【作者】 陈敏；

【导师】 Luis Herrera-Estrella；

【作者基本信息】 南京农业大学 ， 植物营养学， 2023， 硕士

【摘要】 磷是植物生长发育所必需的大量营养元素之一,是构成ATP、核酸、磷脂等细胞生物分子的重要组成部分,也参与生物体能量转移、蛋白质代谢和碳素活化等生物学过程。磷在土壤中多以有机态的形式存在,部分磷被生物固着成有机态,剩余的无机态的磷容易与铁、铝、钙等金属离子形成难溶化合物。大多数土壤存在缺磷的现状,土壤中可供植物吸收的无机态的磷含量较低,不能满足植物正常生长发育的需要。为了应对缺磷,植物在进化过程中形成了一系列适应机制,包括根系构型的改变、产生根系分泌物、形成菌根共生体、诱导高亲和磷转运蛋白的表达等过程来提高根系对磷素的吸收。吸收到植物体内的磷在磷转运体的参与下运输到各组织器官发挥作用。水稻在不同的胁迫条件下会促使不同的基因表达,形成不同的调控网络。磷饥饿作为营养胁迫的一种,其参与水稻磷素调控的分子机制已经得到广泛的研究。近些年,表观遗传成为了研究的热点,染色质可及性的研究也逐步深入。ATAC-seq(assay for transposase accessible chromatin with high-throughput sequencing)技术的出现为研究植物染色质可及性提供了更多的可能,利用该方法可以构建基因组染色质图谱、寻找转录因子,还可以与转录表达联合分析构建调控网络。ATAC-seq技术是利用转座酶对开放染色质区域进行捕获并在开放区域两端接上测序接头,主要过程包括细胞核的准备、文库的构建、测序及生物信息学分析等过程。本研究针对水稻在原有的ATAC-seq文库构建的方法基础上进行优化,且整理了较为详细的实验方法,有利于更高效地提取水稻的细胞核,为构建高质量文库提供参考。为探究低磷处理条件下染色质可及性的变化,本研究用100μM和5μM磷浓度处理的水稻幼苗进行实验,采取正常供磷和低磷浓度处理条件下的水稻叶片构建ATAC-seq文库和RNA-seq(转录组测序)文库,文库测序完成后通过生物信息学软件对水稻ATAC-seq和RNA-seq数据进行分析整理,得到如下结果:1.水稻ATAC-seq文库构建方法中Percoll纯化法较蔗糖密度梯度离心法效率高。在构建ATAC-seq文库的方法上,我们通过不同的试验方法提取细胞核,对比了不同方法的细胞核提取效果,分别尝试了蔗糖密度梯度离心法和Percoll法这两种方法来纯化细胞核,Percoll纯化法比蔗糖纯化法纯化效率更高,操作更为方便,在相同起始材料的情况下获得的细胞核数量更多。纯净的细胞核经DAPI染色后观察并计数,利用实验文库构建所需数量的细胞核,对细胞核进行转座反应,反应获得的DNA片段纯化后经PCR扩增得到足够浓度和总量的DNA片段。为了获得目标DNA产物,用磁珠纯化和分选,用高灵敏度的Pico Green染料染色对文库DNA的浓度进行检测,得到了符合测序条件的ATAC-seq文库。磁珠纯化法能有效获得目的片段。2.构建了水稻低磷处理条件下的水稻ATAC-seq文库,文库质量符合测序要求。为了验证磷处理的浓度有效性,分别测定了100μM和5μM磷浓度处理7天条件下水稻地上部和根的无机磷的浓度,低磷条件下与正常磷处理条件下的水稻相比,无论是地上部还是根都有显著差异,低磷降低了水稻体内磷浓度。除此之外,RT-q PCR检测结果显示IPS1、SPX1、Pht1;2、Pht1;6这些响应磷胁迫的Marker基因表达显著上调,验证了该低磷处理条件的有效性。3.低磷条件下与正常供磷条件下染色质可及性存在差异。利用生物信息学分析软件对测序数据深入分析。文库中插入片段分布在200 bp附近,分析了读数相对于TSS的分布,读数主要分布TSS上下游3000 bp范围内。对峰顶上下游3 kb区域的ATAC-Seq信号进行统计,发现读数集中分布顶峰周围3000 bp内。将差异峰分别比对到不同的功能区域,约有50%的峰在启动子区域分布,外显子区域峰占比3%。4.转录组分析结果显示,不同磷处理之间的差异基因中富集到了参与低磷调控相关的基因。利用软件HISAT2将过滤后的数据比对到水稻参考基因组上,结果显示各样本的唯一比对率大于96%,共富集了2745个基因,其中有521个表达上调,766个表达下调,在上调的基因中有参与低磷调控相关的基因。5.将转录组中的差异基因与差异峰相关基因进行联合分析,272个峰富集基因与690个差异基因中有5个基因相同,染色质相对开放区域与基因表达上调中有2个共同的基因,染色质开放区域与基因表达下调的共有2个基因相同,这可能分别与染色质开放促进转录表达和抑制转录表达有关,染色质相对闭合区域与基因表达上调无交集。综上所述,本研究优化了水稻中ATAC-seq文库构建的方法,构建了磷饥饿条件下水稻全基因组染色质图谱和转录组图谱。根据试验结果,推测低磷触发了水稻染色质可及性的变化,染色质可及区域信号高度富集在转录起始位点附近3kb区域内,染色质可及性的变化与基因表达之间存在关联。本研究为水稻细胞核的提取提供了更优的方法,为后续探索低磷条件下水稻染色质可及性和相关转录机制的深入研究奠定了基础,有利于磷饥饿的分子调控机制和鉴定转录因子的深入研究。更多还原

【Abstract】 Phosphorus is one of the necessary macronutrients for plant growth and development.It is an important component of cellular biomolecules such as ATP,nucleic acid and phospholipid.It is involved in biological processes such as energy transfer,protein metabolism and carbon activation.Phosphorus exists mostly in organic form in soil,part of phosphorus is fixed by organisms into organic state,and the rest of inorganic phosphorus is easy to form insoluble compounds with iron,aluminum,calcium and other metal ions.Phosphorus deficiency exists in most soils,and the content of inorganic phosphorus absorbed by plants is low,which can not meet the needs of normal growth and development of plants.In order to cope with P deficiency,plants have formed a series of adaptation mechanisms during evolution,including changes in root configuration,generation of root exudates,formation of mycorrhizal symbionts,induction of high affinity P transporters and other processes to improve the uptake of P by roots.Phosphorus absorbed into plants is transported to various tissues and organs