【作者】 欧庸彬；

【导师】 柳俊；

【作者基本信息】 华中农业大学 ， 遗传学， 2013， 博士

【摘要】 马铃薯(Solanum tuberosum L.)是世界第四大粮食作物。中国是马铃薯第一生产大国,马铃薯消费正由鲜食为主逐渐向高附加值产品转变。为了延长市场供应期,通常将收获后的马铃薯块茎贮藏在低温条件下,而低温贮藏过程中块茎还原糖含量上升,造成马铃薯加工品质下降。因此,明确低温糖化的调控机制是马铃薯品质研究的难点与热点。前人研究显示,低温条件下转化酶活性上升是造成低温糖化的主要因素。但关于转化酶基因(StvacINV1)在马铃薯块茎低温贮藏中的表达调控机制还不完全明确。本研究基于本实验室前期的工作,通过克隆分析StvacINV1基因的启动子,同时通过分析miRNA及其靶基因,以明确StvacINV1的转录调控和转录后调控机制。主要研究结果如下：1. StvacINV1基因在抗低温糖化和低温糖化敏感型基因型中的表达模式研究选用6个马铃薯基因型作为材料,分析不同基因型的低温糖化敏感程度和StvacINV1表达模式。结果显示,20℃条件下所有基因型在整个贮藏期间的炸片色泽、还原糖含量和蔗糖含量变化幅度较小,但在4℃条件下贮藏的块茎,所有基因型炸片色泽随贮藏时间的延长而逐渐加深,还原糖含量和蔗糖含量上升。色泽指数分析显示,抗低温糖化基因型ND860-2、10908-06、CW2-1薯片色泽指数上升幅度较小；而低温糖化敏感基因型11059-01、ED25和E3薯片色泽指数上升幅度较大。可溶性酸性转化酶活性和StvacINV1转录本丰度分析显示,可溶性酸性转化酶活性在4℃贮藏5d-15d之间上升,而StvacINV1转录本丰度则在4℃贮藏8h-16h即开始上升。低温贮藏后,StvacINV1转录本丰度均高于20℃贮藏的水平,且在栽培种中抗低温糖化基因型和低温糖化敏感基因型间StvacINV1基因的表达模式无规律性的差异。但野生种S. berthaultii (CW2-1)的StvacINV1基因表达水平在低温贮藏5d时明显低于栽培种,暗示该基因型中StvacINV1基因在转录水平的调控可能影响其低温糖化性状。2. StvacINV1基因启动子克隆与功能分析为了明确StvacINV1基因在不同基因型中的表达是否与启动子结构有关,实验选用10个马铃薯基因型,采用高效热不对称PCR技术克隆了StvacINV1基因的启动子。测序结果显示,来自马铃薯不同基因型的启动子序列一致性为94.1%-99.9%,其结构上没有规律性的差异。生物信息学分析显示,StvacINV1基因启动子序列上有9个糖抑制元件、8个赤霉素应答元件。进一步转基因分析表明,该启动子可驱动报告基因在根、茎、叶以及块茎中表达,蔗糖、葡萄糖、果糖抑制启动子活性,而赤霉素和生长素可促进启动子活性。启动子缺失实验证明,该启动子应答蔗糖／葡萄糖、赤霉素、生长素的启动子区域分别为-118至-551、-551至-1021,以及-1021至-1521。在本研究中,低温能够抑制StvacINV1基因启动子活性,与该基因的表达模式存在明显的差异,暗示该基因的表达水平可能受到其它因素的影响,如转录后调节。3.低温糖化相关的miRNA及其靶基因分析研究以抗低温糖化基因型10908-06为材料,分别构建了块茎4℃和20℃贮藏条件下的small RNA文库和降解组文库。通过高通量测序和分析,共获得53个known miRNA、60个novel miRNA、70个miRNA*以及70个靶基因。以测序信息为基础的分析结果表明,36个miRNA/miRNA*在不同贮藏温度条件下呈现差异表达。进一步选择了24个miRNA/miRNA*和55个靶基因通过RT-qPCR进行表达分析,结果显示,共有12miRNA/miRNA*和11个靶基因在4℃和20℃以及抗低温糖化和低温糖化敏感型基因型间表达水平差异显著。12个表达差异显著的miRNA/miRNA*与已进行RT-qPCR分析的靶基因比较分析,有6个miRNA/miRNA*可以匹配上11个靶基因。这11个靶基因中,有3个靶基因在在两个温度及两个基因型间的表达差异达到显著水平,3个靶基因涉及1个miRNA (miR172)和1个miRNA*(miR396a-3p)。miRNA/miRNA*及其靶基因的对比分析证明,miR172在抗低温糖化基因型中,块茎低温贮藏2d显著上调表达,其匹配的靶基因在同一处理的时间内下调表达,靶基因呈现明显的miRNA调控关系。miR396a-3p与其靶基因未呈现明显的调控表达模式。在本研究中,miRNA/miRNA*未发现转化酶靶基因,同时,StvacINV1拘转录本水平与马铃薯低温糖化无显著相关,表明该酶的活性可能是其调控低温糖化的关键因素。这一结论由本实验室最近发现的StvacINV1的翻译后调节机制所证实。但是,本研究所发现的差异表达的miRNA/miRNA*及其靶基因可能为全面了解马铃薯低温糖化的调控机制提供新的线索。更多还原

