马铃薯低温糖化相关淀粉酶基因的功能鉴定及机制解析

发布时间：2018-01-24 10:37

  本文关键词： 马铃薯 低温糖化 淀粉降解 α-淀粉酶 β-淀粉酶 亚细胞定位 块茎　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：马铃薯(Solanum tuberosum L.)是世界上最重要的非谷物粮食作物,在保证人类粮食安全方面具有重要作用。薯片和薯条加工产品在加工业中占据着主要地位,为保证原材料的持续供应,马铃薯块茎经常贮藏于低温条件下(不高于10°C),然而低温贮藏会导致还原糖积累,即为低温糖化现象,使其在油炸加工过程中产生褐化反应,影响产品品质。前人研究表明淀粉降解是低温糖化的主要路径之一,但是并未鉴定到对低温糖化起特异作用的淀粉降解酶基因,淀粉降解途径的低温糖化机理尚不清楚。因此,本研究从全基因组角度对马铃薯α-淀粉酶和β-淀粉酶家族成员进行了鉴定、功能验证及作用机制解析,主要研究结果如下:1.利用马铃薯基因组数据库,鉴定到2个α-淀粉酶、7个β-淀粉酶家族成员,它们不对称地分布在马铃薯染色体上。通过不同物种的α-淀粉酶和β-淀粉酶系统进化树分析,发现淀粉酶进化比较保守,α-淀粉酶被聚为3个亚家族,β-淀粉酶被聚为4个亚家族。马铃薯β-淀粉酶基因家族成员基因结构分析表明,聚在相同亚家族的成员有相似的基因结构,但葡糖基水解酶结构域的比对结果显示,其中一个β-淀粉酶家族成员StBAM9可能不具活性。2.利用具有不同低温糖化抗性的马铃薯基因型,在不同植物组织及不同贮藏温度下的块茎中进行淀粉酶家族成员的表达分析,筛选到3个受低温响应并且在块茎中诱导表达的淀粉酶,分别是α-淀粉酶StAmy23、β-淀粉酶StBAM1和StBAM9。3.为了明确StAmy23、StBAM1和St BAM9在细胞中的作用部位,分别构建了这三个基因融合绿色荧光蛋白的表达载体StAmy23-GFP、StBAM1-GFP、StBAM9-GFP和淀粉粒标记基因融合红色荧光蛋白的表达载体StGBSS-RFP,通过两种方法(基因枪法和农杆菌介导的烟草瞬时表达方法),将目标基因与标记基因进行共表达,并与细胞质标记基因RFP和叶绿素自发荧光叠加分析,首次证明StAmy23定位于细胞质,StBAM1定位于质体基质,St BAM9定位于淀粉粒。此外,又对StBAM1、StBAM9和StGBSS的叶绿体转运肽和截去转运肽的StBAM1、StBAM9和StGBSS进行了亚细胞定位,结果表明,它们的叶绿体转运肽定位于淀粉粒,但是StBAM1叶绿体转运肽不稳定,可能后期转移至基质,与其全长定位于质体基质相关。这些结果说明StBAM1和StBAM9的定位取决于自身的叶绿体转运肽。4.为了研究StAmy23、StBAM1和StBAM9在马铃薯块茎低温糖化中的功能,分别构建了StBAM1和StBAM9基因的RNA干涉表达载体及StBAM1和StBAM9双干涉表达载体,分别遗传转化不抗低温糖化的马铃薯品种鄂马铃薯3号(E3),RNAiStAmy23转基因株系由前人获得。分别测定了转基因株系的叶片淀粉含量及低温贮藏块茎中的糖含量、薯片油炸色泽、淀粉酶活性、淀粉含量等,结果表明,StBAM1和StBAM9参与了白天的叶片淀粉降解,但是在夜晚只有StBAM9与淀粉降解有关,而StAmy23在叶片淀粉降解过程中无显著功能。在低温贮藏的块茎中,与对照相比,干涉St BAM1的转基因块茎和同时干涉StBAM1和St BAM9的转基因块茎中β-淀粉酶活性降低,但是干涉StBAM9的转基因块茎中β-淀粉酶活性没有显著变化。干涉StBAM1和St BAM9均可抑制淀粉降解和还原糖积累,有效改善油炸加工品质,而双干涉转基因的效果则更明显,说明StBAM1和StBAM9可能存在功能叠加。另外,可溶性淀粉含量在干涉StBAM1的转基因块茎中增加,但是在干涉StBAM9的转基因块茎中显著降低,表明StBAM1可能通过水解质体基质中的可溶性淀粉来调节低温糖化,而StBAM9可能直接作用于淀粉粒来调节低温糖化。此外,干涉StAmy23导致转基因低温贮藏块茎中可溶性糖原含量显著增加,还原糖含量降低,可能是StAmy23通过降解细胞质中的可溶性糖原来参与低温糖化的调节。进一步分析不同转基因块茎中还原糖的含量表明,StBAM9在低温糖化中的功能最显著,这3个淀粉酶分别在不同的亚细胞位置水解不同的底物,在马铃薯低温糖化过程中发挥着不同水平的功能,首次揭示了淀粉水解路径在低温糖化中的贡献。5.利用酵母双杂交系统分析了StAmy23、StBAM1、St BAM9分别与淀粉代谢相关蛋白磷酸酶(StLSF1和StLSF2)、葡聚糖水双激酶(StGWD)和淀粉颗粒合酶(StGBSS)的互作关系,结果表明只有StLSF2与St BAM9互作,推测StLSF2可能参与到StBAM9介导的淀粉降解过程中。但原核表达蛋白StBAM1、StBAM9和StLSF2体外活性分析实验证明,StLSF2不影响StBAM1、StBAM9的活性,StBAM1、StBAM9也不影响StLSF2的去磷酸化作用,推测StLSF2可能不是参与StBAM9水解路径的一个重要因子。6.利用马铃薯抗低温糖化材料10908-06、CW2-1和低温糖化敏感材料E3的块茎,经4°C贮藏5天后构建了酵母杂交文库。以StBAM9和截去叶绿体转运肽的StBAM9-P分别作为饵蛋白,进行酵母文库筛选,总共筛选到63个潜在互作蛋白,其中一个潜在互作蛋白即为StBAM1。双分子荧光互补和酵母双杂交互作验证的结果表明,StBAM9与StBAM1互作于淀粉粒表面,首次证明了植物淀粉酶之间存在互作。因此,我们推测StBAM9与StBAM1可能形成一个复合体,将StBAM1从质体基质招募到淀粉粒表面,进行进一步的淀粉降解。
[Abstract]:Potato (Solanum tuberosum L.) is the world's most important non grain crops, which plays an important role in human food security. Potato chips and French fries products occupy the main position in the industry, in order to ensure the continued supply of raw materials, potato tubers often stored in low temperature conditions (not higher than 10 DEG C) however, low temperature storage will lead to the accumulation of reducing sugars, namely low temperature sweetening phenomenon, the browning reaction in the frying process, influence the quality of the product. Previous studies showed that starch degradation is the main path of a low temperature saccharification, but did not play to the identification of starch degrading enzyme gene specific effect on the low temperature low temperature saccharification