水稻ISP基因过表达在延缓叶片衰老中的功能研究

发布时间：2018-02-03 21:02

本文关键词： 水稻衰老转录组分析叶绿素脱落酸　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：衰老是一个高度程序化的复杂过程,受到多种因素影响,且在很大程度上决定了作物的产量和品质。近年来,水稻叶片衰老的相关研究显示,在衰老过程中,叶片组织发生了大量的形态学变化,包括大量水解酶产生、核酸量降低、活性氧等有害物质产生与增多、多种蛋白质被分解、叶绿素降解以及叶片受损,而这些变化均离不开基因的调控作用。研究表明,叶片衰老受多种衰老相关基因调控,如STAY-GREEN、RED CHLOROPHYLL CATABOLITE REDUCTASE 1、NON-YELLOW COLORING 1和NON-YELLOW COLORING 3。本实验室通过对籼稻和粳稻黄化苗蛋白质组的蛋白差异表达分析,鉴定到一个籼稻特异蛋白(Indica Special Protein,ISP),但其具体的功能并不明确。本研究利用转基因技术、转录组测序技术和实时荧光定量PCR技术等研究了ISP基因在延缓水稻叶片衰老中的功能。主要研究内容和结果如下:(1)通过对野生型和ISP过表达植株整个生长周期的表型观察,在生长期,ISP过表达水稻植株表型与野生型类似,并无明显差异;而在成熟期,ISP过表达植株的叶片能够一直保持绿色,野生型叶片在成熟后期呈现变黄枯萎的表型。(2)采用转录组测序的技术分析了野生型水稻和ISP过表达水稻中的基因表达差异。发现ISP过表达植株与野生型植株之间共有889个差异表达基因(FDR≤0.05且log2.0 Fold_change的绝对值≥1),其中543个基因上调表达,346个基因下调表达。通过对转录组测序结果的进一步分析,我们筛选出了一些与植物叶片衰老及叶绿素降解相关基因,过表达植株中这些基因的表达量显著低于野生型,表明过表达ISP基因会抑制某些衰老相关基因的表达。此外,我们发现脱落酸(Abscisic acid,ABA)合成途径中的关键基因也存在明显差异,过表达植株中ABA合成途径相关基因的表达量明显低于野生型植株中的表达量。(3)通过实时荧光定量PCR技术检测了自然条件下野生型及ISP过表达植株中衰老相关基因、叶绿素降解相关基因的表达量水平,同时测定了野生型及ISP过表达植株叶片中叶绿素的含量。结果发现过表达ISP可以抑制年龄依赖型的水稻叶片衰老。(4)黑暗处理野生型和ISP过表达植株的离体叶片,结果发现,黑暗处理的野生型叶片变黄时,ISP过表达的叶片仍然保持绿色。通过实时荧光定量PCR技术检测了黑暗诱导衰老条件下野生型及ISP过表达植株中衰老相关基因、叶绿素降解相关基因的表达量水平,同时测定了野生型及ISP过表达植株叶片中叶绿素的含量。证实了过表达ISP可以抑制黑暗诱导的水稻叶片衰老。(5)采用实时荧光定量PCR技术测定了野生型和ISP过表达植株中ABA合成途径关键基因的表达量水平。结果显示,过表达植株中ABA合成途径相关基因的表达量明显低于野生型植株中的表达量,这一结果证实了转录组测序结果的准确性。此外,我们用50μM ABA激素处理野生型和ISP过表达萌发的种子,发现生长10 d后的ISP过表达植株对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.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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