【作者】 成善汉；

【导师】 谢从华； 柳俊；

【作者基本信息】 华中农业大学 ， 园艺蔬菜学， 2005， 博士

【摘要】 马铃薯是世界第四大粮食作物,用途广泛,其加工产品薯片和炸薯条是风靡全球的食品。为了抑制常温贮藏产生的块茎失水皱缩、病害传播、发芽等现象及延长加工周期,常将马铃薯块茎贮藏在低温条件下。但低温常使块茎中的淀粉加速转化为还原糖,高温油炸加工时,还原糖与块茎中游离的氨基酸发生反应,严重影响炸片或炸条的色泽和品质。块茎“低温糖化”是个复杂的数量性状,涉及淀粉—糖代谢的多种路径,调节和反馈因子多,因此“低温糖化”的机理研究是马铃薯加工品质改良的重要基础工作。本研究拟探索不同温度贮藏条件下马铃薯淀粉—糖代谢的主要酶类与还原糖含量的动态变化关系,明确其主要的调控因子。在此基础上,结合前人的研究结果,分离克隆淀粉—糖代谢关键酶的调控基因,并通过转化马铃薯进行功能验证,以进一步揭示马铃薯块茎淀粉—糖代谢的分子机理,为改良马铃薯加工品质提供理论依据和技术基础。研究取得的主要结果如下: 1.以不同贮藏温度条件下的鄂马铃薯1号(E1)和鄂马铃薯3号(E3)品种为材料,对贮藏块茎还原糖、总糖含量及淀粉-糖代谢中酶活性变化规律进行研究,以明确马铃薯炸片色泽指数的主要影响因素。结果表明,贮藏期间低温是促进块茎还原糖累积的主要影响因素,还原糖含量与炸片色泽具有极显著直线正相关关系。进一步分析表明,在4℃贮藏条件下,腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、尿昔二磷酸葡萄糖焦磷酸化酶(UGPase)和蔗糖合成酶活性(SuSy)与块茎还原糖含量呈显著负指数相关,酸性转化酶(Acid Inv)和碱性转化酶(Alkaline Inv)活性与块茎还原糖的积累呈显著直线正相关,是淀粉—糖代谢过程中影响块茎“低温糖化”的主要因子。 2.转化酶抑制子是一类同源性较低的蛋白质家族,通过转化酶抑制子的调控可能达到抑制转化酶活性,进而调控还原糖积累的效果。本研究根据已报道的烟草转化酶抑制子序列设计特异引物,通过RT-PCR法在烟草T1系中成功分离到液泡转化酶抑制子基因Nt-inhh全长cDNA。序列分析表明,该基因编码区与报道的Nt-inhh基因完全一致。通过构建35S和块茎特异的patatin启动子调控下含有Nt-inhh基因cDNA的表达载体转化马铃薯。分析转基因植株块茎在4℃和20℃贮藏一个月后还原糖含量与液泡酸性转化酶活性表明,Ⅵ活性下降幅度为22.7%(株系A-3)至78.7%(株系B-13),还原糖下降幅度为20%(株系A-17)至80.5(株系A-30),说明Nt-inhh cDNA在马铃薯中的表达成功抑制了液泡酸性转化酶的活性,导致还原糖含量降低。实验还进一步证实了低温贮藏块茎液泡酸性转化酶活性与还原糖含量呈正相关关系。 3.转化酶抑制子是高等植物普遍存在的一类蛋白质家族,本研究用RT-PCR结合5’RACE方法从马铃薯栽培种JH块茎中克隆了转化酶抑制子St-inh cDNA。序列分析表明,St-inh基因编码区全长663bp,编码221个氨基酸。将St-inh基因克隆更多还原

