水稻幼苗缺硫胁迫应答机理研究

发布时间：2018-07-07 09:04

本文选题：缺硫胁迫 + 水稻　；参考：《河南师范大学》2017年硕士论文

【摘要】：水稻是全球最重要的谷类作物之一,同时也是重要饲料、纤维以及生物燃料来源。硫元素(S)作为植物必需营养元素在植物生长、发育、代谢过程以及产量中都起着重要的作用。硫最初主要以硫酸盐的形式从环境中吸收,是蛋白质、维生素、辅酶、谷胱甘肽或铁氧还蛋白等多种物质的组成成分,对还原小分子化合物以及重金属、外源性物质的去毒等起着至关重要的生化作用。近年来由于大量氮肥的施用以及含硫废气的排放限制,土壤缺硫即将成为限制农业生产的一个因素。有关硫的吸收、转运和同化研究得较为清楚。拟南芥中缺硫的应答机理研究得也较为清楚,而水稻对缺硫的应答研究报道较少。因此,本研究将两周龄的水稻幼苗进行缺硫胁迫处理,观察了缺硫胁迫对水稻植株生长和生化水平的影响;并对正常生长与缺硫胁迫一周的水稻叶子和根中转录组进行对比分析,找到了水稻中缺硫胁迫应答基因,并对其进行差异表达分析,从而达到对水稻中缺硫胁迫相关基因表达调控机制探究的目的。具体研究结果如下:(1)生长和生化水平的研究表明,缺硫胁迫两周左右的水稻幼苗POD、CAT、SOD酶活力以及叶绿素含量开始出现显著下降,还原型谷胱甘肽含量在第三周开始出现下降。约在缺硫处理一个月左右植株生长表型表现出根长明显伸长叶片较黄等表型。与拟南芥、油菜、杨树等植物相比,水稻对缺硫胁迫不太敏感。(2)通过对正常及缺硫处理一周的水稻幼苗进行转录组分析,叶中对照组检测到20705条基因,处理组中检测到31200个基因,根中对照组检测到29964个基因,处理组检测到31050条基因,说明在缺硫胁迫下水稻幼苗中有众多基因被诱导表达。应用NOISeq技术对差异表达基因进行检测,叶中筛选出866个差异表达基因,11个基因下调、855个基因上调,根中共有289个差异表达基因,28个基因下调、261个基因上调,且根与叶子中共同的差异表达基因很少。并对13个差异表达基因运用实时荧光定量PCR进行验证,得到了与RNA-seq相似的结果,说明本次高通量测序的可靠性。(3)对差异表达基因进行KEGG、Pathway显著性富集分析表明,在缺硫胁迫下大量与氧化胁迫应答相关的基因发生了差异表达,叶差异表达基因主要与核酸代谢、信号转导等多种代谢通路有关;根中差异表达基因主要与能量代谢、信号转导等多种代谢通路有关。(4)缺硫胁迫下负责硫运输的硫转运蛋白的调控与拟南芥相似,表明水稻采用与拟南芥相似的机制来调控硫酸盐的重新分布;与拟南芥一样,水稻中硫同化途径中的ATPS、APR、MSA1、SDI1、SAM以及miR395靶基因也受到不同程度的调控。但是,在拟南芥中缺硫胁迫可以导致miR395的靶基因ATPS1、ATPS3、ATPS4的表达水平下降,但水稻中ATPS的表达被诱导,这很可能是单子叶和双子叶植物调控机制的差异性。同时预测了Os09g27660很可能是miR395的靶基因。(5)对根和叶中差异表达转录因子分析表明,叶中有21个差异表达转录因子,根中有4个差异表达转录因子,叶中差异表达转录因子数目明显高于根,这些转录因子对于调控缺硫应答基因的表达及植物激素代谢具有重要意义。同时发现多个miRNA靶基因的表达发生明显变化,说明除miR395外其它miRNA很可能也参与了缺硫胁迫的应答。本研究表明缺硫胁迫能促进水稻根的生长;同时水稻幼苗的硫转运机制和同化途径发生了相应的变化来应答硫匮乏的生理状态;缺硫胁迫同时诱导了氧化应激反应,大量的氧化胁迫相关的应答基因被调控;并得到一系列与水稻幼苗缺硫胁迫应答相关的差异表达基因。该研究为水稻缺硫胁迫应答调控网络的进一步研究奠定了基础,也将为提高水稻抗胁迫能力和产量的研究提供理论依据,同时对于如何操纵硫代谢,帮助提高水稻等农作物产量具有重要意义。
[Abstract]:Rice is one of the most important cereals in the world, and is also an important source of feed, fiber and biofuel. Sulfur element (S) plays an important role in plant growth, development, metabolism and production as essential nutrients in plants. Sulphur is originally absorbed from the environment in the form of sulphate, which is a protein, vitamin, The components of a variety of substances such as coenzyme, glutathione or ferrites are essential for the reduction of small molecular compounds and the detoxification of heavy metals and exogenous substances. In recent years, due to the application of large amounts of nitrogen fertilizer and the limitation of the emission of sulfur containing waste gas, the lack of sulfur in soil and soil will be a factor restricting agricultural production. The study of sulfur absorption, transport and assimilation is clearer. The mechanism of response to sulfur deficiency in Arabidopsis is also clear, and the response of rice to sulfur deficiency is less reported. Therefore, the two week old rice seedlings were treated with sulfur deficiency stress, and the effects of sulfur deficiency stress on the growth and biochemical level of rice plants were observed. A comparative analysis was made between the normal growth and the transcriptional group of rice leaves and roots for one week without sulfur stress. The response genes of sulfur deficiency stress in rice were found, and the differential expression analysis was carried out to achieve the purpose of exploring the regulation mechanism of gene expression related to the deficiency of sulfur stress in rice. The specific results are as follows: (1) growth and biochemical levels The results showed that the POD, CAT, SOD enzyme activity and chlorophyll content began to decrease significantly in rice seedlings with two weeks of sulfur deficiency stress, and the content of glutathione was decreased at the beginning of third weeks. Compared with trees and other plants, rice was not sensitive to sulfur deficiency stress. (2) 20705 genes were detected in the control group, 31200 genes were detected in the control group and 29964 genes were detected in the control group, and 31050 genes were detected in the treatment group. A number of genes were induced in rice seedlings. The differential expression genes were detected by NOISeq technology. 