植物叶片淀粉分解机理研究进展

高等植物白天光合作用产物以过渡型淀粉的形式储存于叶片叶绿体,夜晚过渡型淀粉分解为麦芽糖和葡萄糖后转运出叶绿体,用于合成蔗糖、维持叶片呼吸、植物代谢以及生长发育。近年来,通过对拟南芥、水稻、玉米、马铃薯、大豆等植物的研究,发现叶片淀粉分解主要是通过水解和磷酸解两条途径;分解过程受到光照、生物节律和碳源平衡等因素的调控。目前一批重要的植物叶片淀粉分解相关基因已被鉴定出来,随着对这些基因功能研究的不断深入,对植物叶片淀粉分解机理将有更深刻的认识。     

一九九八年我院科研成果鉴定情况

本研究表明,在低温下供试碳源中,可溶性淀粉是平菇菌丝生长的最好碳源,其次是麦芽糖和葡萄糖。不同碳源对平菇胞外多酚氧化酶,愈创木酚氧化酶和漆酶酶活及3种酶总酶活的影响不同。前6周在麦芽糖基质中3种酶总酶活最高。8周时在可溶性淀粉基质中最高。其中1号菌株在甘露醇基质中漆酶酶活最高,2号菌株在麦芽糖基质中漆酶酶活最高。这3种酶与基质中木质素降解密切相关,故在低温下以淀粉为主,搭配麦芽糖等其它碳源,不仅利于平菇菌丝生长,更利于其3种酶活的稳定上升和木质素的快速降解,为子实体的发育积累更多营养。     

低温下五种碳源对平菇菌丝生长和三种胞外酶活的影响

本研究表明,在低温下供试碳源中,可溶性淀粉是平菇菌丝生长的最好碳源,其次是麦芽糖和葡萄糖。不同碳源对平菇胞外多酚氧化酶,愈创木酚氧化酶和漆酶酶活及３种酶总酶活的影响不同。前６周在麦芽糖基质中３种酶总酶活最高。８周时在可溶性淀粉基质中最高。其中１号菌株在甘露醇基质中漆酶酶活最高,２号菌株在麦芽糖基质中漆酶酶活最高。这３种酶与基质中木质素降解密切相关,故在低温下以淀粉为主,搭配麦芽糖等其它碳源,不仅利于平菇菌丝生长,更利于其３种酶活的稳定上升和木质素的快速降解,为子实体的发育积累更多营养。     

Starch accumulation associated with growth reduction at low temperatures in a tropical plant

Growth of Digitaria decumbens is severely reduced by night temperatures of 10 degrees C or below. Ultra-structure of leaves and chemical analyses show a high starch content in chloroplasts of plants illuminated and kept at a temperature of 30 degrees C. This starch disappears after a period in the dark at 30 degrees C, but it remains if the temperature during the dark period is 10 degrees C. The inhibition or slowing of starch translocation out of chloroplasts appears to account for reduced photo-synthesis and growth at low night temperatures.     

普鲁兰酶对不同原料淀粉糖生产的影响

[目的]研究普鲁兰酶对淀粉糖糖分、透射比和过滤速度的影响。[方法]通过对照试验研究普鲁兰酶对淀粉糖糖分以及透射比、过滤速度等的影响,用高效液相色谱确定各种糖的含量,探讨普鲁兰酶对不同原料淀粉糖生产的影响。[结果]普鲁兰酶能提高木薯麦芽糖中麦芽糖的含量,而以玉米和马铃薯淀粉为原料时则提高麦芽三糖的含量;以木薯和玉米淀粉为原料生产低聚异麦芽糖时,普鲁兰酶有利于提高糖的品质。加入普鲁兰酶之后大多数淀粉糖的过滤速度都减慢了,但糖浆透射比增大。[结论]研究结果为制备高品质的麦芽糖和低聚异麦芽糖提供了新的理论依据。     

