【作者】 尹福强；

【导师】 潘光堂；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 烟草是我国重要的经济作物,栽培面积和产量均居世界首位,经济效益高,是我国财政收入的重要组成部分。干旱严重影响烟叶产量和品质,是烟草生产主要的非生物胁迫因素之一。烟草响应干旱胁迫是一个多水平的复杂调控反应,包括了生理生化水平的调节和基因表达网络的分子适应。干旱胁迫下的基因表达调控网络十分重要,尤其是转录因子可用来提高植物对干旱胁迫的耐受性。通过对模式植物的分子生物学和基因组研究,现已发现一些干旱胁迫响应基因和各种参与调节胁迫诱导基因的转录因子。干旱胁迫增加内源脱落酸(ABA)水平,诱导依赖ABA和不依赖ABA的转录调控网络的表达。对拟南芥的相关研究已初步建立了干旱响应转录调控网络,一些重要的启动子元件,如ABA反应元件(ABREs)和耦合元件(CE),也已通过实验验证,但迄今对烟草抗旱的分子机制尚不清楚。此外,microRNAs (miRNAs)也是重要的基因表达调控因子,其主要原理是通过抑制mRNA的表达实现转录后水平的调控。拟南芥中干旱胁迫响应的转录和转录后调控之间存在复杂的相互作用,有些miRNA(如miR159和miR169等)在干旱胁迫应答反应中发挥了重要作用,但这种作用在烟草中仍未被确定。本研究以烟草品种红花大金元为研究材料,经过生理生化指标的检测和筛选,分别对六叶期幼苗进行6h和48h的短期干旱胁迫处理,分析对照和各处理在干旱胁迫下根系mRNA和miRNA的表达动态、鉴定差异表达的miRNA所调控的靶基因并预测干旱胁迫下的基因表达调控网络,旨在揭示烟草苗期在干旱逆境条件下诱导根系mRNA和miRNA的快速响应机理,为阐述烟草苗期耐旱性的分子机制及利用分子标记辅助育种提供理论依据。本研究取得以下结果：1.为选择干旱胁迫下烟草根系基因表达分析的最佳时间点,用20%的PEG6000对烟草六叶期的幼苗进行模拟干旱处理,分别于Oh,3h,6h,12h,24h,48h和96h这7个时间点取其根系(5株烟苗根系均匀混合),其中0h的样品为CK,然后测定其超氧化物歧化酶(SOD)活性、丙二醛(MDA)和脯氨酸(Pro)的含量。结果表明,SOD活性、MDA和Pro含量显着增加,6h和48h两个时间点相对于3h和24h两个时间点,其变化更为显著(P<0.05)。结合胁迫后的表型和生理生化指标的结果,本研究干旱胁迫检测的最佳时间点是6h和48h。2.选择0h,6h和48h的根组织样品作为测序的试验材料,分别命名为NCK(对照组),N6H和N48H。分别对三组样品提取总RNA,采用高通量测序技术构建了数字表达谱(DGE)的文库。经测序,3个文库平均获得约3.37Mb个reads,3个文库中共有21128个基因表达,注释的烟草基因占43.7%,其中3个文库中基因均表达的有13101个基因,仅有1887个基因差异表达,占烟草根系总表达基因的8.9%。qRT-PCR验证结果和测序结果基本一致。利用k-means聚类算法,依照基因功能分类和基因分组对差异基因进行Gene Ontology (GO)功能显著性富集分析,共分成6个类群(P<0.05),主要包含一些编码脂肪酸代谢、酰基转移酶活性、氧化还原酶活性、乙醇代谢及初始乙醇代谢和转移酶活性等的基因；Pathway分析发现有17条通路可能被影响,主要涉及谷胱甘肽代谢、脂肪酸延长、二苯乙烯类化合物和姜辣素的生物合成、次生代谢物的生物合成和苯丙生物合成等。3.根据数据库预测烟草基因组中同源的转录因子,共确定了609个转录因子,DGE的结果表明：所有的转录因子都可以在干旱胁迫下的烟草幼苗根系中检测到。这些根系中的转录因子,有82个差异表达,分属于24个转录因子家族,主要是与抗旱性相关的MYB、NAC和ERF家族,而其他差异表达的转录因子家族(HD-ZIP,NF-YA,NAC, GRAS,TCP)主要参与了发育和分生组织保持,防御／应激信号通路(HSP,WRKY和bZIP),生长素(Aux/IAA)诱导的的激素介导或胁迫介导的信号传导等。53%转录因子在NCK的表达水平最高(G1),而只有10%的转录因子是在N6H的表达水平最高(G2),另外还有37%的转录因子表达高峰在N48H(G3)。此外,我们还确定了一些家族特异性转录因子的表达趋势。C2H2、MYB、WRKY、ERF和Dof家族的转录调节因子分别有3、3、4、3、13和3个基因在NCK中高效表达,GATA、MYB和ERF家族的转录因子在干旱胁迫过程的NCK和N6H时间点分别有1、3和3个基因的表达水平最高；MYB、NAC、MYB相关、NF-YA,HD-ZIP、和ERF家族分别有6、6、2、2、2和2个基因在N6H优先表达。4.通过与已知基因和Nicotiana benthamiana烟草的注释信息进行序列同源性比较,我们发现了276个干旱应答候选基因(DRGs),其中,约40%(110/276)的基因是WRKY、 NAC、ERF和bZIP家族的转录因子。同时,我们也分析了这些干旱应答候选基因(DRGs)的功能,发现了其中有46个是差异表达的干旱应答候选基因(DRGs)。在这些基因中,21个(46%)是分属于NAC (6),MYB (4),ERF (10)和bZIP (1)家族的转录因子；其他干旱应答候选基因(DRGs),如GRF6、ABF1、APX2, SIPK和ZPT2,在干旱胁迫时也呈现不同的表达模式。5.从干旱处理和对照的根组织样品以及正常发育的叶、茎组织中,分离所有小分子RNA,构建small RNA文库并进行高通量测序,测序经生物信息学分析,共检出122个烟草miRNAs。在全部的miRNAs中,保守的miRNAs比非保守的miRNAs的表达量要高得多,如miR166和miR168家族的的表达丰度占总测序片段的57%和16%。然而,仍有43个前人报道的miRNAs未在这些样品中检测到。相对于对照,干旱胁迫的样品中有5个已知的烟草miRNA家族表达量的相对变化(log2root-ck/root-treat)大于2,表明表达差异显著或极显著。虽然miR159, miR169, miR402和miR408的表达在其它植物受干旱胁迫的差异显著,但在本实验中根系样品处理前后表达并没发生明显的改变。6.为了解烟草响应干旱胁迫的miRNA的功能,以前人通过降解组测序得到的已鉴定的烟草miRNA的靶基因为参考,我们的研究仅有27个靶基因GSS序列可以比对到烟草参考基因组中,并与87个转录子相对应。只有两个干旱响应的miRNA家族(miR160和miR395)找到了对应的靶基因。7.在干旱和低温应急反应过程中,许多研究人员提出了响应过程中的复杂的调控网络,涉及到miRNA和其调控的靶基因。本研究在前人的基础上,结合DGE、small RNA的测序数据和其调控的靶基因也绘制了一个简易的调控网络。这个网络主要包含两条通路(依赖ABA和不依赖ABA的通路)。在依赖ABA的通路中,NCED参与了对干旱胁迫的快速和应急反应。其级联转录过程,包括了AREB/ABF,MYB,bZIP, NAC和CBF/DREB1,他们参与了在应激反应中的渐进和适应的过程。SnRK2.6蛋白激酶也参与了在ABA中的信号传导。在不依赖ABA通路中,未知蛋白被认为具有渗透传感器和ERF系统的上游元件的功能。此外,响应的miRNAs (miR160、miR162、 miR394miR395,和miR827)也都表明是其表达存在时间的特异性。这个调控网络的绘制和分析将为以后研究烟草应对干旱,低温和重金属等非生物胁迫反应的基因表达调控提供参考。更多还原

