【作者】 王洋；

【导师】 柏锡；

【作者基本信息】 东北农业大学 ， 植物学， 2014， 硕士

【摘要】 野生大豆可以在相对较严重的盐碱地环境下生长,说明在野生大豆中存在着耐盐和耐碱两套机制,是研究植物盐、碱胁迫生理机制的理想材料。目前对于植物盐胁迫生理和分子机制的研究较多,并且已取得了一定进展,但碱胁迫机制和盐碱混合作用机制的研究却为数不多,尤其是在分子水平上的研究更是知之甚少。为了系统阐明野生大豆盐碱混合胁迫基因表达调控规律,本实验以耐碱能力较强的野生大豆种质G07256(耐碱能力达到pH9.02)为试验材料,采用50mM NaHCO3溶液(pH8.5)模拟盐碱混合胁迫,使用Illumina Solexa测序技术对处理组和对照组野生大豆根部进行转录组测序,并对测序结果进行全面的生物信息学分析,构建连续时间点的野生大豆基因表达谱。本研究有助于加深对植物耐盐碱混合胁迫分子机制的认识,同时也将为作物分子育种提供基因资源奠定理论基础。主要的研究结果及结论如下：1.采用Illumina Solexa技术对NaHC03下的野生大豆进行双端测序,共获得差异表达基因3380个,各时间点差异表达基因数量为1433、1266、1162、1267和632个。所获得差异表达基因其表达量大部分在6h达到峰值,在12h-24h表达量降低。2.野生大豆G07256的基因在NaHC03胁迫处理6h内剧烈表达,胁迫处理12h-24h内基因表达变化幅度减缓,差异基因数量明显降低；其中,下调基因的数量在各时间点基本持平,而在12h时数量明显增加,暗示着胁迫处理6h内是基因早期应答的主要过程,而12h可能是基因应答的关键时刻。3.获得了NaHC03胁迫下差异基因的11个表达模式；对其进行功能分类及通路富集分析发现,野生大豆根部应答过程呈现级联反应,早期主要的应答基因功能及参与的通路为：信号转导、转录调控、防御系统、碳代谢及氨基酸代谢等。4.共差异表达基因功能分析发现,大部分的下调表达基因都是与组蛋白修饰或植物生长过程相关的基因。5.通过差异表达分析及文献挖掘最终获得了28个与NaHC03胁迫响应、并处于应答早期阶段的候选调控基因。随机挑取了12个基因进行realtime-PCR验证,实验结果与Illumina Solexa测序结果相一致,证明测序相对准确,此测序结果可以应用到后续研究中。6.对NaHCO3胁迫下野生大豆测序数据进行可变剪接分析,主要的剪接方式为3’端可变剪接(exon3’alternative),其次是外显子缺失(exon deletion)类型,胁迫3h时,这两种可变剪接类型的数量增至最多,随后数量下降至24h再次升高,且高于对照组剪接类型数量。该结果为后续深入讨论混合盐碱胁迫影响可变剪接方式的研究奠定了一定的基础。转录本的特征分析中发现了不同时间段的新转录本,包含了许多表达丰度较低的新转录本,对于补充野生大豆全基因组数据具有重要意义；对野生大豆进行SNP相关分析,挖掘得到了1324个非同义突变的SNP,为野生大豆与栽培达到在表型及性状差异的研究上奠定了理论基础。更多还原

【Abstract】 Wild soybean(Glycine soja) could survive in a relative severer sodic soil regions which may illustrate that there may exist two kinds of mechanism to respond to these unfavorable environmental stimuli. Hence, wild soybean is an ideal material to study the physiological mechanism of plants to salt and alkali stress. In recent years, tremendous progress has been made in understanding plant response to external stimuli. However, most researches mainly focused on the salt stress, and very limited progress was found for alkali or saline-alkali mix stress, especially on molecular function.The wild soybean(G07256) used in this study with a strong resistant ability against alkai stress, which can germinate and set seed in the salt-alkaline soil at pH9.02.In order to understanding its response of molecular mechanism to the salt-alkali mix stress, we used50mM NaHCO3solution(pH8.5) to simulate the salinity mixed stress. Through Illumina solexa sequencing technology, we obtained the wild soybean root genes expression profile under NaHCO3stress. This study could helps us to investigate the alkaline resistant genes and better understanding the molecular mechanisms of plants under salt-alkali mixed stress, as well as to provide theoretical basis for genetic resources in plant molecular breeding.The main results and conclusions are as follows:1. Through Illumina Solexa sequencing, we obtained3380differential expressed genes under the NaHCO3stress. We altogether identified1443,1266,1162,1267,1162differential expressed genes from control groups and treatment groups. These genes’ expression quantities peaked at6h under NaHCO3stress while decreased at12h to24h.2. Within6hs under NaHCO3stress, the genes of Glycine soja (G07256) expressed vigorously while between12h to24h, the express of it became slowly, and the number of differential expressed genes decrease sharply. Among them, the number of down-regulated genes were almost the same in different times, while about12h,, the number of it became sharply increased. We suggested that under NaHCO3stress6h is an early response time point, while the12h is the turning point of genetic response.3. We obtained11gene expression patterns under NaHCO3stress; making gene function classification and pathway enrichment analysis, we found that wild soybean root response presented a cascade process.The main pathways and functions were as follows: hormone signal transduction, transcriptional regulation,immune system, carbohydrate metabolism pathways.4. Co-differentially expressed genes function analysis showed that the function of most of the down-regulated genes were associated with histone modification or plant growth process related.5. Realtime-PCR validation showed that the relative expression quantities of12genes are consistent with Illumina solexa sequencing results. This proves that sequencing results is relatively accurate and we could use it in the following study.6. Analysis the alternative splicing of wild soybean under NaHCO3mixed stress, we found that the main alternative splicing type is exon3’alternative, the following is exon deletion. Under NaHCO3stress with3h, the number of these two kinds of alternative splicing type achieved most then dropped until24h. This finding laid a foundation about the research about the salinity-alkalinity stress influence alternative splicing.7. The characteristics of the transcirptome analysis found many novel transcripts which contains so many low expression abundance new transcripts. It means great significance for supplement of wild soybean genome sequence data. In SNP related analysis, we mined1324non-synonymous mutations SNP, which laid a theoretical foundation on wild soybean and cultivated research about their phenotypic traits and differences.更多还原