酶法制取高麦芽糖浆的研究

高麦芽糖浆(HMS)是以玉米淀粉为原料采用酶法生产淀粉糖的一种新型淀粉糖生产技术。淀粉经过α-淀粉酶作用,快速搅拌混合,迅速升温而液化。最佳液化条件是:α-淀粉酶用量4μ/g,温度85℃,pH值6.0,时间15分钟。液化液无色透明,含不溶物质少。糖化采用β-淀粉酶与异淀粉酶同时作用。最侄糖化条件为:β-淀粉酶用量120μ/g,异淀粉酶用量30μ/g,温度60℃,pH5.1,时间48小时。糖化液色泽浅黄,糊精含量较少,易于过滤。精制后的糖液近无色。糖液调整pH值后,可在真空下或常温常压下进行浓缩。分析薄板层析结果表明:DE值为81.6的糖液,含高麦芽糖69.7％(麦芽糖量10.9μg/ml糖浆,麦芽三糖量4.3μg/ml糖浆)。此糖浆即为高麦芽糖浆。     

糖对稻米淀粉黏滞性的影响

利用快速粘度分析仪测定葡萄糖、果糖、麦芽糖和蔗糖对稻米淀粉黏滞性的影响,结果显示：随着糖浓度的增加,稻米淀粉峰值粘度下降;随着葡萄糖、果糖、麦芽糖的增加,黏滞谱特征值的最终粘度、热浆粘度和消减值显著降低,而蔗糖对热浆粘度基本没有影响;葡萄糖、果糖使崩解值显著增加,而麦芽糖使崩解值先下降后上升,蔗糖使崩解值显著下降;4种糖都使糊化温度显著增加。     

十一份甘薯种质资源的可溶性糖含量测定与差异分析

HPLC-ELSD法测定植物可溶性糖具有灵敏度高、检出限低、线性范围大等优点。为了了解甘薯品种的可溶性糖组分和糖化特点,对11份甘薯重要种质资源的生薯和熟薯可溶性糖含量进行了测定和差异分析。结果表明,甘薯的生薯和熟薯均含有果糖、葡萄糖、蔗糖和麦芽糖,试验群体的平均含量分别为58.41 mg·g^-1和123.05 mg·g^-1。在生薯可溶性糖中,蔗糖含量最高,占可溶性糖的45%左右,麦芽糖含量最低,仅占可溶性糖的10%左右。在熟薯可溶性糖中,麦芽糖含量最高,其中约90%来源于蒸熟过程中淀粉的转化,增量可占熟薯可溶性糖的50%以上。熟薯中的果糖、葡萄糖、蔗糖主要来源于生薯可溶性糖,在甘薯蒸熟过程中变化较小。因此,甘薯的熟薯可溶性糖含量高低是由麦芽糖含量决定的。浙薯13蒸熟糖化效果好,熟薯可溶性糖和麦芽糖含量分别为179.64 mg·g^-1和138.44 mg·g^-1,均显著高于其余10个品种。红皮白心是一个生薯高糖品种,生薯可溶性糖和果糖含量均居参试品种之首。     

水分胁迫对水稻籽粒灌浆及淀粉合成有关酶活性的影响

 【目的】探索不同水稻抗旱品种在水分胁迫条件下籽粒灌浆特性及有关淀粉酶活性的变化规律。【方法】选用抗旱性不同的3个杂交水稻品种,分别在正常供水和水分胁迫条件下,研究水稻茎鞘物质运转、籽粒灌浆和淀粉合成有关酶活性。【结果】水分胁迫下,抽穗期和成熟期茎鞘干物重以及抽穗后茎鞘物质输出量、输出率、转化率均下降;籽粒生长潜势、最大灌浆速率和平均灌浆速率降低,籽粒生长活跃期缩短,各品种达最大灌浆速率的时间提前;籽粒灌浆过程中可溶性淀粉合成酶（SSS）、Q酶和ADPG焦磷酸化酶3种酶活性都不同程度的被抑制。各性状抗旱性强的品种均比抗旱性弱的品种减少的幅度小。抽穗期茎鞘干物重、抽穗后茎鞘干物质输出量、输出率和转化率均与籽粒产量呈显著或极显著正相关。【结论】在水分胁迫下抗旱性强的品种具有较高的茎鞘物质输出率和转化率、籽粒灌浆速率及淀粉合成有关酶活性,是产量降低较少的生理基础。     

LAAT-1 is the lysosomal lysine/arginine transporter that maintains amino acid homeostasis