【Abstract】 Tobacco is an important agricultural and economic crop in China, and its cultivation area and yield in China were the highest in the world. Moreover, Tobacco is an important part of Chinese fiscal revenue. Drought seriously impacts on tobacco yield and quality, which is one of main environmental stresses resulting in reduction of yield loss.To survive under drought stress in their rooted lifestyle, tobacco has evolved a considerable degree of drought stress response, which is a complex trait regulated at multiple levels including the adjustment of various biochemical and physiological processes and molecular adaption to gene regulatory network. The regulatory networks of gene expression under drought and cold stress are critical, and that particular transcription factors can be employed to enhance drought stress tolerance in plants.Decades of research into the effects of drought on model plant physiology and development have generated a wealth of information by molecular biology and genomics. It has identified some drought responsive genes (DRGs) under drought stress and some transcription factors (TFs) involved in regulation of these DRGs. Drought stress increases endogenous abscisic acid (ABA) levels and induces ABA-dependent and ABA-independent transcriptional regulatory networks. Many of these responses can be mimicked by external application of ABA. Drought-responsive transcriptional networks have been primarily developed from related studies in Arabidopsis, and some important promoter elements were confirmed experimentally, such as ABA-responsive elements (ABREs) and coupling elements (CE). However, the networks that underlie these responses in tobacco have not been extensively characterized. In addition to DRGs and these TFs mentioned above, microRNAs (miRNAs) are short (20-22nt), endogenously expressed, non-translated RNAs that function in posttranscriptional gene regulation. There is a complex interplay between transcriptional and posttranscriptional regulation of drought response in Arabidopsis, for instance, some miRNAs (miR159and miR169) play an important role in drought stress response. Unfortunately, it has not been extensively characterized in tobacco.In the study, the roots of a flue-cured tobacco (Nicotiana tabacum L.) cultivar, Honghua Dajinyuan (a drought-tolerant cultivar) were treated in time-course drought stress and then detected by physiological analyses. Only these samples at6h and48h treatment and normal sample were used for analyses of mRNA and miRNA expressing profiling, identification of their target regulated by specially-expressed miRNAs, developing and mapping the transcriptional and posttranscriptional gene regulatory network. We aim to unveil the roles of mRNAs and miRNAs in rapidly responding to drought in tobacco root cell, which will give sight to mRNA and miRNA regulation mechanism under drought stress and help breeder breed excellent drought resistance inbred lines by the marker-assisted selection of DRGs.1. To explore the optimal time point under drought stress for gene expression analysis, uniform seedlings of tobacco with six leaves were challenged to drought stress treatments at six time points (3,6,12,24,48, and96h) with20%PEG6000. We measure three physiological indexes, Superoxide dismutase (SOD) activities and proline (PRO) and malondialdehyde (MDA) contents. These results suggest that SOD activities, PRO, and MDA contents all significantly increased at6h and48h relative to3h and24h, respectively, and the optimal time point for drought stress assays are6h and48h. Our experiments using tobacco roots with experiments at two time points and control were named as NCK (control), N6H, and N48H, respectively.2. Total RNA was isolated from the frozen root samples and DGE library preparation was then performed in