本文選題：缺硫脅迫 + 水稻��； 參考：《河南師范大學(xué)》2017年碩士論文

【摘要】：水稻是全球最重要的谷類作物之一,同時也是重要飼料、纖維以及生物燃料來源。硫元素(S)作為植物必需營養(yǎng)元素在植物生長、發(fā)育、代謝過程以及產(chǎn)量中都起著重要的作用。硫最初主要以硫酸鹽的形式從環(huán)境中吸收,是蛋白質(zhì)、維生素、輔酶、谷胱甘肽或鐵氧還蛋白等多種物質(zhì)的組成成分,對還原小分子化合物以及重金屬、外源性物質(zhì)的去毒等起著至關(guān)重要的生化作用。近年來由于大量氮肥的施用以及含硫廢氣的排放限制,土壤缺硫即將成為限制農(nóng)業(yè)生產(chǎn)的一個因素。有關(guān)硫的吸收、轉(zhuǎn)運和同化研究得較為清楚。擬南芥中缺硫的應(yīng)答機理研究得也較為清楚,而水稻對缺硫的應(yīng)答研究報道較少。因此,本研究將兩周齡的水稻幼苗進行缺硫脅迫處理,觀察了缺硫脅迫對水稻植株生長和生化水平的影響;并對正常生長與缺硫脅迫一周的水稻葉子和根中轉(zhuǎn)錄組進行對比分析,找到了水稻中缺硫脅迫應(yīng)答基因,并對其進行差異表達分析,從而達到對水稻中缺硫脅迫相關(guān)基因表達調(diào)控機制探究的目的。具體研究結(jié)果如下:(1)生長和生化水平的研究表明,缺硫脅迫兩周左右的水稻幼苗POD、CAT、SOD酶活力以及葉綠素含量開始出現(xiàn)顯著下降,還原型谷胱甘肽含量在第三周開始出現(xiàn)下降。約在缺硫處理一個月左右植株生長表型表現(xiàn)出根長明顯伸長葉片較黃等表型。與擬南芥、油菜、楊樹等植物相比,水稻對缺硫脅迫不太敏感。(2)通過對正常及缺硫處理一周的水稻幼苗進行轉(zhuǎn)錄組分析,葉中對照組檢測到20705條基因,處理組中檢測到31200個基因,根中對照組檢測到29964個基因,處理組檢測到31050條基因,說明在缺硫脅迫下水稻幼苗中有眾多基因被誘導(dǎo)表達。應(yīng)用NOISeq技術(shù)對差異表達基因進行檢測,葉中篩選出866個差異表達基因,11個基因下調(diào)、855個基因上調(diào),根中共有289個差異表達基因,28個基因下調(diào)、261個基因上調(diào),且根與葉子中共同的差異表達基因很少。并對13個差異表達基因運用實時熒光定量PCR進行驗證,得到了與RNA-seq相似的結(jié)果,說明本次高通量測序的可靠性。(3)對差異表達基因進行KEGG、Pathway顯著性富集分析表明,在缺硫脅迫下大量與氧化脅迫應(yīng)答相關(guān)的基因發(fā)生了差異表達,葉差異表達基因主要與核酸代謝、信號轉(zhuǎn)導(dǎo)等多種代謝通路有關(guān);根中差異表達基因主要與能量代謝、信號轉(zhuǎn)導(dǎo)等多種代謝通路有關(guān)。(4)缺硫脅迫下負責(zé)硫運輸?shù)牧蜣D(zhuǎn)運蛋白的調(diào)控與擬南芥相似,表明水稻采用與擬南芥相似的機制來調(diào)控硫酸鹽的重新分布;與擬南芥一樣,水稻中硫同化途徑中的ATPS、APR、MSA1、SDI1、SAM以及miR395靶基因也受到不同程度的調(diào)控。但是,在擬南芥中缺硫脅迫可以導(dǎo)致miR395的靶基因ATPS1、ATPS3、ATPS4的表達水平下降,但水稻中ATPS的表達被誘導(dǎo),這很可能是單子葉和雙子葉植物調(diào)控機制的差異性。同時預(yù)測了Os09g27660很可能是miR395的靶基因。(5)對根和葉中差異表達轉(zhuǎn)錄因子分析表明,葉中有21個差異表達轉(zhuǎn)錄因子,根中有4個差異表達轉(zhuǎn)錄因子,葉中差異表達轉(zhuǎn)錄因子數(shù)目明顯高于根,這些轉(zhuǎn)錄因子對于調(diào)控缺硫應(yīng)答基因的表達及植物激素代謝具有重要意義。同時發(fā)現(xiàn)多個miRNA靶基因的表達發(fā)生明顯變化,說明除miR395外其它miRNA很可能也參與了缺硫脅迫的應(yīng)答。本研究表明缺硫脅迫能促進水稻根的生長;同時水稻幼苗的硫轉(zhuǎn)運機制和同化途徑發(fā)生了相應(yīng)的變化來應(yīng)答硫匱乏的生理狀態(tài);缺硫脅迫同時誘導(dǎo)了氧化應(yīng)激反應(yīng),大量的氧化脅迫相關(guān)的應(yīng)答基因被調(diào)控;并得到一系列與水稻幼苗缺硫脅迫應(yīng)答相關(guān)的差異表達基因。該研究為水稻缺硫脅迫應(yīng)答調(diào)控網(wǎng)絡(luò)的進一步研究奠定了基礎(chǔ),也將為提高水稻抗脅迫能力和產(chǎn)量的研究提供理論依據(jù),同時對于如何操縱硫代謝,幫助提高水稻等農(nóng)作物產(chǎn)量具有重要意義。
[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.
【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q945.78;S511

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