【作者】 张迟；

【导师】 谢从华；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2007， 博士

【摘要】 马铃薯是世界第四大粮食作物，用途广泛，其加工产品炸片和炸条是重要的油炸食品。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，通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褐化反应，严重影响油炸产品的色泽和品质，甚至对人体健康产生不良影响。因此，控制“低温糖化”已经成为马铃薯采后生理学研究的热点。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。在植物体内，蔗糖降解只有两个途径：蔗糖合成酶途径和转化酶途径。转化酶，又称蔗糖转化酶，可分为中性(或碱性)转化酶和酸性转化酶，其中，酸性转化酶又包括液泡转化酶和胞壁转化酶。前人的研究表明，酸性转化酶活性与还原糖／蔗糖比率成显著正相关，从而成为淀粉—糖代谢中的关键酶之一。因此，进一步明确转化酶在调控马铃薯低温糖化过程中的作用，对于马铃薯淀粉—糖代谢的调控进而提高马铃薯低温贮藏下的加工品质具有重要的理论和实践意义。本研究旨在克隆马铃薯内源酸性转化酶基因，并通过构建组成型启动子CaMV35S和块茎特异表达启动子CIPP驱动的反义基因(或片段)载体以及RNA干涉载体转化马铃薯，对内源酸性转化酶基因的表达进行调控，进一步明确其在淀粉—糖代谢过程中的作用，揭示酸性转化酶在调控马铃薯低温糖化中的关系。主要结果如下：1．以发表的酸性转化酶基因的序列设计引物，采用RT-PCR的方法，从低温处理的马铃薯品系JH块茎cDNA中克隆到全长为1920bp的特异条带，DNA序列测定结果显示，目标片段为inv的完整的编码序列，为一个开放读码框，编码639个氨基酸。Blastn结果显示，该片段与已知的酸性inv基因cDNA S70040(番茄)以及液泡转化酶cDNA X70368.1(马铃薯)、Z12025(番茄)和Z12026(醋栗番茄)的同源性分别达到95％，98％，96％和96％。其中，Expasy蛋白结构预测的结果显示，氨基酸序列的34到56有一个跨膜结构域KIISGIFLSSFLLLSVAFFPILN，同时，在N端1-49存在一个信号结构，在614的位置存在一个蛋白剪切位点GAS，120-124和299-303为inv基因的两个保守位点NDPNG和WECVD，在靠近N端的序列中还存在几处糖基化位点。上述结果初步证明，所克隆的cDNA为马铃薯液泡转化酶基因，该序列已经在GenBank注册，登记号为AY341425。2．通过PCR扩增和限制酶切，将克隆的cDNA分为靠近5’端(397bp)，包含有保守位点的中间区段(625bp)以及靠近3’端(955bp)的三个片段。四个片段(包括全长)分别与35S和CIPP启动子构建成反义载体转化马铃薯。对转基因块茎进行低温贮藏(设置常温贮藏对照)后测定酸性转化酶活性和还原糖含量，结果显示，不同插入片段大小对酶活性和还原糖含量的影响没有显著差异。其中，35S启动子的转基因株系中，低温(4℃)贮藏下，酸性转化酶活性2005年的平均下降幅度从为29％-47％，2006年平均降幅为10％-13％；还原糖含量2005年的平均降幅为19％-26％，2006年为29％-34％。室温(2012)贮藏下，2005年的酸性Inv活性降幅均在60％以上，但还原糖含量变化不大；而2006年的还原糖含量下降均达到30％以上，酸性Inv活性无显著变化。两类启动子转基因株系的抑制效率存在显著差异。4℃贮藏条件下，CIPP启动子驱动的转基因植株的酶活性下降53％(贮藏6周)和21％(贮藏10周)，还原糖含量下降35％(贮藏6周)和67％(贮藏10周)；但是室温贮藏条件下，不同启动子的转基因株系的平均酶活和还原糖含量都没有明显差异。试验结果证明，所使用的cDNA片段均能达到相似的反义抑制效果。而采用块茎特异表达的CIPP启动子的转化植株，其块茎的酸性转化酶活性和还原糖含量在低温贮藏下的抑制效果均优于组成型表达的35S启动子。3．研究还构建了RNA干涉载体并转化马铃薯，并对转基因植株和试管薯进行酸性转化酶活性的测定。结果表明，在测定的5个株系中，植株的酸性转化酶活性均有不同程度的下降，其中除Ni-1外，酶活下降均在65％以上，最大降幅为78％(Ni-4)，平均下降59.3％。而同时测定的抑制效果较好的反义转基因株系中，酸性转化酶活性最大降幅为84％(ES4-3)，平均下降58.4％，表明RNAi技术在马铃薯抗“低温糖化”基因工程中也同样具有较好的应用潜力。4．Northern分析表明，转基因植株和块茎中反义RNA的表达量在不同的启动子之间存在一定的差异，其中，35S启动子所驱动的株系在植株和块茎中均有强烈的表达，CIPP启动子所驱动的株系只在块茎中呈现强烈的表达，而在植株的表达几乎没有，也再次证实了CIPP启动子的块茎特异性。RNA干涉的转基因植株中内源酸性转化酶的mRNA表达量也明显降低。Northern分析进一步说明酸性转化酶的反义抑制和RNA干涉显著降低了植株内源酸性转化酶的mRNA的表达，证明Invertase可能主要在转录后水平上调控块茎低温糖化。5．对抑制效果较好的株系进行块茎低温贮藏以后，测定了其蔗糖和淀粉含量。结果表明，在低温贮藏(4℃)条件下，株系ES4-3的抑制效率达到最高，酸性转化酶活性降低了72％，还原糖含量下降了68.7％，同时引起蔗糖含量的上升(含量增加92.4％)和淀粉的积累(淀粉含量增加7.2％)。同时，EC4-5和EC19-5的酸性转化酶活性也分别表现出33.4％和23.9％的下降，还原糖含量分别下降8.69％和69.5％，从而导致蔗糖含量分别增加了7.6％和12.2％。总体上，转基因株系中酸性转化酶活性和还原糖含量的大量变化能够引起蔗糖分解显著降低，但对淀粉的合成并没有明显的促进。在室温贮藏(20℃)条件下，除个别株系(EC4-5)外，各测定指标变化不大。证明低温导致的块茎还原糖积累可能主要来自于蔗糖分解途径，而不是淀粉降解。6．选取测定结果较好的转基因块茎及其非转基因对照和加工品种对照，低温贮藏后进行炸片试验。结果显示，转基因块茎的炸片色泽指数较非转基因对照均有所降低，同一株系不同生产年份的块茎炸片色泽变化较大，虽然未作重复，但从趋势上看来，转基因株系块茎的炸片色泽指数比非转基因对照E3降低，并且基本在＜3的范围内，与加工品种Atlantic的炸片色泽指数基本相当，证明通过抑制酸性转化酶基因的表达，可以有效的降低还原糖含量，达到改良油炸加工品质的目的。