parallel by using the Illumina gene expression sample preparation kit. The sequence reads of all libraries ranged from3.33M (million) to3.39M, and averaged about3.37M. We examined the dynamics of gene expression under drought stress using DGE data, and only1,887out of21,128genes that were differentially expressed among NCK, N6H, and N48H, represented8.9%of the root transcriptome. A sample of22transcripts with significant differences in gene expression was randomly selected for validation via qRT-PCR, which were consistent with that obtained from DGE. We used Gene Ontology annotation to assign genes to functional categories and grouped genes by expression dynamics using the K-Means clustering algorithm. We identified six clusters which contained many genes that encode enzymes for fatty acid metabolism, transferase (transferring acyl groups), oxidoreductase, ethanol metabolism, primary alcohol metabolism and transferase, etc. In addition, biological pathways influenced by drought were evaluated by enrichment analysis of all differentially expressed genes. Significantly enriched metabolic pathways and signal transduction pathways were also identified. A total of17pathways, including contained glutathione metabolism, fatty acid elongation, stilbenoid, diarylheptanoid and gingerol biosynthesis, biosynthesis of secondary metabolites were affected based on the above mentioned six clusters (P＜0.05).3. A primary objective was to identify genes that encode TFs and resolve the dynamics of accumulation of TFs under drought stress in our DGE data. To test this, we retrieved putative orthologs of tobacco genes based on information from the EnsemblCompara gene trees at solgenomics.net, plantgdb.org, and http://www.ncbi.nlm.nih.gov/. We then queried known plant TFs in the Plant Transcription Factor Database (v2.0, http://planttfdb.cbi.edu.cn/) and identified609tobacco TFs with sequence similarities to known plant TFs. Furthermore, all TFs can be detected in roots of tobacco seedlings responding to drought stress. Of these TFs in root tissue,82were differentially expressed during time-points and belong to24TF families. These TFs associated with functions in drought tolerance (MYB, NAC, and ERF), while others played roles in development and meristem maintenance or identity (HD-ZIP, NF-YA, NAC, GRAS, and TCP), defense/stress signaling pathways (HSP, WRKY, and bZIP), hormone-mediated or stress-mediated signaling by auxin (AUX/IAA). The abundance of most of these TFs (53%) was at the highest levels in NCK (G1), whereas only10%was at the highest levels in N6H (G2). The reminder (37%) indicated peak expression in N48H (G3). We also identified family-specific expression trends. Members of the C2H2(3genes), MYB (3), bHLH (4), WRKY (3), ERF (13) and Dof (3) families of transcriptional regulators were highly expressed in NCK. Several GATA (1), MYB (3) and ERF (3) TFs accumulated to the highest levels during the stress-response phase from NCK to N6H. Transcriptional regulators including MYB (6), NAC (6), MYB-related (2), NF-YA (2), HD-ZIP (2) and ERF (2) were preferentially expressed in N48H.4. We identified276candidate DRGs in tobacco with sequence similarity to known genes and Nicotiana benthamiana annotation. Interestingly, about40%(110out of276genes) were TFs including WRKY, NAC, ERF, and bZIP families. In the present study, we also investigated the roles of these candidate DRGs, and found46differentially expressed DRGs under drought stress. Out of the54differentially expressed DRGs,21(46%) were