一种微量、快速测定植物种子β-淀粉酶活性的方法

针对3,5-二硝基水杨酸（DNS）法的缺点,以对硝基苯酚麦芽五糖（PNPG5）为底物,建立并改进了植物种子β-淀粉酶活性测定的方法。本方法具有微量和快速的特点;进一步通过对4种植物材料种子的酶活性测定以及α-淀粉酶的干扰评估实验,证实了该法可以运用于植物种子β-淀粉酶活性的专一性测定。     

中国大麦地方品种的α-淀粉酶酶活性研究

为了研究中国大麦地方品种种质资源α-淀粉酶的遗传变异,发掘具有高α-淀粉酶酶活性的材料。本试验以257份中国地方品种为试验材料,利用3,5-二硝基水杨酸法(DNS)和降落数值法(FN)测定了2010年和2011年收获的发芽种子与干种子中的α-淀粉酶活性。分析了不同年份间及发芽种子与干种子中的酶活性的相关性。结果表明,中国大麦地方品种的α-淀粉酶酶活性存在广泛的遗传变异,酶活性最高的品种几乎是酶活性最低品种的4倍,且多数品种的α-淀粉酶活性在发芽后第5天或第6天达到最高。发芽种子的α-淀粉酶酶活性在年份之间的相关性达到极显著水平,说明发芽种子的α-淀粉酶酶活性主要受遗传因素控制;发芽种子与干种子的酶活性之间相关性不显著,表明干种子测定的降落数值不能用来预测大麦品种的α-淀粉酶活性。     

不同生态环境中铜绿丽金龟幼虫肠道细菌 产消化酶活性比较

目的测定不同生态环境中铜绿丽金龟幼虫肠道细菌产消化酶活性。方法采用平板透明圈法和分光光度计法。结果平板透明圈法测得废弃菜园和花生田中铜绿丽金龟幼虫肠道细菌产蛋白酶和淀粉酶活性差异无统计学意义(F=1.089、0.4963,P0.05),而细菌的产纤维素酶活性有显著差异,其中花生田铜绿丽金龟幼虫肠道中分离到的粘质沙雷菌产酶活性显著高于其他菌株;分光光度计测得的不同生态环境中铜绿丽金龟幼虫肠道细菌的产蛋白酶、淀粉酶和纤维素酶活性差异均有统计学意义(F=461.565、42.349、18.7673,P0.05)。从变异系数来看,分光光度计法测得的蛋白酶、淀粉酶和纤维素酶的变异程度较低,而脂肪酶测定时平板透明圈法的变异系数较低。结论平板透明圈法和分光光度计法均可用于铜绿丽金龟幼虫肠道细菌产消化酶活性测定。通过分析研究比较,分光光度计法适于测定细菌的产蛋白酶、淀粉酶和纤维素酶活性,平板透明圈法适于测定细菌的产脂肪酶活性。     

大麦成熟籽粒中b-淀粉酶水平与麦芽糖化力的相关分析

对10个二棱啤酒大麦品种和10个F₂杂种的成熟籽粒进行了B-淀粉酶活性和麦芽糖化力的分析。结果发现：B-淀粉酶水平与麦芽糖化力之间相关极显著（r=0.991）。B-淀粉酶和a-淀粉酶之间以及a-淀粉酶与糖化力之间的相关系数分别为0.499和0.55,其相关密切程度不如B-淀粉酶和糖化力。因此,在啤酒大麦育种程序中,测定籽粒中b-淀粉酶的活性水平可以预测麦芽糖化力的高低。     

大麦成熟籽粒中b-淀粉酶水平与麦芽糖化力的相关分析

对10个二棱啤酒大麦品种和10个F₂杂种的成熟籽粒进行了B-淀粉酶活性和麦芽糖化力的分析。结果发现：B-淀粉酶水平与麦芽糖化力之间相关极显著（r=0.991）。B-淀粉酶和a-淀粉酶之间以及a-淀粉酶与糖化力之间的相关系数分别为0.499和0.55，其相关密切程度不如B-淀粉酶和糖化力。因此，在啤酒大麦育种程序中，测定籽粒中b-淀粉酶的活性水平可以预测麦芽糖化力的高低。     