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. To provide a uniform flow of tubers to fresh market and processing from harvest, cold storage at about 4℃is usually adopted, which could reduce the excessive loss of moisture, development of rots and sprout growth. Cold storage, however, causes a large accumulation of reducing sugar(RS) that reacts with the amino acids under high temperature and resulted in undesirable dark brown color of the products. The starch-sugar conversion involves multiple metabolic pathways with multi-regulating and-feedback regulating factors. Therefore, mechanism of"cold-sweetening"is a basic issue for improving potato post-harvest quality. Only the invertase and reversible sucrose synthase reactions catalyze known paths of cleavage of sucrose in vivo. Invertases(Inv) include acid invertases which can be subgrouped as vacuolar and cell wall invertases, and neutral(or alkaline) invertases. Previous report indicated a strong positive correlation between acid invertase activity and the ratio of RS and sucrose, which was accounted for the conversion of starch-sugar. The present research focus on the cloning of complete CDS(coding sequence) of endogenous acid invertase gene, regulation of its expression through RNAi and antisense strategy, and their influence on RS content. The main results obtained are as following:1. The complete CDS of invertase gene was obtained from a tetraploid potato clone JH, which was considered resistant to cold-sweetening in previous work, by RT-PCR with specific primers derived from the complete reported CDS(Genebank accession No. L29099). The result of sequencing showed that the over-length of the CDS was 1920 bp encoding 639 amino acids with 95% homology to the cDNA of acid invertases in tomato(S70040), and was 98%, 96% and 96% similar to that of the vacuolar invertases in potato(X70368.1), tomato(Z12025) and Lycopersicon pimpinellifolium(Z12026). The protein prediction analysis showed a transmembrane region(34-56 in amino acids) and an N-term signal sequence(1-49). Along with the sequence there were two conservative sites, NDPNG(120-124) and WECVD(299-303), and several potential glycosylation sites, suggesting that the cloned cDNA was protentially vacuolar invertase gene.2. Performed with PCR amplification and digestion, the cloned cDNA yielded three fragments which represents 5’-end(397 bp), the conservative portion(625 bp) and 3’-end(955 bp). The above fragments and together with the full-length CDS were individually cloned into pBI121 vector controlled by the constitutive Cauliflower Mosaic Virus(CaMV) 35S promoter and pBIC vector controlled by patatin class I promoter(CIPP) in antisense orientation. Analysis of stored transgenic tubers revealed that, controlled by 35S promoter, when stored at low temperature(4℃), expression of antisense acid invertase repressed the endogenous invertase activity of the four transgenic lines(ES4, ES6, ES9 and ES19) that were stored for 6 weeks by 33%, 29%, 41% and 47% and by 10%, 13%, 13% and 12% for 10 weeks period. These variations led to significant RS content decrease by 26%, 23%, 23% and 19% for 6 weeks and by 34%, 29%, 34% and 32% for 10 weeks. When storage temperature increased to 20℃, the average decrease of endogenous acid invertase activity was over 60% for 6 weeks storage, while no remarkable alteration in RS content during this period. However, more than 30% reduction in RS content and non-detectable variation in acid invertase activity was observed for 10 weeks storage. Comparison between transgenic tubers harboring 35S and CIPP promoters showed that, stored at 4℃, antisense