TFs which belonged to NAC (6), MYB (4), ERF (10), and bZIP (1) families. Other DRGs, such as GRF6, ABF1, APX2, SIPK, and ZPT2, have different expression patterns in response to drought stress.5. The samples of our small RNA libraries were used based on the result of physiological index measurement as follows:equal quantities (10ug) of total RNA isolated from tobacco roots treated with two time points (6and48h) were mixed together to construct the drought-treated small RNA library (Root-treat), and total RNA prepared from the control roots sample was used to construct the control small RNA library (Root-ck). Here,122tobacco miRNAs were detected in our sequencing datasets. Conserved miRNAs were far more abundant than non-conserved miRNAs in our libraries as reported previously. MiR166and miR168were the most abundant miRNA families which accounted for about57%and16%of the total sequence reads from the known miRNAs datasets, respectively. However,44experimentally identified tobacco miRNA families (33miRNAs) were not detected in our dataset. Comparison of the normalized sequence reads of the miRNAs between the two libraries indicated that five known tobacco miRNA families had relative changes (log2root-ck/root-treat) greater than two and thus might be differentially or extremely differentially expressed. However, miR159, miR169, miR402, and miR408sequence reads displayed no meaningful changes between two libraries even though their expression had been reported to be affected by drought stress treatments in other plants.6. MiRNAs regulate gene expression at the posttranscriptional level by repressing mRNA expression, and some miRNA families were experimentally verified to be responsive to salt or drought stress in plants in recent research. To understand the functions of drought-responsive miRNAs in tobacco, we got the complete list of targets of tobacco miRNAs identified by degradome sequencing in a recent study, and only27target_GSS sequences can be mapped to the tobacco reference genome and correspond to87transcripts. Unfortunately, the targets of only two drought-responsive miRNAs (miR160and miR395) were obtained.7. With the availability of regulatory networks of gene expression in drought and cold stress responses, an integrated gene regulatory network has been proposed for the molecular mechanisms of the response of tobacco roots to drought stress using differentially expressed DRGs, the changed expression profiles of miRNAs and subsequent target transcripts as a basis. Two pathways (ABA-dependent and ABA-independent) can shed light into cell mechanisms involved in stress signaling and/or adaptation at transcriptional regulation. In the ABA-dependent pathway, NCED1was involved in rapid and emergency responses to drought stress. Left parts of Figure7show transcription cascades that were involved in slow and adaptive processes in stress responses, such as those involving AREB/ABF, MYB, bZIP, NAC and CBF/DREB1. SnRK2.6protein kinases were also involved in ABA signaling. In the ABA-independent pathway, unknown proteins were thought to function as an osmo-sensor and function upstream of the ERF system. In addition, the responsive miRNAs (miR160, miR162, miR394, miR395, and miR827) also showed a transitory expression model. Right parts of Figure7show proposed regulation cascades after drought in tobacco roots. This network analysis will also serve as a reference for future studies on tobacco responses to various stresses, such as to drought, cold and heavy metals.更多还原