Molecular cloning and functional identification of invertase isozymes from green bamboo Bambusa oldhamii

Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.     

Expression and characterization of sweet potato invertase in Pichia pastoris

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.     

外源脱落酸增强甘薯幼苗耐盐性的作用

【目的】本文系统研究了不同浓度NaCl胁迫及外源脱落酸(ABA)对NaCl胁迫下甘薯幼苗生根及一些生理生化特性的影响,探讨了外源ABA对盐胁迫下甘薯幼苗的缓解效应,为增强盐碱地甘薯耐盐性、 提高产量提供理论依据。【方法】以甘薯种植品种徐薯25为实验材料,在装有石英砂的具孔塑料盆中放入培养室自然光照/昼夜温度[(261)/(171)℃]中培养,并进行不同浓度NaCl处理以及对NaCl 300 mmol/L胁迫甘薯幼苗叶片喷施ABA溶液,连续处理7 d后,测定生根数,使用CIRAS-1型便携式光合仪测定光合作用指标、 植物效率分析仪测定叶绿素荧光参数、 采用比色法测定丙二醛、 脯氨酸、 可溶性糖含量和超氧化物歧化酶活性、 用原子吸收分光光度计测定Na+、 K+、 Ca2+含量,利用SPSS13.0和Excel软件对数据进行处理分析。【结果】低浓度 NaCl胁迫(50 mmol/L)对甘薯幼苗影响较小; 随着盐度的增加,甘薯生根不断减少,相对电导率、 丙二醛(MDA)、 脯氨酸和可溶性糖含量持续增加,甘薯叶片超氧化物歧化酶(SOD)活性呈先增加后降低趋势; 叶片净光合速率(Pn)、 蒸腾速率(Tr)、 气孔导度(Gs)、 光系统Ⅱ(PSⅡ)最大光化学效率(Fv/Fm)、 捕获的激子将电子传递到电子传递链中超过QA的其他电子受体的概率(0)、 用于电子传递的量子产额(E0)逐渐降低,放氧复合体活性(Vk)和用于热耗散的量子比率(D0)不断增加; 叶片中Na+含量增加,K+、 Ca2+和K+/Na+水平降低。高浓度(300 mmol/L)NaCl胁迫下,甘薯幼苗的正常生理代谢受到显著抑制。适当增加外源ABA浓度,能够显著缓解NaCl胁迫造成的伤害作用,以ABA浓度为70 mol/L的缓解效果最好。【结论】外源ABA可显著促进盐胁迫下甘薯幼苗生根,维持细胞膜的稳定性,降低膜脂过氧化程度,调节植物细胞的渗透和离子动态平衡,使甘薯幼苗叶片维持较高的Fv/Fm、 0、 E0和较低的Vk、 D0,缓解PSⅡ光抑制的程度,改善植物的光合作用,提高植物的耐盐性。因此,喷施70 mol/L ABA是缓解NaCl胁迫效应,提高甘薯幼苗耐盐性的一种有效方法。     

Invertase inhibitors from sweet potato (Ipomoea batatas): purification and biochemical characterization

Two proteinaceous invertase inhibitors, designated ITI-L and ITI-R, were purified to electrophoretic homogeneity. ITI-L was purified from acetone powder of sweet potato leaves through sequential steps entailing buffer extraction, acid treatment, DEAE-Sephacel ion-exchange chromatography, and Sephacryl S-100 gel filtration. ITI-R was purified from sweet potato tuberous roots by sequentially applying buffer extraction, Con A-Sepharose affinity chromatography, DEAE-Sephacel ion-exchange chromatography, Sephacryl S-200, and Superose 12 gel filtration. The optimal pHs for interaction between ITI-L and ITI-R and acid invertase from sweet potato leaves were 5.5 and 5.0, respectively. The molecular masses of ITI-L and ITI-R were 10 and 22 kDa, respectively, as estimated by both gel filtration and SDS-PAGE. Both inhibitors were thermostable (90% of the activity remained after incubation at 100 degrees C for 20 min), and Western blotting showed them to be immunologically related.     

紫甘薯对硒的吸收和累积特征

以紫甘薯为试验材料,采用盆栽试验的方法研究了基施硒酸钠[Se(VI)]和亚硒酸钠[Se(IV)]条件下,紫甘薯对外源硒的吸收累积规律,并比较了施用两种不同价态硒的紫甘薯富硒效果。结果表明:两种硒源均可显著提高紫甘薯各器官含硒量,且紫甘薯含硒量均随施硒量的增加而增大。当土壤施硒量为Se 8 mg/kg时,施用硒酸钠、亚硒酸钠收获期薯块的硒含量(干基)分别达到6.69、0.88 mg/kg。紫甘薯生育期40 d时各器官硒含量叶茎薯块,130 d时硒含量叶薯块茎。当硒酸钠施用量为Se 4 mg/kg时,紫甘薯薯块中的硒累积量最高达923.81μg/株,硒在紫甘薯块根中的分配率可达67%~70%,硒酸钠处理下,紫甘薯对硒的吸收利用率远远高于亚硒酸钠处理。综合紫甘薯含硒量和施硒量对生长的影响结果分析,施用硒酸钠和亚硒酸钠均能增加紫甘薯薯块的硒含量,紫甘薯对硒酸钠敏感性高于亚硒酸钠,生产过程中应充分考虑施用硒酸钠对作物造成的毒害。     

甘薯茎叶多酚分离纯化及降血糖活性研究进展

甘薯茎叶为甘薯地上蔓生部分,一年能多次采收,与甘薯块茎产量相当.甘薯茎叶中的多酚类物质含量丰富,主要为酚酸类和黄酮类物质,具有抗氧化、降血糖、抗菌消炎、抗肿瘤、保护肝脏和免疫调节等作用,且甘薯茎叶多酚类物质的降血糖作用已成为天然产物活性研究领域的热点之一.本文综述了甘薯茎叶多酚类物质的提取和分离纯化方法、酚酸类和黄酮类...     

