 参考：《沈阳农业大学》2016年博士论文

【摘要】：小麦是我国第二大口粮作物,其产量和品质与国家粮食安全和人民生活水平息息相关。盐胁迫是小麦生产的主要限制因素之一,在转录和翻译水平上严重影响了小麦的光合作用、呼吸代谢、水分状况和各种酶的变化等生理生化过程。因此在分子水平上阐明小麦对盐胁迫的响应机理,鉴定耐盐关键基因,分析相关蛋白的修饰水平,对小麦抗逆育种具有重要的意义。试验以耐盐小麦品种青麦6号和盐敏感材料中国春为材料,对人工生长箱培养1周的小麦幼苗进行盐胁迫处理,利用Illumina测序平台对盐胁迫前后6、12、24和48h的小麦根系进行高通量测序,利用生物信息学方法对测序结果进行系统对比研究,并对部分同源基因响应盐胁迫时的差异表达进行了分析。同时,我们利用LC-MS/MS联用分析技术和生物信息学方法分析小麦乙酰化和琥珀酰化蛋白质参与调控的生理进程进行了研究。主要结果如下：1.从全基因组水平上研究了小麦根系响应盐胁迫的基因表达模式,鉴定到36804个差异表达基因,青麦6号和中国春盐胁迫前后基因表达存在显著差异。处理前青麦6号和中国春大约55.4%和50.1%的部分同源基因之间差异表达,而盐胁迫后其比例上升到62.6%和59.9%,其差异表达模式通过实时定量PCR得到了验证。通过分析盐胁迫后的上调表达的基因发现6h和12h盐胁迫处理后青麦6号上调基因数目少于中国春,但48h后显著高于中国春,下调表达基因的表达规律正好相反。基因GO富集分析显示,青麦6号在"cell growth"、"response to ABA stimulus"、"potassium ion transport"、"establishment of localization"和"response to salt stress"等代谢途径显著富集,而中国春在“cell death”、"jasmonic acid biosynthetic process"等代谢途径富集显著。2.利用结构域搜索的方法,在小麦基因组中注释到3718个转录因子基因,分布在51个基因家族,其中1583个在盐胁迫下表现出显著差异表达。胁迫响应转录因子在不同家族中分布不均衡,差异表达转录因子最多的7个家族(Myb、bZIP、bHLH、NAC、 C2H2、AP2/ERF和WRKY)占所有差异表达转录因子总数的47%。表达模式聚类分析显示,差异表达的转录因子可以聚类到两个群,分为20种不同的表达模式。其中第8群和第9群(160个转录因子)中,青麦6号盐响应转录因子表达丰度高于中国春。3.利用生物信息学方法,将小麦根系盐胁迫响应基因定位到IWGSC发布小麦参考基因组上,发现其在染色体上的分布并不是随机排列的,而是很多盐胁迫相关基因成簇分布。通过预测,我们一共鉴定到142个盐胁迫基因基因簇,并且发现它们在A、B、D部分同源群上部分不均匀,其中D基因组(57)中比A(36)和B(49)具有更高的盐响应热点。另外通过基因注释发现,同一基因簇中的串联基因编码相类似的蛋白,但是其响应模式具有明显差异,表明复制是导致盐响应的热点区形成的原因之一,但其功能可能发生了分化。4.对小麦赖氨酸乙酰化和琥珀酰化之间的重叠进行第一个全面分析,发现在277个蛋白质中有416个特异的乙酰化修饰位点,在173个蛋白中有330个特异的琥珀酰化修饰位点。根据GO注释、KEGG途径和结构域富集结果进行综合分析,表明小麦的乙酰化和琥珀酰化修饰作用可以对细胞内的各种代谢途径与过程进行调节与控制。试验结果还表明有26个参与光合作用和卡尔文循环的蛋白质发生了这2种赖氨酸酰化修饰,21个琥珀酰赖氨酸位点有相同的乙酰化位点,33个蛋白的乙酰化和琥珀酰化发生变性,占琥珀酰化的蛋白质的7.9%,表明小麦琥珀酰化和乙酰化之间存在重叠。这33种蛋白中的7种酶参与了卡尔文循环,这表明这两种类型的修饰可能在调节光合作用和碳固定代谢过程中发挥重要作用。本研究为探索在小麦及所有植物的赖氨酸乙酰化和琥珀酰化的生理作用提供了重要参考。
[Abstract]:Wheat is the second largest grain crop in China. Its yield and quality are closely related to national food security and people's living standard. Salt stress is one of the main limiting factors for wheat production. The physiological and biochemical processes, such as photosynthesis, respiration, water status and changes of various enzymes, are seriously affected at the level of transcription and translation. It is important to clarify the response mechanism of wheat to salt stress at the molecular level, identify the key salt tolerant genes and analyze the modification level of the related proteins, which is of great significance to the resistance breeding of wheat. The salt stress of the wheat seedlings with salt tolerant wheat variety green wheat 6 and salt sensitive material for 1 weeks in the artificial growth box was carried out in the experiment. The Illumina sequencing platform was used to sequence the wheat root system of 6,12,24 and 48h before and after salt stress, and the sequencing results were systematically compared with the bioinformatics method, and the differential expression of some homologous genes in response to salt stress was analyzed. At the same time, we used LC-MS/MS combined analysis technology and biological information. The physiological processes of wheat acetylation and succinylated protein were studied. The main results were as follows: 1. the gene expression patterns of wheat root response to salt stress were studied at the whole genome level, and 36804 differentially expressed genes were identified, the expression of gene expression in green wheat 6 and spring salt stress in China was significant. Differences. There was a difference in the expression of some homologous genes between 55.4% and 50.1% of green wheat 6 and China spring before treatment, while the proportion of salt stress increased to 62.6% and 59.9%, and its differential expression pattern was verified by