【作者】 李娇；

【导师】 田曾元；

【作者基本信息】 郑州大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 本文以对干旱胁迫敏感的Ye478(选自8112和5003的二环系,我国玉米单交种推广的优良自交系,Ye478)和耐早的H21(选自美国先锋种子公司杂交种P78599,我国西北干旱和半干旱地区骨干系,H21)玉米自交系为材料,在苗期进行干旱胁迫处理,对利用solexa高通量测序获得的转录组数据进行基因表达的可变剪接分析,通过对比干旱胁迫与正常对照植株基因可变剪接方式与程度的差异,了解发生可变剪接事件的基因参与的生物学途经；通过反转录PCR等手段扩增出一些基因的部分剪接变体；通过高通量测序分析和qRT-PCR分析了解玉米可变剪接重要的调控者SR蛋白基因家族干旱胁迫下表达规律,筛选干旱胁迫相关的候选SR成员,揭示其与玉米干旱胁迫应答基因表达调控的内在联系。通过对比我们发现：1.总体上,干旱逆境会诱导部分基因的可变剪接,并通过可变剪接反映玉米的受旱程度。干旱逆境会对植物的各种生理状态有所影响,植物体内的各种与水分相关的生理生化反应会发生改变。由于Ye478的耐旱能力远低于H21,所以其在干旱胁迫下发生的可变剪接数目远多于耐旱品系H21。受干旱程度的影响,植物可变剪接数目会出现上升受环境逆境影响植物会增加基因组的多样性大大增加此外,可变剪接多样性的改变一定程度上反映了玉米受胁迫影响的程度；耐早品系H21在干旱条件下所发生的可变剪接改变要小于Ye478,由于可变剪接可以影响基因转录后的命运,这可以理解为Ye478受干旱影响更严重,关闭了大量非生活所必需的基因的表达。2.可变剪接对玉米干旱逆境适应性有一定的影响。H21和Ye478两个品系在干旱逆境下可变剪接的差异表明：部分基因的可变剪接可以增加玉米的耐旱能力在H21对照和Ye478、H21干旱胁迫下发生类似可变剪接的基因,可能与玉米干旱适应性相关。3.在所有两个玉米自交系中,发生的可变剪接的基因大部分在干旱和非干旱胁迫下均发生可变剪接。H21中2/3和Ye478中4/5在正常水分条件下发生可变剪接的基因在干旱胁迫下仍然发生可变剪接。由干旱诱导而发生可变剪接的基因占Ye478和H21干旱中所有发生可变剪接的基因中1/3左右,表明一部分基因可变剪接是玉米行使生理功能所必需的,不会受干旱等环境因素的影响。4.在干旱胁迫下发生可变剪接的基因多数集中在NTP结合、糖代谢以及蛋白合成转运等相关途径中这表明,玉米干旱胁迫下发生可变剪接的基因主要为能量和蛋白代谢相关基因。这表明蛋白和能量的代谢是玉米在干旱逆境下最主要的生理途径,其可能与玉米在干旱胁迫下抗旱性相关的重要通路。5.镁离子结合蛋白是H21在干旱胁迫特异发生可变剪接的重要蛋白成员,这表明镁离子结合蛋白的可变剪接可能与H21的干旱抗逆性相关。6.ABA信号转导通路是玉米干旱胁迫下重要的通路,是触发植物逆境胁迫反应主要传递者。ABA诱导蛋白对植物逆境胁迫具有重要作用。ABA诱导蛋白,不论在H21还是在Ye478中都由于干旱而发生大量的可变剪接。多种转录因子和在H21和Ye478干旱条件下出现相似的可变剪接变化。此外R蛋白及部分剪接因子家族也发生可变剪接,这些可变剪接已被实验验证。7.SR蛋白是玉米中最重要的剪接因子之一,大量SR剪接因子表达量的总体趋势与全基因组可变剪接事件变化的趋势基本一致；可以用大量SR剪接因子的表达量的改变趋势来反应全基因组可变剪接的改变,因而可以用大量剪接因子的表达量变化来表示玉米干旱耐受性。8.部分SR蛋白家族成员与玉米的逆境胁迫应答密切相关。RSZ. ASF/SF2等家族可能与玉米细胞凋亡相关能够在一定程度上反映玉米受胁迫的程度,而SC35亚家族的部分成员在各个转录组中的不同表达显示了其与玉米干旱耐受性的亲密关系。更多还原