with the participation of phosphorus transporters.Under different stress conditions,rice can promote the expression of different genes and form different regulatory networks.As a kind of nutrient stress,the molecular mechanism of phosphorus starvation involved in phosphorus regulation in rice has been extensively studied.In recent years,epigenetic inheritance has become a focus of research,and the study of chromatin accessibility has been gradually deepened.The emergence of ATAC-seq(assay for transposase accessible chromatin with high-throughput sequencing)technology provides more possibilities for studying chromatin accessibility in plants.This method can be used to construct genome chromatin map,search transcription factors,and construct regulatory network in combination with transcriptional expression analysis.ATAC-seq technology uses transposase to capture inputs in open chromatin regions and attach sequencing joints at both ends of open chromatin regions.The main processes include nuclear preparation,library construction,sequencing and bioinformatics analysis.In this study,the method of constructing rice ATAC-seq library was optimized,and a more detailed experimental method was arranged,which was conducive to more efficient extraction of rice nucleus,and provided a reference for constructing high-quality libraries.In order to explore the changes of chromatin accessibility under different phosphorus treatment conditions,this study used rice treated with 100μM and 5μM phosphorus concentrations to construct ATAC-seq libraries and RNA-seq(transcriptome sequencing)libraries from rice leaves treated with different phosphorus concentrations.After the library sequencing was completed,the rice ATAC-seq and RNA-seq data were analyzed and sorted by bioinformatics software,and the following results were obtained:1.Percoll purification method was more efficient than sucrose density gradient centrifugation method in the construction of rice ATAC-seq library.In terms of the method of constructing ATAC-seq library,we extracted nucleus by different test methods,compared the effect of nuclear extraction by different methods,and respectively tried two methods of sucrose density gradient centrifugation and Percoll method to purify nucleus.Percoll purification method was more efficient and convenient than sucrose purification method.The number of nuclei obtained with the same starting material is higher by Percoll.After DAPI staining,the pure nuclei were observed and counted,and the required nuclei of library was constructed,and the nuclei were transposed.The DNA fragments obtained from the reaction were purified and amplified by PCR to obtain sufficient concentration and total amount of DNA fragments.In order to obtain the target DNA products,the DNA concentration of the library was detected by magnetic bead purification and sorting,and the concentration of DNA was detected by high sensitivity Pico Green dye.Magnetic bead purification method can effectively obtain the target fragments.2.Rice ATAC-seq library was constructed under the condition of rice low phosphorus treatment,and the quality of the library met the sequencing requirements.In order to verify the effectiveness of phosphorus treatment,the concentrations of inorganic phosphorus in the aboveground