水稻苯丙氨酸氨解酶基因在大肠杆菌BL21 DE3中的表达

目的 :探索水稻苯丙氨酸氨解酶基因在大肠杆菌中的表达及其规律 ,为治疗苯丙酮尿症奠定基础。方法 :应用基因工程技术 ,将水稻苯丙氨酸氨解酶的cDNA(rPAL - 1-cDNA)重组入大肠杆菌高效表达质粒pET - 2 8c ,通过异丙基硫代 - β -D -半乳糖苷 (IPTG)诱导 ,在大肠杆菌中获得表达。 结果 :SDS -聚丙烯酰胺凝胶电泳表明 ,诱导 1,3,5 ,7h后 ,表达蛋白量占菌体总蛋白分别为 2 1.40 % ,30 .6 0 % ,35 .40 % ,35 .43% ,融合蛋白His6 -rPAL分子量为 78.6kDa ,主要以包涵体形式存在。结论

苯丙氨酸氨解酶的包被及其对家兔肠道苯丙氨酸吸收的影响

目的考察苯丙氨酸氨解酶(L-phenylalanine ammonia-lyase, PAL)的包被条件,并考察其对家兔肠道苯丙氨酸吸收的影响. 方法绿豆芽 (Phaseolus radiatus L.)来源的PAL通过光照、硫酸铵盐析和不同的柱层析等制备过程, 使PAL纯化到电泳纯.酶活性的测定用紫外分光光度法,苯丙氨酸含量采用荧光比色法.结果 PAL被纯化了74.6倍,并经聚丙烯酰胺凝胶 (PAG) 固定化(T ∶C=8 ∶30).该固定化酶比活0.153 U·g-1, 固定化百分数为64%. 将PAG固定化酶在人工胃液和人工胰液中保温不同时间后,发现该固定化PAL在1 h后全部失活. 将PAG固定化的PAL过120目尼纶筛网制成微粒,再用硝酸纤维膜包被,在人工胃液中不同时间保温,发现酶活性可维持2.5 h,包被的酶颗粒在光镜下呈球形,直径2～11.4 μm,自然干燥保存1个月不破坏.用硝酸纤维膜包被的PAL与L-苯丙氨酸同时注入家兔小肠腔内,与正常对照比较,PAL对苯丙氨酸有明显消除作用,吸收量减少,血苯丙氨酸浓度上升缓慢.结论 该包被条件温和,PAL损失减少,预期可作为口服的酶制剂应用于临床.

苯丙氨酸氨解酶抗白血病作用的实验研究

本文采用MTT比色分析法对比观察了从绿豆中分离提纯的苯丙氨酸氨解酶(PAL)对小鼠白血病L_(1210)细胞和人白血病K_(562)细胞的作用。结果表明:(1)PAL对白血病细胞有明显的抑制作用,2.8μ/ml的酶作用48小时后抑制率分别达62.1％和82.6％,与空白对照组相比有显著性差异(P<0.01)。(2)随酶剂量的增加,抑制效果明显增强,同样作用48小时,0.70μ/ml的酶其抑制率分别为5.2％和14.1％,当酶增加为3.5μ/ml,可分别达77.1％和85.8％(P<0.001)。

苦荞发芽过程中促进黄酮合成的因素初探

研究苦荞发芽过程中黄酮类化合物的变化规律。苦荞发芽过程中通过改变温度、光照等条件和喷洒乙烯利处理,能够明显刺激次级代谢产物黄酮的生成和提高苯丙氨酸氨解酶(PAL)活性,PAL活性与黄酮含量变化基本上是相符的,PAL活性升高,黄酮合成量也增加。

苦荞内生菌的分类鉴定及对黄酮合成的影响

从苦荞中分离得到2株内生菌N3和XJ01,通过形态观察、生理生化实验和16SrDNA系统进化分析,鉴定这2株苦荞内生菌属于肠杆菌科泛菌属。用内生菌及其多糖类提取物对萌发苦荞进行处理,结果表明,N3和XJ01提取物能诱导苦荞样品中苯丙氨酸氨解酶的活性升高,进而促进萌发苦荞的黄酮合成。内生菌提取物的最佳使用浓度均为50mg/L,经N3提取物处理的苦荞黄酮含量最高,达到4.59%,比对照提高15.3%。

真菌诱导子对苦荞萌发过程中黄酮合成的影响

将源于苦荞种子表面的真菌K10、K11和K18制成诱导子,并对萌发苦荞进行诱导处理。结果表明,K11和K18能诱导苦荞样品中苯丙氨酸氨解酶的活性升高,进而促进萌发苦荞的黄酮合成。K11和K18诱导子的最佳使用浓度为200 mg/L和50 mg/L,诱导子处理后,苦荞种子萌发第6天的黄酮含量最高,分别达到5.13%和4.39%,比对照提高24.5%和6.6%。