Defective catabolite export from lysosomes results in lysosomal storage diseases in humans. Mutations in the cystine transporter gene CTNS cause cystinosis, but other lysosomal amino acid transporters are poorly characterized at the molecular level. Here, we identified the Caenorhabditis elegans lysosomal lysine/arginine transporter LAAT-1. Loss of laat-1 caused accumulation of lysine and arginine in enlarged, degradation-defective lysosomes. In mutants of ctns-1 (C. elegans homolog of CTNS), LAAT-1 was required to reduce lysosomal cystine levels and suppress lysosome enlargement by cysteamine, a drug that alleviates cystinosis by converting cystine to a lysine analog. LAAT-1 also maintained availability of cytosolic lysine/arginine during embryogenesis. Thus, LAAT-1 is the lysosomal lysine/arginine transporter, which suggests a molecular explanation for how cysteamine alleviates a lysosomal storage disease.     

Program clocks in small mammals

Complex patterns of time, direction, and speed of running by small nocturnal mammals in activity wheels sometimes are duplicated almost exactly from night to night. These activity pattern repetitions disclose: (i) previously unknown capabilities of biological clocks to act as sequence programmers for behavior; (ii) that animals can retain a record of the sequence and timing of their activities covering an entire night; and (iii) that the activities of one night can bias an animal toward similar behavior on subsequent nights.     

麦芽糖转运相关基因的表达对阿维菌素合成的影响

阿维菌素是由阿维链霉菌(Streptomyces avermitilis)发酵产生的大环内酯类化合物,是一种应用广泛的生物农药。对阿维链霉菌中麦芽糖转运系统基因的表达量对阿维菌素合成的关系进行了初步研究。克隆malEFG基因至链霉菌整合型和多拷贝表达载体,分别转化阿维链霉菌野生型菌株ATCC31267和阿维菌素高产菌株GB165中,结果不同的阿维链霉菌转化子阿维菌素的产量都有一定程度的提高。野生型菌株阿维菌素转化子的产量是出发菌株的3~3.2倍,高产菌株的转化子阿维菌素的产量提高了40%~60%。     

pH-dependent gating in a FocA formate channel

The formate transporter FocA was described to switch its mode of operation from a passive export channel at high external pH to a secondary active formate/H(+) importer at low pH. The crystal structure of Salmonella typhimurium FocA at pH 4.0 shows that this switch involves a major rearrangement of the amino termini of individual protomers in the pentameric channel. The amino-terminal helices open or block transport in a concerted, cooperative action that indicates how FocA is gated in a pH-dependent way. Electrophysiological studies show that the protein acts as a specific formate channel at pH 7.0 and that it closes upon a shift of pH to 5.1.     

The high-affinity E. coli methionine ABC transporter: structure and allosteric regulation

The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.     

Cloning and expression of a rat brain GABA transporter

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.     

天达V901草菇菌丝生长条件的筛选与优化

采用单因素试验和正交试验相结合的方法,研究了天达V901草菇菌丝在不同营养条件和不同培养条件下的生长状况。结果表明,适宜草菇菌丝生长的碳源有葡萄糖、纤维素、可溶性淀粉、麦芽糖和蔗糖,但对草菇菌丝生长最有利的碳源是可溶性淀粉;适宜草菇菌丝生长的最佳氮源是蛋白胨;最佳碳氮比是10∶1;最佳可溶性淀粉浓度是5.0%;钾对草菇菌丝生长有明显促进作用。草菇菌丝生长的最适温度为35℃;最适pH值为7.5。     

苗期夜温对番茄畸形果发生的影响

以“川番杂交一号”番茄为材料,苗期进行不同的温度处理,研究幼苗茎尖的生理生化变化、调查田间畸形果发生情况。结果表明:苗期降低夜温,可使幼苗茎尖的可溶性蛋白质、可溶性糖和淀粉含量明显地提高,同时增强了POD、PPO、CAT和NR的活性。低夜温处理后,番茄畸形果的发生率、平均级数、综合指数均有显著或极显著地提高,尤其是在6℃的低夜温处理后,第一、二果穗及两果穗平均的畸形果发生率高达56.21%、53.97%、55.05%。对于畸形果类型而言,降低夜温对椭圆果和乱形果的发生率没显著影响,对裂果、指突、孪生果和混发果的发生率有显著影响。     