粘虫中肠α-淀粉酶活性测定方法的参数优化

针对粘虫Mythimna separata中肠α-淀粉酶筛选了11种不同参数组合的3，5－二硝基水杨酸活性测定方法，并对其中最适组合的各个参数进行了优化。结果表明：在离体测定条件下，粘虫中肠α-淀粉酶活性的最优化测定参数为0.03 mol/L 磷酸盐缓冲液（pH 8.0，含有55 mmol/L NaCl）、温度45℃、吸收波长480 nm。Ca²⁺对α-淀粉酶活性具有抑制作用。该优化法能够显著降低粘虫、德国小蠊Blattella germanica、黄粉虫Tenebrio molitor、淡色库蚊Culex pipiens pallens和家蝇Musca domestica等昆虫α-淀粉酶的米氏常数K_m值，且粘虫和德国小蠊α-淀粉酶的V_max值增大，但黄粉虫、淡色库蚊和家蝇α-淀粉酶的V_max值均明显减小。结果说明，在该优化体系下，粘虫α-淀粉酶与底物的亲和力增强，最大反应速度增大，测定酶活性的准确性和灵敏度显著提高；同时该优化体系也可作为测定德国小蠊α-淀粉酶活性的优化方法，但不适合作为黄粉虫、淡色库蚊和家蝇α-淀粉酶的最优化测定方法。     

Escherichia Coli核糖体蛋白质突变对lac Z表达的影响

测定了Escherichia coli P90CF[rec A ara D (lac pro B) F’ lac I, pro A+ proB+]及其依赖链霉素突变体(StrDA)、抗链霉素突变体(StrRA)、和回复突变体的b-半乳糖苷酶活性。发现StrDA的酶活性大大低于野生型的酶活性，而StrRA的酶活性与野生型的接近。不同的回复突变体，酶活性差别很大，最高的与野生型的接近，大多数阶于野生性与StrDA之间，少数低于StrDA。测定了E. coli A19及其衍生株的b-半乳糖苷酶活性，并与其lcI857成斑率相比较。在VT977(S8+S3+S18+L6+L24+L27)和VT567(S8+L27)中，它们的成斑率分别是野生型的6.02倍和3.56倍，而b-半乳糖苷酶活性只有野生型的19.23%和28.02%。相反，VT711(S8+S4+L6+L24)的成斑率只是野生型的6×10-7，而b-半乳糖苷酶活性仍达野生型的33.20%。     

塔里木兔和家兔的消化酶活性比较

为比较塔里木兔(Lepus yarcandensis)和家兔(Oryctolagus curiculus)消化能力的差异,探索塔里木兔对野外生存环境食物的适应机制,测定了塔里木兔和家兔胰腺及肠道的消化酶活性,包括淀粉酶(碘-淀粉比色法)、纤维素酶(3,5-二硝基水杨酸法)、脂肪酶(比浊法)及胰蛋白酶(紫外线吸收法)。用SPSS 15.0软件对实验数据进行统计学分析。采用one-way AVNOVA和多因素方差分析对比分析了塔里木兔和家兔消化酶活性。结果表明:1)塔里木兔肠道的淀粉酶活性明显高于家兔。其中,塔里木兔十二指肠、空肠及回肠的淀粉酶活性极显著高于家兔(P0.01)。淀粉酶活性在塔里木兔和家兔空肠最高。2)塔里木兔肠道的纤维素酶活性高于家兔。其中,塔里木兔盲肠的纤维素酶活性极显著高于家兔(P0.01),其回肠的纤维素酶活性显著高于家兔(P0.05)。纤维素酶活性在塔里木兔和家兔盲肠最高。3)家兔胰腺和肠道的脂肪酶活性明显高于塔里木兔。其中,家兔胰腺、十二指肠、空肠及回肠的脂肪酶活性显著高于塔里木兔(P0.05)。与消化道相比,脂肪酶活性在家兔和塔里木兔胰腺最高,肠道中空肠的脂肪酶活性最高。4)家兔胰腺和肠道的胰蛋白酶活性高于塔里木兔。其中,家兔空肠和十二指肠的胰蛋白酶活性显著高于塔里木兔(P0.05)。胰蛋白酶活性在家兔和塔里木兔空肠最高。可见,与家兔相比,塔里木兔对淀粉类、纤维类物质有较强的消化能力,对脂肪类的消化能力较弱。其中,淀粉酶活性和纤维素酶活性升高,脂肪酶活性和胰蛋白酶活性降低。这可能是塔里木兔适应贫瘠、匮乏的食物环境的重要原因之一。     