866 differentially expressed genes were screened in the leaves. 11 genes were down and 855 genes were up-regulated. There were 289 differentially expressed genes in the root, 28 genes, 261 bases due to up regulation, and the common differentially expressed genes in the roots and leaves. 13 differentially expressed genes were verified by real time fluorescence quantitative PCR, and the results similar to RNA-seq were obtained to demonstrate the reliability of this high throughput sequencing. (3) the KEGG of differentially expressed genes and the significant enrichment analysis of Pathway showed that the genes related to the response of oxidative stress response to large quantities under sulfur deficiency stress occurred The differentially expressed genes are mainly related to metabolic pathways such as nucleic acid metabolism and signal transduction. The differentially expressed genes in the root are mainly related to various metabolic pathways such as energy metabolism and signal transduction. (4) the regulation of sulfur transporter in sulfur transport under sulfur stress is similar to that of Arabidopsis thaliana, indicating that rice is similar to Arabidopsis. To regulate the redistribution of sulfate; like Arabidopsis thaliana, the target genes of ATPS, APR, MSA1, SDI1, SAM and miR395 in the sulfur assimilation pathway in rice are also regulated in varying degrees. However, the lack of sulfur stress in Arabidopsis can lead to the decline in the expression level of the target gene ATPS1, ATPS3, ATPS4 of the miR395, but the expression of ATPS in rice is induced, It is likely that the regulation mechanism of mono cotyledonous and dicotyledonous plants is different. At the same time, it is predicted that Os09g27660 is probably the target gene of miR395. (5) analysis of differential expression transcription factors in roots and leaves shows that there are 21 differentially expressed transcription factors in leaves and 4 differentially expressed transcription factors in the roots, and the number of differentially expressed transcriptional factors in leaves is significantly higher At the root, these transcription factors are important for regulating the expression of sulfur deficient response genes and plant hormone metabolism. At the same time, it is found that the expression of multiple miRNA target genes changes obviously, indicating that other miRNA except miR395 may also participate in the response to sulfur deficiency stress. This study shows that the stress deficiency can promote the growth of rice root; The mechanism of sulfur transport and the assimilation pathway of rice seedlings changed to respond to the physiological state of sulfur deficiency; the oxidative stress response was induced by sulfur deficiency stress, and a large number of response genes related to oxidative stress were regulated, and a series of differentially expressed genes related to the response to sulfur deficiency stress in rice seedlings were obtained. The further study of the response regulation network for the response to sulfur deficiency stress will provide a theoretical basis for improving the resistance to stress and yield of rice, and it is of great significance to how to manipulate the sulfur metabolism and improve the yield of rice.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945.78;S511

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