【Abstract】 The potato (Solarium tuberosum L.) is the fourth food crop with multiple uses in the world, of which potato chips and fries are fashionable. The harvested tubers of potato are stored at low temperature to prevent losses caused by spoilage spreading, sprouting and shrinkage, and to extend the processing period of industries. Cold storage, however, induces the accumulation of reducing sugar (RS) due to the conversion of starch to reducing sugar that can react with the free amino acid of tubers at high temperature frying. This results in a dark-coloured, bitter-tasting and undesirable product unfiting for human consumption. The statch-sugar conversion involves multiple metabolic pathway with multi-regulaing and -feedback regulating factors. Therefore, mechanism of "low temperature sweetening (LTS) " is a basic research for improving potato post-harvest quality. The aim of the present research reported is exploit the dynamic relationship between the content of RS and activities of the main enzymes involved in the starch-sugar conversion of the tubers stored at different temperatures and clarify the key factors affecting the accumulation of RS. Moreover under the basis of the present analysis and previous research, to clone the genes regulating of key factors and to elucidate its function by transformation in order to obtain further understanding of the starch-sugar conversion mechanism with purpose to provide academic evidences and technological supports for improving the processing quality of potato tubers. The main results obtained are as following:1. The experiment was designed, via storing potato tubers of cvs. E-Potato 1 and E-Potato 3 in different temperature, to explore the variation patterns of reducing sugar and total sugar (TS) contents and enzyme activities that involved in the pathway of starch-sugar metabolism aiming at to identify the main factors that influence the chip color. The results showed that low temperature in storage was a main factor that accelerated the accumulation of RS of the stored tubers and there was a very significant linear relationship existed between RS content and chip color index of the tubers. Further analysis elucidated that when tubers stored at 4℃, the activities of ADP glucose pyrophosphorylase (AGPase), UDP glucose pyrophosphorylase (UGPase) and sucrose synthase (SuSy) were negatively exponential to the RS content significantly while that of acid invertase and alkaline invertase were significantly linear to RS content. It suggested that these enzymes could play main roles in the cold sweetening of potato tubers through regulating starch-sugar metabolism.2. The invertase inhibitor is a protein family with low homology. Activity regulation of the invertase inhibitor to invertase may bu efficient to reduce theaccumulation of the reducing sugars through its effect on the activities of invertases. A full cDNA sequence of vacuolar invertase inhibitor gene was amplified from leaves of tobacco Tl strain by RT-PCR and using the primers designed on the base of published sequence of NCBI. Nt-inhh (vacuolar invertase inhibitor), assession number in GenBank AY594179. Analysis of Blast showed that the cloned cDNA shares 100% homology with Nt-inhh of tobacco. The sense-orientation vectors containing Nt-inhh cDNA regulated by 35S or patatin promoter were constructed and introduced successfully into potato E3 by the PCR detection and northern blot analysis. The transgenic tubers were harvested and stored at 4°C or 20 "C for 1 month in order to analyse the variation in reducing sugar content and vaculoar invertase (VI) activity. The results showed that VI activity are inhibited by range from 22.7% (strain A-3) to 78.7% (strain B-13) and RS content decrease by range from 20% (strain A-17) to 80% (strain A-30). This experiment further identified that the activity of invertase was significantly linear to RS content.3. Invertase inhibitor is a protein family found in higher plant speices. A full length cDNA encoding invertase inhibitor was isolated by RT-PCR combined with 5’ RACE from potato tubers of cv. JH and designated as St-inh. The encoding region of St-inh is of 663bp encoding a protein of 221 amino acids. The DNA fragment including St-inh cDNA was cloned into the vector pET28a (+) and expressed successfully in E. coli. Co-incubation of the proteins produced by St-inh in E. coli and the invertase extracts from potato tubers of cv. El and JH and tomato fruits showed that the invertase activities of potato tubers and tomato fruits decreased by 34.3%, 21% and 33.8% respectively. Moreover, the reaction between expression proteins and CWI and VI extracts from cv. E3 leaves resulted in the decrease of CWI and VI activities. These results indicated that products of St-inh protein had a function of invertase inhibitors. The analysises of the nucleotide and amino acid sequences using T-COFFEE demonstrated that St-inh cDNA was of over 95% homologous to Kunitz-type C and there was a typical domain of Kunitz-type protein [L, I, V, M]-X-D-X-[E, D, N, T, Y]-[D, G]-[R, K, H, D, E, N, Q]-X-[L, I, V, M]-X (5) -Y-X-[L, I, V, M]. Therefore, it was conjectured that St-inh could be a member of Kunitz- type gene family. Clustalw analysis for amino acid sequences showed that invertase inhibitor is a protein family with very low homology. St-inh has 14.2% to 21.0% homologous to Ara-inh^ Nt-inhh^ Nt-inh^ To-inh^ Pa-inh^ Ip-inh^ Ci-inh and Zm-inh, indicating that St-inh is a new invertase inhibitor gene from potato.4. Low temperature promoter (LTP) was isolated from E3 genomic DNA by PCR. Two Agrobacterium transformation vectors were constructed, which contained a sense St-inh gene under the regulation of 35S and LTP and introduced successfully into potatoE3 by the PCR detection and northern blot analysis. The analysis of RS content and invertase activities from the tubers stored at 4°C and 20 °C for 1 month indicated that the maximal extent of RS decreased by 80.3% (Strain E-l) and VI activity decreased by 54.2% (strain D-7) with average reduction in RS content by 21.0% and VI activity by 19.0%. This experiments further identified that the activity of VI was significantly linear to RS content. The results reconfirmed that invertase is a main factor influencing low temperature sweetening which can be altered by regulating the expression of the invertase inhibitor, hence reducing the content of RS. Northern blot results revealed that the expression of St-inh increase especially in the the control of LTP but which did not lead to the difference of RS inhibition compared with 35S, suggesting that either constitutive or induced expression of St-inh had similar function in regulating RS accumulation of stored potato tubers.更多还原