saccharification. The mechanism of starch degradation pathway is not clear. Therefore, the research for the identification of potato alpha amylase and beta amylase family members from a genome-wide perspective, functional verification and mechanism System analysis, the main results are as follows: 1. using the potato genome database to identify 2 alpha amylase, beta amylase family members of 7, they are distributed asymmetrically in potato chromosome. By different species of alpha amylase and amylase phylogenetic analysis found that evolutionary conservative amylase, amylase can be divided into 3 subfamilies, beta amylase were clustered into 4 subfamilies. Potato beta amylase gene family member gene structure analysis showed that poly have similar gene structure in members of the same subfamily, but the ratio of glycosyl hydrolase domain showed that one member of the family of beta amylase StBAM9 may not be activated by.2. with different potato genotypes of low temperature sweetening resistance, analysis of expression of the amylase family members in different plant tissues and different temperatures in the tubers, screening 3 by low temperature response and induced expression in tuber amylase, respectively, alpha amylase StAmy23, beta amylase StBAM1 and StBAM9.3. to determine StAmy23, StBAM1 and St BAM9 site in the cell, respectively. The expression vector StAmy23-GFP was constructed, the three fusion gene of green fluorescent protein expression vector StGBSS-RFP StBAM1-GFP. StBAM9-GFP and starch granule marker gene fusion of red fluorescent protein, by two methods (the expression of biolistic and Agrobacterium mediated method, the instantaneous tobacco) target gene and marker gene co expression, and spontaneous fluorescence overlay analysis and cytoplasmic marker gene RFP and chlorophyll, for the first time that StAmy23 was located in cytoplasm, located in StBAM1 St BAM9 is located in the plastid stroma, starch grains. In addition, the StBAM1, and the chloroplast transit peptide transporter StBAM9 and StGBSS on StBAM1, StBAM9 and StGBSS. The subcellular localization results showed that the chloroplast transit peptide located in the starch grains, but StBAM1 chloroplast transit peptide is not stable, the latter may be transferred to the substrate, and its full-length located in the plastid matrix. These results indicate that StBAM1 and StBAM9 positioning depends on the chloroplast transit peptide.4. from the body in order to study the StAmy23, StBAM1 and StBAM9 in the low temperature saccharification of potato tubers, we constructed StBAM1 and StBAM9 gene RNA expression vector and StBAM1 interference and StBAM9 interference expression vector, genetic transformation were not anti cold sweetening potato varieties e-potato 3 (E3), RNAiStAmy23 transgenic lines obtained by previous researchers. The sugar content and starch content in leaves low temperature storage tubers of transgenic lines were determined in the fried potato chips color, amylase activity, the results showed that the starch content, StBAM1, and StBAM9 in the daytime The leaf starch degradation, but at night only StBAM9 and StAmy23 and degradation of starch, starch degradation in leaves had no significant function. In the low temperature storage tubers, compared with the