水稻种子萌发过程中α 淀粉酶与萌发速率关系的分析

用丙烯酰胺凝胶电泳技术和3,5_二硝基水杨酸（DNS）测定还原糖法,对不同萌发时间农垦58F、农垦58S和农垦58种子胚中α_淀粉酶同工酶及其酶活性变化进行研究。0～36小时萌发过程中,农垦58F和农垦58S α_淀粉酶活性高于农垦58。胚中检测出五条酶。A2、A4和A5为上述种子共有酶带。A3为农垦58F和农垦58S特异性酶带。A1为农垦58特异性酶带。据此认为α_淀粉酶对水稻种子萌发速度起重要调控作用。A1和A3可能是调控种子发芽快慢的重要酶分子。     

水稻种子萌发过程中α—淀粉酶与萌发速率关系的分析

用丙烯酰胺凝胶电泳技术和3,5－二硝基水杨酸（DNS）测定还原糖法,对不同萌发时间农垦58F,农垦58S和农垦58种子胚中α－淀粉酶同工酶及其酶活性变化进行研究,0－36小时萌发过程中,农垦58F和农垦58Sα-淀粉酶活性高于农垦58。胚中检测出五条酶,A2,A4和A5为上述种子共有酶带,A3为农垦58F和农垦58S特异性酶带,A1为农垦58特异性酶带,据此认为α-淀粉酶对水稻种子萌发速度起重要调控作用,A1和A3可能是调控种子发芽快慢的重要酶分子。     

转trxS基因大麦发芽种子水解酶活性的变化

利用转基因技术是改良大麦品种品质的有效途径.研究了转trxS基因对大麦种子发芽过程中水解酶活性的影响,结果表明转基因种子中α-淀粉酶、自由态β-淀粉酶和极限糊精酶的活性比未转基因种子高;转基因种子醇溶蛋白和谷蛋白中巯基的含量提高,说明该基因能够表达,为大麦育种和品质改良提供新的途径.     

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.     

Identification of high levels of type 1 and type 2A protein phosphatases in higher plants.

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.     

Survey of plants for enterokinase inhibitors

A radioisotopic assay procedure was developed for the assay of enterokinase inhibitors. This assay was used to test a number of plant species for enterokinase inhibiting activity. Enterokinase inhibiting activity was found in 5 of the tissues surveyed. These included peanut seeds, leaves of two tomato varieties, and tubers of two potato cultivars. Peanut seeds contained protein enterokinase inhibitors that were separated by gel filtration on Sephadex G-50 after quantitative recovery by affinity chromatography on anhydrotrypsin-Sepharose. Enterokinase inhibiting activity was found to accumulate in tomato leaves as a result of wounding. It was concluded that some plants contain enterokinase inhibitors.     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

Screening for new hydroxynitrilases from plants

We established a simple HPLC method to determine the activity and stereochemistry of the chiral mandelonitrile synthesized from benzaldehyde and cyanide, and applied it to screen for hydroxynitrile lyase (HNL) activity of plant origin. A total of 163 species of plants among 74 families were examined for (R)- and (S)-HNL activities using the method. We discovered that homogenate of leaves of Baliospermum montanum shows (S)-HNL activity, while leaves and seeds from Passiflora edulis, and seeds from Eriobotrya japonica, Chaenomles sinensis, Sorbus aucuparia, Prunus mume, and Prunus persica show (R)-HNL activity. Partially purified (R)-HNLs from Passiflora edulis and Eriobotrya japonica acted not only on benzaldehyde but also on aliphatic ketone. The enantiomeric excess of (R)-methylpropylketone cyanohydrin synthesized from 2-pentanone using homogenate from leaves of Passiflora edulis was 87.0%, and that of (R)-mandelonitrile synthesized by homogenate from seeds of Eriobotrya japonica was 85.0%.     

Antisense expression of 3-oxoacyl-ACP reductase affects whole plant productivity and causes collateral changes in activity of fatty acid synthase components

