【作者】 林原；

【导师】 谢从华；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2013， 博士

【摘要】 马铃薯(.Solanum tuberosum L.)是世界第四大粮食作物,在保障食品安全中具有重要地位。为了减小常温贮藏产生的失水皱缩、病害传播,腐烂及发芽等情况并延长加工周期,在农业生产中通常将收获的块茎贮藏于低温下,但是在低温贮藏过程中,马铃薯块茎低温糖化所积累的还原糖导致油炸加工过程中产品色泽变深,口感变苦及有害物质如丙烯酰胺的积累,从而严重影响了加工品质。因此,马铃薯低温糖化是块茎油炸加工业的瓶颈。诸多研究表明,低温下块茎中转化酶酶活性持续上升是造成低温糖化的重要因素。尽管马铃薯存在特异转化酶抑制蛋白,但关于转化酶活性调控机制还不完全明确。基于此,在本实验室的前期工作的基础上,本研究拟通过分析转化酶StvacINV1和转化酶抑制子StInvInh2B的互作蛋白组,从中筛选出与转化酶StvacINV1活性调控相关的互作蛋白,并进行功能分析和鉴定,以明确StvacINV1的翻译后调控机制。主要研究结果如下：1. StvacINV1/StInvInh2B蛋白在低温糖化抗/感基因型中的互作蛋白组分析用马铃薯抗低温糖化(S. berthaultii, CW2-1)和低温糖化敏感(S. tuberosum,AC035)基因型作为材料,分别构建酵母双杂交文库。以转化酶StvacINVl和其特异的转化酶抑制子StInvInh2B为饵蛋白,分别从两个杂交文库中筛选各自的互作蛋白组,并通过不同的假阳性排除方法筛选出潜在的互作蛋白。研究结果表明,StvacINVl从CW2-1文库中筛选到24个潜在互作蛋白,从AC035文库中筛选到了3个潜在互作蛋白；而StInvInh2B从CW2-1文库中筛选到8个潜在互作蛋白。潜在互作蛋白组生物信息学分析发现,除了筛选得到StvacINVl新的抑制子外,还发现StvacINVl和SflnvInh2B能分别和同一个蔗糖非酵解-1相关激酶(Sucrose non-fermenting-1(SNFl)-related protein kinases) SbSnRKl的α和β亚基(SbSnRKla, SbSnRK1β)互作,从而说明马铃薯StvacINV1的翻译后调节可能是一种多蛋白互作的模式。2. StvacINV1-StInvInh2B-SbSnRK1复合体及其对StvacINV1活性的体外调控研究在烟草活体细胞内对StvacINV1, StlnvInh2B和SnRK1的互作进行了双分子荧光互补验证,表明这三个蛋白质之间存在互作,推测其可能组成一个蛋白质复合体并能调控其中StvacINV1的活性。通过真核或原核表达,获得了有活性的StvacINV1, StInvInh2B以及SbSnRKlα和SbSnRK1βp亚基的重组蛋白,体外的酶活性实验结果显示,SbSnRK1β能封阻StInvInh2B的转化酶抑制功能,而SbSnRK1α的磷酸化水平则能调控SbSnRK1β的功能。本研究首次揭示并证明StvacINV1-StInvInh2B-SbSnRK1组成了一个蛋白质复合体,并通过SbSnRK1a的磷酸化水平,对StvacIN1的活性进行精细调控。3. SbSnRK1a基因在马铃薯低温糖化中的作用为了研究SbSnRK1a在马铃薯低温糖化过程中的功能,本研究分别构建了SbSnRK1a的超量和干涉载体,对低温糖化敏感的基因型鄂马铃薯3号(E3)进行了遗传转化,并分别获得了15超量和8个干涉转基因株系。根据基因表达水平,分别对其中5个超量和4个干涉株系的相关生理生化指标进行了测定,结果表明超量株系块茎在低温贮藏30天后,转化酶活性降低80～95%,还原糖含量也降低90%左右,油炸薯片的色泽显著降低；而干涉株系呈现相反的趋势。与此同时,淀粉-糖代谢的其它酶类和代谢产物相关测定表明,除转化酶活性及其代谢直接产物(蔗糖、还原糖)有明显变化外,其他酶活性及代谢产物并无明显变化。这些结果证明SbSnRK1a基因参与蔗糖分解途径,通过调控转化酶活性影响块茎中还原糖的积累。本研究结果同时显示,SbSnRK1a对改良马铃薯“低温糖化”性状具有潜在的应用价值。更多还原