Activities of lipoxygenase and phenylalanine ammonia lyase in poplar leaves induced by insect herbivory and volatiles

A study was conducted to explore the defense response in woody plants after insect herbivory. The activities of two enzymes, lipoxygenase （LOX）, a key enzyme ofjasmonate （JA） pathway, and phenylalanine ammonia lyase （PAL）, a rate-limiting enzyme of phenyl- propanoid pathway, were measured in the leaves of one-year-old poplar （Populus simonii × P. pyramidalis ＇Opera 8277＇） cuttings after Clostera anachoreta larvae attack. The results show that the increased activities of LOX and PAL were found not only in the leaves wounded by C. anachoreta larvae but also in their tipper systemic leaves, indicating that JA and phenylpropanoid pathways were activated, and the defense response was stimulated systemically. The increase in LOX and PAL activities in neighboring intact poplar cuttings sug- gested that there exists the interplant communication between poplar plants mediated by the herbivore-induced volatiles. Methyl jasmonate （MeJA） was also proved to be an airborne signal to induce defense response in P simonii × P pyramidalis ＇Opera 8277＇ cuttings.

Germination Characteristics and Secondary Metabolism Regulation of Scutellaria baicalensis Georgi Seeds under Different Temperatures

[Objective] To study the germination characteristics and secondary metabolism of Scutellaria baicalensis Georgi seeds under different temperatures.[Method]The activities of superoxide dismutase（SOD）and peroxidase（POD）were determined with lactoflavine-NBT method and guaiacol-colorimetry assay,respectively.The catalase（CAT）,ascorbate peroxidase（APX）,phenylalanine ammonia-lyase（PAL）and cinnamic acid 4-Hydroxylase（C4H）were determined with ultraviolet spectrophotometry.The secondary metabolites were detected by High Performance Liquid Chromatography（HPLC）.[Result]The germination percentage,germination potentiality and germination index were seriously affected by low or high temperature.The proper germination temperature was 20-25 ℃.The activities of SOD,POD and CAT were significantly decreased as comparison to suitable temperature.The activities of PAL and C4H were also significantly decreased as comparision to suitable temperature which was accordance to the secondary metabolites.There was a positive correlation between the flavonoids content and the PAL and C4H activity（r=0.956,r=0.951,P0.05）.[Conclusion]The quality of the skullcaps could be improved by improving the activity of PAL and C4H.The proper temperature for the seed germination and the formation of secondary metabolites was 20 ℃.

Germination Characteristics and Secondary Metabolism Regulation of Scutellaria baicalensis Georgi Seeds under Different Light Intensities

[Objective] This study aimed to investigate the primary and secondary metabolisms during the germination of Scutellaria baicalensis Georgi seeds under different light intensities. [Method] The activities of CHL, soluble sugar, PAL, C4H and CHS were determined with ultraviolet spectrophotometry. The secondary metabolites were detected by High Performance Liquid Chromatography （HPLC）. [Result] The results indicate that the germination of Scutellaria baicalensis Georgi seeds is not sensitive to light and the seedlings were very sensitive to light. The CHL, soluble sugar, PAL, C4H and CHS continuously increased with light intensity. The content of secondary metabolites also increased. [Conclusion] Light increased the formation of leaf photosynthetic pigment, thereby affecting the primary metabolites. The activities of PAL, C4H and CHS significantly increased with the development of light intensity. Finally the secondary metabolites of medicinal plants increased sharply. Therefore, the quality of Scutellaria baicalensis Georgi materials can be improved by increasing the light intensity moderately.

Changes of Primary and Secondary Metabolism during the Seed Germination of Scutellaria baicalensis Georgi

[Objective] To investigate the primary and secondary metabolism during the seed germination of Scutellaria baicalensis Georgi. [Method] The activities of SOD and POD were determined with lactoflavine-NBT method and guaiacol-colorimetry as- say, respectively. The activities of CAT, APX,PAL and C4H were determined with ultraviolet spectrophotometry. The contents of secondary metabolites were detected by High-performance Liquid Chromatography （HPLC）. [Result] The results indicate that the soluble sugar content decreased during the first 5 days and then increased when the cotyledons formed. The contents of PAL, C4H and CHS continuously in- creased at different stages during the seed germination process. The secondary metabolites also showed the consistent variation trend. In addition, the contents of secondary metabolites had significant positive correlation with the key enzyme activi- ty. [Conclusion] The formation of secondary metabolites is significantly positively cor- related with the key enzyme during the seed germination process. Therefore, the key enzyme activity can be enhanced by adopting appropriate measures to improve the secondary metabolites, thereby obtaining high-quality medicinal materials.