real-time quantitative PCR. By analyzing the up-regulated gene of salt stress, it was found that the number of green wheat 6 was up after 6h and 12h stress treatment. The number of genes was less than that of spring in China, but after 48h, it was significantly higher than that of China spring. The expression of down regulated genes was the opposite. The gene GO enrichment analysis showed that the metabolic pathways such as "cell growth", "response to ABA stimulus", "potassium ion transport", "establishment ion" and "establishment" and other metabolic pathways were significantly enriched. In the spring of China, in the "cell death", "jasmonic acid biosynthetic process" and other metabolic pathways, a significant.2. use domain search method was used. In the wheat genome, 3718 transcription factor genes were annotated and distributed in 51 gene families. 1583 of them showed significant differential expression under salt stress. The stress response transcription factors were not The 7 families (Myb, bZIP, bHLH, NAC, C2H2, AP2/ERF and WRKY) with the most differentially expressed transcriptional factors (Myb, bZIP, C2H2, AP2/ERF and WRKY) accounted for the 47%. expression pattern of all the total differential expression transcription factors. The differentially expressed transcription factors could be clustered into two groups and divided into 20 different expression patterns. Among them, eighth and ninth groups (16 In the 0 transcription factors, the expression abundance of the salt response transcription factor of the green wheat 6 was higher than that of the Chinese Spring.3. bioinformatics method. The salt stress response gene of wheat root system was located to IWGSC to publish the wheat reference genome, and it was found that the distribution of the genes on the chromosomes was not arranged randomly, but a lot of salt stress related genes were distributed. Over prediction, we have identified 142 salt stress gene clusters and found that they are partially inhomogeneous on A, B, and D homologous groups, and the D genome (57) has a higher salt response than A (36) and B (49). There is a distinct difference, indicating that replication is one of the reasons for the formation of salt response hot spots, but its function may have occurred in the first comprehensive analysis of the overlap between the acetylation and succinylation of wheat lysine, and found that there were 416 specific acetylation modification sites in the 277 proteins and 330 of the 173 proteins in the 277 proteins. A specific succinylation site. Based on the GO annotation, the KEGG pathway and the domain accumulation results, the results showed that the acetylation and succinylation of wheat could regulate and control the various metabolic pathways and processes in the cells. The results also showed that 26 eggs involved in photosynthesis and the Calvin cycle. These 2 lysine acylation modifications occur in white matter, 21 succinyl lysine sites have the same acetylation sites, 33 proteins have acetylation and succinylation, accounting for 7.9% of succinylated proteins, indicating that there is a overlap between succinylation and acetylation of the wheat. The 7 enzymes of the 33 proteins participate in the Calvin cycle. These two types of modification may play an important role in regulating photosynthesis and carbon immobilization. This study provides an important reference for the exploration of the physiological role of lysine acetylation and succinylation in wheat and all plants.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1
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本文编号：1882772