cDNA driven by CIPP promoter repressed the endogenous invertase activity by 53% for 6 weeks storage and by 21% for 10 weeks period, which turned down the RS content by 35% in tubers stored for 6 weeks and by 67% for 10 weeks, although little variation were observed between the two promoters at 20℃. No significant difference was detected on the acid Inv activity and RS content among transgenic tubers expressing antisense cDNA varied in fragment size indicating a similar efficiency of inhibition. However, the tuber-specific promoter, CIPP, performed a higher efficiency of inhibition than did the constitutive 35S promoter.3. RNAi vector inserted with portion of inv cDNA was also introduced into potato plants. Successfully expression of RNAi vector in potato plants aroused a range of variations in transgenic lines by an average of 59.3% reduction in acid invertase activity, in which the best repression was found in Line Ni-4 by 78% decrease in invertase activity. Comparatively, the average of 58.4% reduction was found on invertase activity in well inhibited antisense transgenic lines, in spite of the occurrence of 84% decrease(Line ES4-3). The little difference suggested a powerful potential of RNAi technology.4. Analysis of expression of antisense mRNA showed a large accumulation of antisense mRNA in both transgenic plants and tubers driven by 35S promoter whereas only in transgenic tubers controlled by CIPP promoter, indicating again the tissue-specific expression of CIPP. However, the accumulation of mRNA of endogenous acid invertase in RNAi transgenic plant was obviously decreased compared with the control plant. Since the reduction of endogenous acid inv mRNA were observed both in antisense and RNAi transgenic plants, the regulation of Invertase in cold-sweetening of tubers was probably focused on transcriptional levels.5. Further analysis of selected transgenic plants indicated that, under low temperature storage, the strongest suppression was found in tubers of the transformant ES4-3 which accounted for 28% of enzyme activity of the control. This reduction in Inv activity led to a 68.7% decrease in the accumulation of reducing sugar, further resulting in a 92.4% and 7.2% increase in the amount of sucrose and starch content respectively compared with wild-type tubers. Anther available transgenic lines EC4-5 and EC19-5 achieved remarkable reduction in Inv activity, reaching 66.6% and 77.1% of the control respectively, while the former revealed a relative little decrease(8.69%) in RS content contrast to the latter(69.5%), resulting a moderate elevation of 7.6% and 12.2% in sucrose. In general, low level of RS accumulation at low temperature was resulted from less sucrose conversion without obvious stimulation of starch synthesis, indicating that cold-induced accumulation of reducing sugar was mainly derived from sucrose cleavage rather than starch degradation.6. Chips were prepared from good repressed transgenic tubers and untransformed control tubers and processing variety’Atlantic’ stored at low temperature(4℃). Comparison of chip color index showed a decrease trend in transgenic chips in contrast to the control tuber and a large variation between two production seasons.更多还原