Prediction Model of Secondary Substances in Anthocyanins Synthesis of Purple Corn

The aim of this study was to assay the polyphenols,flavonoid,polyphenol oxidase and phenylalnine ammonialyase which were relative to the anthocyanins synthesis of purple corn. The optimization of multiple linear regression model of anthocyanins synthesis was y=4.383 86-0.205 45x1＋5.479 638x2＋0.195 575x4. According to standard partial regression coefficient testing,the result indicated that polyphenols content was negatively correlated with anthocyanins and the relative influence to anthocyanins synthesis was-42.7%; flavonoid content and activity of polyphenol oxidase were positively correlated with anthocyanins of purple corn and the relative influence to anthocyanins synthesis were 71.45% and 73.32% respectively. There was no positive correlation between the activity of phenylalnine ammonialyase and anthocyanins of purple corn. The establishment of multiple linear regression model of anthocyanins synthesis was to provide theory foundation of producing anthocyanins in laboratory.

医药

苹果酸酶工程技术生产工艺术编号:Y97—1—1苹果酸在工业、医药和食品工业上有广泛的用途,生产方法有化学合成法和发酵法,合成法只能生产 DL—苹果酸;发酵法生产 L—苹果酸,

生理 生化 生态

W970825 小麦和玉米根依赖光的质子排出[刊,英]/Gorr,G.…//Botanica Acta.-1995,108(4).-351～357(WBTA,1996,13(3),1929]W970826 引导物和抑制物对离体质膜磷脂酶 C 的影响与小麦叶片苯丙氨酸氨解酶活性相关[刊,英]/

Germination Characteristics and Secondary Metabolism Regulation of Scutellaria baicalensis Georgi under Different Illumination Time

This study aimed to investigate the primary and secondary metabolism of Scutel aria baicalensis Georgi during seed germination process under different il umination time. [Method] Chlorophyl （CHL） content, soluble sugar content, phenylalanine ammonia lyase （PAL） activity and cinnamate-4-hydroxylase （C4H） ac-tivity were determined with ultraviolet spectrophotometry. The secondary metabolites were detected by high performance liquid chromatography （HPLC）. [Result] The re-sults indicated that the germination of S. baicalensis seeds was not sensitive to light and the seedlings were very sensitive to light. CHL content, soluble sugar content, PAL activity and C4H activity increased continuously with the il umination time. The secondary metabolites showed a similar trend. [Conclusion] Il umination time promoted the formation of leaf photosynthetic pigments, thereby affecting the synthesis of primary and secondary metabolites and the activities of PAL and C4H. Therefore, the quality of S. baicalensis can be improved by regulating the il umina-tion time appropriately.

Cloning, Expression and Analysis of pal, a Positive Gene Involved in Capsaicin Biosynthesis in Capsicum chinense Jacq

The cDNA sequence of Capal gene was cloned from Capsicum chinense Jacq by RT-PCR and sequenced. Bioinformatics analysis showed that Capal en- codes a putative polypeptide of 683 amino acids with a calculated molecular mass of 74.2 kD and a theoretical pl of 6.9. Multiple sequence alignments and phyloge- netic tree analyses showed that Capal protein of C. chinense is similar to that of Capsicum annuum var. conoides, with an overall sequence similarity of 96%. The prokaryotic expression vector pET-32a-pal was constructed and induced to express in E. coil BL21. The SDS-PAGE analysis showed that the relative molecular mass of the induced new protein is about 74 kD, which was basically identical with that predicted by DNAMAN software （74.3 kD）, Real-time PCR analysis showed that ex- ogenous jasmonic acid （JA） promoted pal expression. The accumulation of capsaicin in pepper was analyzed by high performance liquid chromatography （HPLC）, and the results indicated that exogenous jasmonic acid （JA） can promote the synthesis of capsaicin. This study will provide valuable experimental basis for the research of transcription regulation and explaining the gene function of pal.

Promotive Effects of Alginate-Derived Oligosaccharides on the Inducing Drought Resistance of Tomato

In order to determine the role of alginate-derived oligosaccharides (ADO) in drought stress resistance of tomato (Ly-copersicon esculentum Miller) seedlings, the leaves were exposed to different concentrations of ADO (0.05%, 0.10%, 0.20%, 0.30% and 0.50%) after drought stress was simulated by exposing the roots to 0.6 molL-1 PEG-6000 solution for 6 h. Changes in biomass, electrolyte leakage and malondialdehyde (MDA), free proline, total soluble sugars (TSS) and abscisic acid (ABA), the enzyme activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) were measured to investigate the effects of ADO treatment. The results showed that the treatment with an ADO concentration of 0.20% exhibited the highest performance of drought stress resistance in the tomato seedlings by decreasing the electrolyte leakage and the concentration of MDA, increasing the contents of free proline, TSS and ABA, and increasing the activities of CAT, SOD, POD and PAL after treatment with ADO. It is suggested that changes in electrolyte leakage, MDA, osmotic solutes, ABA, anti-oxidative enzyme and PAL activities were responsible for the increased drought stress resistance in tomato seedlings. To our best knowledge, this is the first report of the effect of ADO treatment on enhancing the drought stress resistance of tomato seedlings.