【Abstract】 Potato (Solalnum tuberosum L.) is the fourth most important food crop in the wold and China ranks the first with its potato production. The global consumption of potato is shifting from staple food to value-added processed products. Potato tubers are often stored at low temperature to prevent sprouting and minimize disease losses. However, low temperature leads to accumulation of reducing sugars in potato tubers, a process known as the cold-induced sweetening (CIS). Reducing sugars react with free amino acids during frying for potato chips or French fries, resulting in unacceptable color change and acrylamide formation, which cause major economic and healthy concerns. Potato vacuolar acid invertase that hydrolyzes sucrose into glucose and fructose has been confirmed to play an important role in CIS. StvacINVl, which encods a potato vacuolar acid invertase, is induced by low temperature in potato tubers and is a main contributor to invertase activity. However, its expression regulatory mechanismemains to be elucidated.To disclose the factors that regulating the StvacINVl transcription, the promoter of StvacINV1was cloned and analyzed. Futhermore, the microRNAs (miRNA) in connection with CIS were analyzed by deep sequencing of small RNA libraries and degradomes. The main results are as follows:1. Six potato genotypes with distint CIS resistance were selected for dynamic analysis of sugar accumulation and invertase expression patterns in tubers during one month of low temperature storage. The significant increase of sucrose content was found to occur earlier than the raise of reducing sugar content in all the genotypes tested. The activity of soluble acid invertase increased accompanied with sugar accumulation, and the transcripts of StvacINV1increased rapidly in tubers exposed to cold treatment. However, no distinct StvacINVl expression pattern could be clarified between the CIS-resistant and the CIS-sensitive genotypes.2. The5’-flanking sequence of StvacINV1was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINVl promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINVl promoter was suppressed by sucrose, glucose, fructose and cold, while it was enhanced by indole-3-acetic acid (IAA) and giberellic acid (GA3). Further deletion analysis clarified that the promoter regions from-118to-551,-551to-1021, and-1021to-1521were responsive to stimuli of sucrose/glucose, GA3and IAA, respectively. The events of reduction in the promoter activity under low temperature implied other regulatory mechanisms for StvacINV1expression, such as post-trnascriptional regulation.3. Two small RNA and two degradomes libraries were constructed from the potato (10908-06) tubers stored at20℃or4℃for deep sequencing. A total of53known miRNAs,60novel miRNAs and70miRNA*s were identified by small RNA sequencing, and70miRNA-targeted genes were predicted by degradome sequencing. The expression level of36miRNAs/miRNA*s showed significant changes in response to cold. Twenty-four miRNAs/miRNA*s and55targets were selected for the expression profiling. The results showed that12miRNAs/miiRNA*s were differencially expressed between20℃and4℃, and between CIS-resistant and the CIS-sensitive genotypes. Among them,11targets were matched by six miRNA/miRNA*, including three genes whose expressions were significantly different between the temperature treatments and between the potato genotypes. Of these three genes, a gene encoding APETALA2is the target of miR172, and the other two genes are the target of miR396a-3p. miR172was induced by2d-cold-storage in the tubers of10908-06along with a down-regulation of its target, indicating the expression of the APETALA2gene is possibly modulated by miR172. Since no miRNAs were identified to match the StvacINV1gene, and the aboundance of StvacINV1mRNAs was not significantly related to CIS, present results suggest that the StvacINV1activity may be essential for potato CIS. This conclusion reinforces the finding that a post-translational regulation of StvacINVl plays key roles in the process of potato CIS. However, the identified miRNA/miRNA*s, as well as their target genes, may provide new clues for approaching a full understanding of the regulatory mechanisms of potato CIS.更多还原