外源ABA对木薯叶片内源激素及淀粉合成相关基因的影响

在盆栽条件下,用外源脱落酸(Abscisic acid,ABA)处理块根形成期的木薯品种,测定处理及对照在不同时刻叶片中ABA等激素变化、ABA合成与失活以及淀粉合成途径关键基因的表达变化。结果表明:1)正常条件下木薯叶片内源ABA水平及ABA合成途径基因均存在明显的夜间低而白天较高的昼夜节律,ABA信号转导相关基因也表现出下午的高峰表达,它们与淀粉合成途径基因的夜间表达一般具有相反的趋势。2)ABA处理后,木薯叶片内源ABA含量迅速下降,与观察到的ABA合成途径基因表达下降与后期ABA失活基因激活表达相一致,同时发现细胞分裂素中的玉米素核苷(Zeatin riboside,ZR)和生长素(Indole acetic acid,IAA)水平发生改变,预示木薯具有迅速恢复ABA平衡态的机制,并且与其他类激素存在协同作用。3)与此同时,外源ABA处理明显抑制淀粉合成上游基因表达,特别是夜晚高峰期的表达,而对下游基因的抑制相对较小。说明这些基因及相关酶类不同程度受到ABA的调控;ABA明显倾向于调控淀粉合成上游相关基因;外源ABA处理改变了叶片临时淀粉合成速率。     

野生紫平菌丝生长营养物质优化研究

采用单因素试验方法,研究野生紫平菌丝在不同营养条件下的生长状况。结果表明：培养基中适宜菌丝生长的碳源是麦芽糖,其次是蔗糖和淀粉;氮源是蛋白胨,其次是酵母浸粉;适宜的碳氮比是25∶1;母种培养基中需添加复合维生素B 10~15 mg/L,硫酸镁3.0~4.5 g/L。最适培养基配方为：麦芽糖20.36 g,蛋白胨2.64 g,硫酸镁4 g,复合维生素B 15 mg,琼脂20 g,加水至1 000 m l。     

低温对朵丽蝶兰成花过程中碳水化合物及糖转运蛋白基因表达的影响

 [目的]蝴蝶兰（Phalaenopsis）原产热带,其成花过程中的“温敏”现象已成为制约蝴蝶兰产业发展的重要因素。朵丽蝶兰（Doritaenopsis hybrid）是朵丽兰（Doritis）与蝴蝶兰（Phalaenopsis）的杂交后代,是目前商业应用最广泛的蝶兰种类之一。研究其在相对低温诱导下的营养生长特性及相关基因的表达模式,对明确蝶兰花芽分化的生理生化机制,并深入理解蝶兰成花“温敏”现象的机理,推动整个蝴蝶兰产业的发展有着重要的意义。 [方法]本研究对朵丽蝶兰（Doritaenopsis hybrid）进行高温（30℃/25℃）和低温（22℃/18℃）处理,测定其叶片的生长和叶绿素荧光,以及碳水化合物的含量变化,同时对朵丽蝶兰（Doritaenopsis hybrid）‘温敏’SSH文库中分离的两个糖转运蛋白基因（片段）在相对低温处理下的表达特性进行分析, [结果]结果显示,低温处理的整个阶段,蝶兰叶面积的增长显著低于高温处理,并且在低温处理的前四周（28d）,叶片的光能转化效率和PSII的活性明显下降,叶片的淀粉含量急剧下降,还原糖含量持续增加,蔗糖含量在第28d前后表现为先增后减,其中,在低温处理21d至35d中,DhST1的mRNA表达与蔗糖含量的变化一致,而DhSUT1则表现为持续的下降,但二者在抽出的花梗中都有较高的表达量。[结论]低温明显诱导朵丽蝶兰叶片营养生长的减缓,并在处理的开始阶段对光合系统产生一定抑制,同时,碳水化合物含量及相关的糖转运蛋白基因对低温表现出明显的响应,但相关基因表达模式的差异说明在低温诱导的花芽形成过程中承担着不同的重要作用,为进一步明确蝴蝶兰成花“温敏”现象的分子机理提供科学依据。