Brassica napus cv Westar plants were transformed with 3-oxoacyl-ACP reductase (KR) in antisense orientation, driven by either the cauliflower mosaic virus 35S promoter or a seed-specific acyl carrier protein promoter to determine the effects on plant productivity and on the activity of other fatty acid synthase (FAS) components. In plants with altered KR activity, total seed yield was reduced in all cases. In less severely affected plant lines, seeds had a normal appearance and composition but the yield of seeds was reduced by approximately 50%. In more severely affected lines, reductions in both seed fatty acid content and the number of seeds produced per plant were evident, resulting in a 90% reduction in fatty acid synthesized per plant. These phenotypes were independent of the promoter used. In severely affected lines, a large proportion of seeds showed precocious germination, and these had a reduced oleate content and increased levels of polyunsaturated 18-carbon fatty acids, compared with normal seeds of the same line. This reduction in 18:1 fatty acids was mimicked on imbibition of seeds with a normal appearance, indicating a preferential use of oleate moieties in precocious germination events. The reduction in activity of KR was mirrored for a second fatty acid synthase component, enoyl-ACP reductase, indicating a mechanism to maintain the ratio of fatty acid synthase components throughout embryogenesis.     

Change of thiamin-binding protein and thiamin levels during seed maturation and germination in sesame

Thiamin-binding proteins (TBPs) occur in many types of plant seeds. The biochemical and structural properties such as subunit structure and affinity for thiamin of the proteins have been characterized. However, the change of TBP and thiamin during seed maturation and germination is little known. Sesame (Sesamum indicum L.) seeds have unique albumin TBPs, because the other TBPs from plant seeds are generally globulins. In this study, we studied the change of the TBP and thiamin levels in sesame seeds. The protein content and thiamin-binding activity of the seeds increased with seed development after flowering. Immunological analysis using an antibody against the TBP of sesame seeds showed that the protein was accumulated in seeds during maturation. The thiamin content of the seeds increased with seed development after flowering. On the other hand, the thiamin-binding activity decreased during seed germination when TBP was degraded. The thiamin content of the seeds decreased during the germination. However, the amount of thiamin phosphate in the seeds during germination was little changed. These results suggested that thiamin was accumulated and stored as a complex with TBP in sesame seeds.     

Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase

In this work we test the hypothesis that yield of rice ( Oryza sativa L.) can be enhanced by increasing endosperm activity of ADP-glucose pyrophosphorylase (AGP), a key enzyme in starch biosynthesis. The potential for increases in yield exist because rice initiates more seeds than are taken to maturity and possesses excess photosynthetic capacity that could be utilized if there were more demand for assimilate. Following an approach already shown to be successful in wheat, experiments were designed to increase demand for assimilate by increasing the capacity for starch synthesis in endosperm. This was accomplished by transforming rice with a modified maize AGP large subunit sequence ( Sh2r6hs) under control of an endosperm-specific promoter. This altered subunit confers upon AGP decreased sensitivity to allosteric inhibition by inorganic phosphate (Pi) and enhanced heat stability, potentially leading to higher AGP activity in vivo. The Sh2r6hs transgene increased AGP activity in developing endosperm by 2.7-fold in the presence of Pi. Increases in AGP activity in transgenic seeds compared with controls were maximal between 10-15 days after anthesis. Starch content of individual seeds at harvest was not increased, but seed weight per plant and total plant biomass were each increased by more than 20%. Increased endosperm AGP activity thus stimulates setting of additional seeds and overall plant growth rather than increasing yield of seeds already set. Results demonstrate that deregulation of endosperm AGP increases overall plant sink strength, leading to larger, more productive plants in a manner similar to that in wheat having similar genetic modification.     

Effect of laser light treatment on some biochemical and physiological processes in seeds and seedlings of white lupine and faba bean

The aim of present study was to determine the changes of some biochemical and physiological processes, which occurred in seeds and seedlings of white lupine and faba bean after pre-sowing treatment with laser beams. It was found this treatment of seeds considerably increased the activity of amylolytic enzymes in seeds of both plants. The greatest differentiation of the enzymatic activity was noticed after 120?h from the time of sowing but the activity of these enzymes in the seeds of both tested plants was similar and it had the same course in time. The irradiated seeds of white lupine and faba bean had higher fresh weight at the time of imbibition than the seeds which were not treated with laser beams. It resulted in earlier and more uniform germination. The concentration of free radicals increased considerably in the seeds pre-treated with laser beams and the largest increase in seeds of both plant species was noticed after five exposures to laser beams. Treating seeds with laser beams considerably increased the amount of indole-3-acetic acid (IAA) in the germinating seeds. Three exposures of seeds caused the largest increase of this plant hormone content in the seeds. The activity of IAA in faba bean was slightly higher than in white lupine seeds. Pre-sowing stimulation with laser had a positive influence on the growth and development of seedlings, which had longer hypocotyl and roots in comparison to seedlings which grew from non irradiated seeds.