【Abstract】 Potato (Solanum tuberosum L.), the fourth important food crop in the world, is vital to global food security. After harvest, potato tubers are often stored at low temperature to prevent sprouting and minimize the disease losses. However, low temperature leads to accumulation of reducing sugars (mainly glucose and fructose) in the tubers known as cold-induced sweetening (CIS). During frying in high temperature, the reducing sugars react with free amino acids and result in unacceptable color change and acrylamide formation in potato chips or French fries, which cause major economic losses and healthy concerns. We previously reported that the vacuolar invertase (VI, one of the most critical enzymes in CIS) can be inhibited by endogenous invertase inhibitor (VIH). However, VI activity regulation is still unclear. In present study, proteins associated with StvacINV1activity were captured from potato and the mode of post-translational regulation of VI activity was further clarified both in vivo and in vitro. The main achievements are as below.1. The protein-protein interaction (PPI) protomics of StvacINVl and StInvInh2B in potato CISTo understand the target proteins of VI and VIH, the two yeast-two hybrid (Y2H) libraries were constructed for potato CIS-resistant wild species S. berthaultii acc. CW2-1and CIS-sensitive cv. AC035, respectively. The StvacINV1and StInvInh2B were employed as the baits for Y2H libraries screening, and two protein-protein interaction (PPI) protomics were obtained after false positive analysis. One contained27potential interaction partners of StvacINV1(24from CW2-1library and3from AC035), and is the other incuded8potential target proteins of StInvInh2B (all from CW2-1library). Noticeably, not only putative invertase inhibitors were captured by StvacINV1, but also the a and β subunits of sucrose non-fermenting-1-related protein kinase1(SnRKl), were selected by both StvacINV1and StInvInh2B. The results suggested that, in addition to the invertase inhibitors, other proteins may also contribute to the VI activity modulation at poat-translational level. 2. Confirmation of StvacINV1-StInvInh2B-SbSnRKl complex in plant and the regulatory mode for VI activityThe StvacINV1-StInvInh2B-SbSnRKlprotein complex was reconfirmed in tobacoo cells using BiFC assay. All proteins of the complex including StvacINV1, StInvInh2B, and a and β subunit of SbSnRK1were individually expressed in vivo and purified proteins were obtained for further analysis. The StvacINV1protein was mixed in each possible combinations with StInvInh2B, SbSnRKla and SbSnRK1β. The residual activity of StvacINV1in these protein mixtures was measured and used to indicate the effects of each protein on the alteration of VI activity. The results demonstrated that inhibitory function of StInvInh2B to StvacINV1was suppressed by SbSnRK1β, and phosphorylation of SbSnRKla counteracted SbSnRK1β roles which led to restore of the function of StInvInh2B and, hence, inactivation of StvacINV1. A subtle regulatory mode of VI activity is established.3. SbSnRK1a plays critical roles in potato CISTo justify if the roles of SbSnRKla played in the protein complex have impacts on potato CIS, the SbSnRK1a gene was transformed into a CIS-sensitive potato variety E-Potato3(E3) by Agrobacterium-mediated over-expression (OE) and RNA interference (Ri). In total,15OE-and8Ri-lines were obtained. Depends on the SbSnRK1a transcripts,5OE-and4-Ri lines were further analyzed in potato CIS. After the cold storage, all the OE tubers had chip color remarkably lighter than wild type E3, whereas the Ri tubers showed darker color. This phenomenon was in accordance with the changes of invertase activity that decreased by83～95%in OE tubers and increased by30-100%in Ri tubers after stored at4℃for30days, suggesting that transcriptional elevation of SbSnRK1a remarkably improved chip color by suppressing the invertase activity. This result provides in-depth understanding of the regulation of invertase cleavage pathway in the process of potato CIS.更多还原