Change of 3 Kinds of Enzyme Activity and Soluble Sugar in 3 Chinese Cabbage Varieties with Different Resistance Level During the Process of Plasmodiophora brassicae Infecting Their Roots

There are different levels of resistance in Chinese cabbage varieties against clubroot.The content of catalase(CAT),peroxidase(POD),phenylalanin ammonia-lyase(PAL),Super Oxide Dismutase(SOD)and soluble sugar activities of 3cultivars with different clubroot-resistant levelswere detected after inoculation in this study.The results suggest that the changing rates of CAT,PAL and SOD contents of resistance cultivars after inoculation were higher than those of susceptible cultivars(Baigengbaiye>Zaobaicaitai>Huangjinxiaobaicai);the POD activities of resistance cultivars reached a peak on an earlier day than those of susceptible cultivars;the increasing rate of soluble sugar of susceptible cultivars was higher than that of the resistance cultivars,and ended with two peaks.Therefore,the four enzymes(CAT,POD,PAL and SOD)and soluble sugar may be used as physiological and biochemical reference indexes for the resistance identification to clubroot after inoculation with P.brassicae.

Alteration in Biochemical Response in Sesamum indicum upon Different Plant-Pathogen Interactions

Sesamum, an important oil yielding crop suffers a huge loss in its yield due to attack of large number of fungal pathogens. In semi-arid regions Sesamum is mainly affected by two major plant-pathogenic fungus viz. Macrophomina phaseolina and Fusarium oxysporum. The aim of the study was to analyze the metabolic alterations in Sesamum after infection with both pathogens. This accomplished by individually by （the word estimating is not quantitative） the levels of total phenolic compounds and the activities of phenylalanine ammonia lyase （PAL） of one week old plants. The PAL showed high activity in infected plants, revealing the active phase in the synthesis of secondary metabolites in the Sesamum plant after infection. As a consequence, in infected plants the contents of polyphenols along with salicylic acid （SA） considerably exceeded when compared to control plants. This in vivo study of M. phaseolina and F. oxysporum infection reveals the differences of resistance levels in sesame against these two pathogens. These results provide important information regarding the plant-pathogen interactions and also forfor Sesamum improvement programs seeking the adaptation to diverse range of fungal attack along with adverse environmental factors.

Effects of four metal Ions on phenylalanine ammoni-alyase activities and flavonoids content of ginkgo biloba leaves

The enzyme activities of phenylalanine ammonia-lyase from six years old Ginkgo biloba leaves in the potted orchard were induced by four groups of metal ions respectively. The PAL activities and flavonoids content were measured by the UV-Vis spectrophotometer. Changes of the whole year were also studied. The results showed that four groups of metal ion treatments had significant effects on the PAL activities and flavonoids contents in Ginkgo leaves. The treatments with 0.1% and 0.3% FeSO4, 0.1%, 0.2% and 0.3% ZnSO4, 0.1%, 0.3% and 0.5% MnSO4, 0.1% and 0.4% CuSO4 not only enhanced flavonoids contents, but also extended the optimum harvest dates of ginkgo leaves. The optimum combination was 0.1% FeSO4, 0.2% ZnSO4, 0.3% MnSO4, and 0.4% CuSO4. It could improve flavonoids content up to 1.619 4%.

Effects of 2,5-Norbornadiene （NBD） on Physiological and Biochemical Responses of Fresh-cut Winter Squash

Ethylene is one of the most important plant hormones that regulate physiological responses, maturity and senescence of postharvest fruits and vegetables. The determination of its role in the wound-healing response of fruits and vegetables is helpful in understanding the wound responses. 2,5-Norbornadiene （NBD）, inhibitor ethylene binding were used to determine the effect of ethylene action on wound-induced response of fresh-cut winter squash, a kind of vegetables known to produce ethylene markedly after cutting. The effect of NBD on the physiological and biochemical changes of fresh-cut winter squash was investigated in this experiment. The results showed that NBD had great effects on physio-biochemical metabolism of fresh-cut winter squash. Ethylene production was inhibited significantly by NBD. NBD also had an inhibition effect on polyphenol oxidase （PPO）, peroxidase （POD） and phenylalanine ammonia lyase （PAL） activity. The effect of NBD on lipoxygenase （LOX） activity and superoxide dismutase （SOD） was not obvious. NBD treated sample showed a more rapid increase in catalase （CAT） activity, which may help in healing the damage of the tissue. These information are important in understanding the fundamental biology of wound response and in the development of new technology to hasten wound-healing and, thereby, to minimize costly wound-related problems in fresh-cut industry.