control, reduce the beta amylase activity in transgenic tubers and BAM1 interference St StBAM1 and St BAM9 interference transgenic tubers, but transgenic tubers interference in StBAM9 beta amylase activity did not change significantly. StBAM1 and St interference BAM9 inhibited the degradation of starch and reducing sugar accumulation, improve the quality and effect of the frying process, transgenic double interference is more obvious, indicating that StBAM1 and StBAM9 may have the function of superposition. In addition, the soluble starch content increased in transgenic tubers interference in StBAM1 however, in transgenic tubers interference StBAM9 decreased significantly, indicating that StBAM1 may through the hydrolysis of soluble starch plastid matrix to adjust the temperature of sugar However, StBAM9 may act directly on the starch grains to adjust temperature saccharification. In addition, StAmy23 interference leads to soluble glycogen content in transgenic tubers increased significantly in low temperature storage, reducing sugar content reduced, soluble sugar may be adjusted by the StAmy23 degradation in the cytoplasm in the original low temperature saccharification. Further analysis showed that the reducing sugar content of different transgenic tubers in the most significant StBAM9 in the low temperature saccharification function, the 3 amylase respectively in the subcellular location of hydrolysis of different substrates, play the function of different levels in the process of low temperature sweetening in potato starch hydrolysis, reveals the first path in the low-temperature sweetening the contribution of.5. by yeast two hybrid system of StAmy23, StBAM1 St, BAM9 were associated with the metabolism of starch protein phosphatase (StLSF1 and StLSF2), dextran water dikinase (StGWD) and starch synthase (StGBSS). The interaction between StLSF2 and St, the results show that only BAM9 interaction, suggesting that StLSF2 may participate in the degradation of starch in the process of StBAM9 mediated. But the prokaryotic expression of protein StBAM1, StBAM9 and StLSF2 in vitro analysis experiments prove that StLSF2 does not affect the activity of StBAM1, the activity of StBAM9, StBAM1, StBAM9 does not affect the dephosphorylation of StLSF2 the role, suggesting that StLSF2 may not participate in the hydrolysis of StBAM9 path is an important factor of.6. using the cold induced sweetening material 10908-06, CW2-1 and low temperature sweetening sensitive material E3 tuber by 4 ~ C after 5 days storage constructed yeast hybrid library. With StBAM9 and truncated the chloroplast transit peptide StBAM9-P as bait protein, yeast library a total of 63 screening, screening of potential interacting proteins, including a potential interacting protein is StBAM1. bimolecular fluorescence complementation and yeast two hybrid interaction test showed that StBAM9 and StBAM 1, on the surface of starch granules, it is the first time that there is interaction between plant amylase. Therefore, we speculate that StBAM9 and StBAM1 may form a complex, and StBAM1 can be recruited from plastid matrix to starch granule surface for further starch degradation.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S532

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