【Abstract】 In this manuscript we will to explain the alternative splicing of the whole transcriptomes in seedling stage.under drought stress in two maize (zea mays sp. L) inbred lines, the sensitivity to drought stress inbred lines Ye478(from8112and5003of the system, the most popular inbred line in China) and drought tolerance oneH21(from the P78599, pioneer Seed Company, USA, widely plant in Northwest China and half arid zones).Drought stress treatment is in the seedling stage, analysis the data of alternative splicing in whole transcnptomeobtained using high-throughput Solexa sequencing for gene expression, through the comparison of differences of genes alternative splicing events and degree under drought stress and normal condtion, to understand the variable shear biological and molecular pathway; by means of reverse transcription PCR amplified part of splice variants of some genes; by high-throughput sequencing analysis and qRT-PCR analysis to understand the expression of corn important regulatorsof alternative splicing, SR protein gene family. Screening of drought related candidate SR members, and reveal the internal relationship between its response gene expression and regulation of maize drought stress. The follows are the result:1. Overall, drought stress will be alternative splicing induced gene reflect the drought degree of Maize.Drought stress physiological state of the plant will have an impact, all kinds of plant body and water related physiological and biochemical changes. Ye478because of the drought resistance ability is far lower than the H21, so the number of alternative splicing occurs under drought stress than drought tolerant lines H21. Affected by drought, plant alternative splicing number will rise by environmental stresses affecting plant will increase the diversity of genome is greatly increased in addition, alternative splicing diversity variable change to some extent reflects the corn stress influence degree; alternative splicing drought-tolerant strains H21occurred in drought conditions change to less than Ye478, due to alternative splicing can influence gene transcription of the fate, it can be understood as Ye478affected by drought is more serious, shut down the expression of a large number of required for non housekeeping genes.2. Alternative splicing has certain influence on maize drought adaptability. There are some difference variable under the condition of drought stress between H21and Ye478:alternative splicing genes could increase maize drought tolerance similar alternative splicing genes in H21and Ye478drought stress while it occurs alternative splicing in H21control group, may be associated with Maize Drought adaptability.3. In all two maize inbred lines, most alternatively splicedgenes occurAlternative splicingin both drought and non-drought stress occurred alternative splicing.2/3in H21and4/5in Ye478alternatively spliced genesin normal moisture conditions still alternative splicing under drought stress. Induced by drought and alternatively spliced genes accounted for about Ye478and H21drought all alternatively spliced genes in1/3, shows a portion of a gene alternative splicing is maize physiological functions necessary, are not affected by the drought and other environmental factors.4. The majority of the alternatively spliced genes under drought stress related pathways in NTP binding, sugar metabolism and protein synthesis, suggesting that transport, alternative splicing occurs in Maize under drought stress genes were mainly protein and energy metabolism related genes. This indicates that the protein and energy metabolism is the main physiological pathway in Maize under drought stress, the stress of drought resistance of important pathways associate with Maize under drought stress.5. Magnesium ion binding protein is an important member of H21under drought stress specific alternatively spliced proteins, suggesting that the magnesium ion binding splicing protein may be associated with drought resistance for H21.6. The ABA signal transduction pathway of Maize under drought stress, is triggering main transmission of the plant stress response. Induction of ABA protein plays an important role in plant abiotic stress. ABA induced protein, either in H21or in Ye478ocurrs a large number of alternative splicing due to drought. Many transcription factorshave similar alternative splicing changes in the H21and Ye478under drought condition. In addition, R protein and splicing factor family also have alternative splicing, these alternative splicing has been verified by experiment.7. The SR protein is one of the most important splicing factors in maize, the change trend of whole genome alternative splicing, is consistent with the trend of SR splicing factors expression; whole genome splicing changes can be replaced by the expression of a large number of SR splicing factors, so it can be used to change the amount of splicing factor expression to express the maize drought tolerance.8. The SR protein family member and the stress response is closely related to maize. RSZ, ASF/SF2families may be associated with maize cell apoptosis can reflect the corn stress to a certain extent, and the different expression of some members of the SC35subfamily of transcription in each group shows its close relationship with maize drought tolerance.更多还原