part and root of rice were measured under the condition of 100μM and 5μM phosphorus treatment for 7 days,respectively.There were significant differences between the aboveground part and root of rice under the condition of low phosphorus treatment and the condition of normal phosphorus treatment,and low phosphorus reduced the phosphorus concentration in rice.In addition,the results of RT-q PCR showed IPS1,SPX1 and Pht1;2,Pht1;6.The expression of these Marker genes in response to P stress was significantly up-regulated,which verified the effectiveness of the low P treatment condition.3.There are differences in chromatin accessibility under different low phosphorus conditions.Bioinformatics software was used to analyze the sequencing data.The size of inserted fragments in the library peaked at 200 bp,and the distribution of readings relative to TSS was analyzed.The R readings were mainly distributed in the upper and lower reaches of TSS at 3000 bp.The ATAC-seq signals in the upper and lower 3 kb region of the summit were counted,and it was found that the readings were concentrated within 3000 bp around the summit.The difference peaks were compared to different functional regions,about 50%of the peaks were distributed in the promoter region and 3%in the exon region.4.Transcriptome analysis showed that genes involved in low phosphorus regulation were enriched in differential genes between different phosphorus treatments.HISAT2software was used to compare the filtered data to the rice reference genome,and the results showed that the unique comparison rate of each sample was greater than 96%,and a total of2745 genes were enriched,of which 521 were up-regulated and 766 were down-regulated.Among the up-regulated genes,there were genes involved in the regulation of low phosphorus.5.The combined analysis was conducted on the genes of the regions of differential genes and differential peak ratio in the transcriptome.The 272 peak enriched genes shared 5 genes with 690 differential genes,the relatively open region of chromatin shared 2 genes with up-regulated gene expression,and the open region of chromatin shared 2 genes with down-regulated gene expression.This may be related to the promotion and inhibition of transcription expression by open binding of chromatin,and the relatively closed region of chromatin has no intersection with the up-regulation of gene expression.In conclusion,this study optimized the method of constructing ATAC-seq libraries in rice,and constructed whole-genome chromatin and transcriptome maps of rice under phosphorus starvation conditions.According to the results of the experiment,it was speculated that low phosphorus triggered the change of chromatin accessibility in rice,and the signal in the chromatin accessibility region was highly enriched in the 3kb region near the transcription initiation site.There is an association between changes in chromatin accessibility and gene expression.This study provides a better method for nuclear extraction from rice,lays a foundation for further research on chromatin accessibility and related transcription mechanisms in rice under low phosphorus conditions,and is conducive to further research on molecular regulatory mechanisms of phosphorus starvation and identification of transcription factors.更多还原