本文關鍵詞： 水稻 衰老 轉錄組分析 葉綠素 脫落酸　出處：《江蘇大學》2017年碩士論文　論文類型：學位論文

【摘要】：衰老是一個高度程序化的復雜過程,受到多種因素影響,且在很大程度上決定了作物的產(chǎn)量和品質。近年來,水稻葉片衰老的相關研究顯示,在衰老過程中,葉片組織發(fā)生了大量的形態(tài)學變化,包括大量水解酶產(chǎn)生、核酸量降低、活性氧等有害物質產(chǎn)生與增多、多種蛋白質被分解、葉綠素降解以及葉片受損,而這些變化均離不開基因的調控作用。研究表明,葉片衰老受多種衰老相關基因調控,如STAY-GREEN、RED CHLOROPHYLL CATABOLITE REDUCTASE 1、NON-YELLOW COLORING 1和NON-YELLOW COLORING 3。本實驗室通過對秈稻和粳稻黃化苗蛋白質組的蛋白差異表達分析,鑒定到一個秈稻特異蛋白(Indica Special Protein,ISP),但其具體的功能并不明確。本研究利用轉基因技術、轉錄組測序技術和實時熒光定量PCR技術等研究了ISP基因在延緩水稻葉片衰老中的功能。主要研究內容和結果如下:(1)通過對野生型和ISP過表達植株整個生長周期的表型觀察,在生長期,ISP過表達水稻植株表型與野生型類似,并無明顯差異;而在成熟期,ISP過表達植株的葉片能夠一直保持綠色,野生型葉片在成熟后期呈現(xiàn)變黃枯萎的表型。(2)采用轉錄組測序的技術分析了野生型水稻和ISP過表達水稻中的基因表達差異。發(fā)現(xiàn)ISP過表達植株與野生型植株之間共有889個差異表達基因(FDR≤0.05且log2.0 Fold_change的絕對值≥1),其中543個基因上調表達,346個基因下調表達。通過對轉錄組測序結果的進一步分析,我們篩選出了一些與植物葉片衰老及葉綠素降解相關基因,過表達植株中這些基因的表達量顯著低于野生型,表明過表達ISP基因會抑制某些衰老相關基因的表達。此外,我們發(fā)現(xiàn)脫落酸(Abscisic acid,ABA)合成途徑中的關鍵基因也存在明顯差異,過表達植株中ABA合成途徑相關基因的表達量明顯低于野生型植株中的表達量。(3)通過實時熒光定量PCR技術檢測了自然條件下野生型及ISP過表達植株中衰老相關基因、葉綠素降解相關基因的表達量水平,同時測定了野生型及ISP過表達植株葉片中葉綠素的含量。結果發(fā)現(xiàn)過表達ISP可以抑制年齡依賴型的水稻葉片衰老。(4)黑暗處理野生型和ISP過表達植株的離體葉片,結果發(fā)現(xiàn),黑暗處理的野生型葉片變黃時,ISP過表達的葉片仍然保持綠色。通過實時熒光定量PCR技術檢測了黑暗誘導衰老條件下野生型及ISP過表達植株中衰老相關基因、葉綠素降解相關基因的表達量水平,同時測定了野生型及ISP過表達植株葉片中葉綠素的含量。證實了過表達ISP可以抑制黑暗誘導的水稻葉片衰老。(5)采用實時熒光定量PCR技術測定了野生型和ISP過表達植株中ABA合成途徑關鍵基因的表達量水平。結果顯示,過表達植株中ABA合成途徑相關基因的表達量明顯低于野生型植株中的表達量,這一結果證實了轉錄組測序結果的準確性。此外,我們用50μM ABA激素處理野生型和ISP過表達萌發(fā)的種子,發(fā)現(xiàn)生長10 d后的ISP過表達植株對ABA具有超敏性。
[Abstract]:Aging is a complex process which is highly stylized, are affected by many factors, and largely determines the yield and quality of crops. In recent years, the research of rice leaf senescence showed that during aging, the morphological changes of a large number of leaf tissue, including a large number of hydrolase production, reduce the amount of nucleic acid, activity oxygen and other harmful substances and a variety of protein was increased, decomposition, degradation of chlorophyll and leaf damage, but these changes cannot do without regulation of genes. The results showed that leaf senescence by various senescence associated gene regulation, such as STAY-GREEN, RED CHLOROPHYLL CATABOLITE REDUCTASE 1 protein expression analysis of differences of indica and Japonica Rice etiolated seedling protein group by NON-YELLOW COLORING 1 and NON-YELLOW COLORING 3. in the laboratory, the identification of a specific protein (Indica Special Protein indica, ISP), but its The function is not clear. In this study, the use of transgenic technology, transcriptome sequencing and real-time fluorescence quantitative PCR of ISP gene in delay senescence in function. The main research contents and results are as follows: (1) the phenotype of wild-type and ISP overexpressing plants throughout the growth period of observation, in the growth period, the overexpression of ISP in rice plant phenotype similar to wild type, there is no obvious difference; but in the mature period, over expression of ISP plants can keep green leaves in the wild type phenotype showed yellowing withered in the mature period. (2) the transcriptome sequencing technology of wild type rice and ISP. The expression of differentially expressed genes in rice. The over expression of ISP between plants and wild type plants have a total of 889 differentially expressed genes (FDR and log2.0 = 0.05 Fold_change absolute value is more than 1), of which 543 genes were up-regulated, 34 6 genes were downregulated. Through further analysis of the transcriptome sequencing results, we screened out some related plant leaf senescence and chlorophyll degradation gene expression, expression of these genes in plants was significantly lower than that of the wild type, showed that over expression of some aging related gene expression inhibition of ISP gene will. In addition, we found that abscisic acid (Abscisic acid, ABA) key genes in the biosynthesis pathway are distinct, over expression of related genes in plant ABA synthesis pathway was significantly lower than the expression in wild type plants. (3) by real-time fluorescence quantitative PCR detection of ISP and wild type plants under natural conditions over expression of aging related genes, expression level of chlorophyll degradation related gene, wild type and ISP over expression of chlorophyll content in leaf was measured. Results showed that overexpression of ISP can inhibit the years Age dependent senescence of rice leaves. (4) the dark treatment of wild type and ISP expression of leaves in vitro, found that plants, wild type leaves dark processing yellow leaves, ISP expression remained green. By real-time fluorescence quantitative PCR technique to detect dark and wild type ISP induced aging condition overexpression of plant senescence related genes, the expression level of chlorophyll degradation related gene, wild type and ISP over expression of chlorophyll content in leaf was measured. Confirmed that overexpression of ISP inhibited dark induced senescence of rice leaves. (5) wild type and ISP expression level of expression of ABA synthesis in plants way of key genes were determined by real-time fluorescence quantitative PCR. The results showed that over expression of genes expression in plant ABA synthesis pathway was significantly lower than that of the expression in wild type plants, the results The accuracy of transcriptome sequencing was achieved. In addition, we used 50 micron M ABA hormone to deal with wild type and ISP over expressing germinated seeds. It was found that ISP overexpressing plants after 10 d growth had hypersensitivity to ABA.

【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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