玉米叶绿体铁氧还蛋白1的原核表达及部分性质分析

叶绿体铁氧还蛋白(Fd)通过活性中心的铁硫簇传递还原力,在各种氧化还原途径中起重要作用.本研究中,氨基酸序列比对显示玉米中5种Fd的叶绿体导肽同源性很低,而去除导肽的成熟蛋白氨基酸序列具有很高的同源性.采用RT-PCR技术从玉米幼叶总RNA中克隆了编码成熟Fd1的基因.并分别插入pQE 80和p28SUMO表达载体,转...     

蚯蚓菌根互作对土壤酶活、 甘薯根系生长及养分吸收的影响

【目的】蚯蚓和丛枝菌根真菌处于不同的营养级,但在促进植物生长和提高土壤肥力等方面却都发挥着积极作用。单独对土壤微生物或土壤动物的研究较多,但对土壤微生物与土壤动物之间相互作用的研究很少。因此研究它们对土壤和植物生长的作用可为挖掘土壤生物的潜力和提高土壤生物肥力提供依据。【方法】采用盆栽试验,研究了蚯蚓(Eisenia fetida)与丛枝菌根真菌(Rhizophagus irregularis)互作对甘薯生长和养分吸收的影响。试验采用两因素完全随机试验设计,分为接种和不接种菌根真菌及添加和不添加蚯蚓。试验共4个处理: 不加菌根和蚯蚓(CK); 接种菌根真菌(AM); 添加蚯蚓(E); 添加蚯蚓和菌根真菌(E+AM),每个处理4次重复。调查了甘薯养分吸收、 根系形态及土壤养分变化,采用Canoco4.5软件对土壤生物与植物对应关系进行RDA (redundancy analysis)分析。【结果】接种菌根真菌显著提高了甘薯地上和地下部生物量(P0.05),而添加蚯蚓的处理仅提高了甘薯地上部生物量。同时添加蚯蚓和菌根的处理显著提高了甘薯地上地下部生物量,并且高于其他三个处理(P0.05)。与对照相比,接种菌根真菌显著提高了土壤磷酸酶活性(P0.01),增幅近一倍; 同时提高了土壤磷的植物有效性,土壤有效磷含量下降了30%左右。添加蚯蚓后土壤脲酶活性从5.45 mg NH+4-N/g显著增加到8.71 mg NH+4-N/g,土壤碱解氮的含量从5.82 mg/kg显著增加到6.89 mg/kg (P0.05)。RDA分析表明蚯蚓菌根互作对甘薯地上和地下部氮磷含量、 根表面积、 根体积、 根平均直径和根尖数均存在显著的正交互效应。蚯蚓菌根互作通过调控土壤酶和改变土壤养分有效性促进甘薯对土壤氮磷养分的吸收。【结论】蚯蚓(Eisenia fetida)通过调控土壤脲酶和碱性磷酸酶增加了土壤中氮磷的有效性从而促进甘薯地上部生长。丛枝菌根真菌(Rhizophagus irregularis)通过调控土壤磷酸酶和增加植株地上地下部吸磷量从而促进甘薯生长。添加蚯蚓或接种菌根真菌均能增加根系吸收面积和根体积从而促进甘薯对养分的吸收。蚯蚓和菌根真菌相互作用通过调控土壤酶和改变土壤养分有效性以及促进根系发育从而互补的促进甘薯养分吸收和生长。     

Effects of Sodium on Potassium Nutrition in Three Tropical Root Crop Species

The potassium (K) nutrition and high K requirement of tropical root crops may be affected by their sodium (Na) status, as has been observed in a number of plant species. Solution culture was used to study the effects of K and Na supplies in tannia [Xanthosoma sagittifolium (L.) Schott.], sweet potato [Ipomoea batatas (L.) Lam.] and taro [Colocasia esculenta (L.) Schott]. At low K supply, Na ameliorated symptoms of K deficiency and increased growth in tannia, and to a lesser extent in sweet potato, but not in taro. None of the species responded to Na at adequate K supply. Differences in response to Na were attributed to differences in Na translocation to plant tops. At maximum Na supply, the Na concentration in index leaves averaged 1.82% in tannia, 0.205% in sweet potato, and 0.0067% in taro. An increase in the supply of Na resulted in a shift in the critical K concentration for deficiency (i.e., 90% of maximum yield) in index leaves from 2.9% to 1.2% in tannia, and from 4.8% to 2.5% in sweet potato. The critical K concentration in taro was 3.3%, irrespective of Na supply. To overcome the problem in tannia and sweet potato of determining the critical concentration relevant to a leaf sample of unknown K status, a relationship was established for each species relating the critical K concentration to the concentration of Na in the index leaves.     

Natural abundance of 15N in sweet potato, pumpkin, sorghum and castor bean: possible input of N2-derived nitrogen in sweet potato

Two combinations of plant species, sweet potato (three cultivars) and pumpkin, and sweet sorghum (three cultivars) and castor bean were grown separately in three plots of alluvial soil from June to September 1996. The shoots (leaves plus stems) of sweet potato and pumpkin, and the whole tops (leaves plus stems and grains) of sweet sorghum and castor bean were harvested twice, once in August and once in September in order to analyze their natural abundance of ¹⁵N (δ¹⁵N). The δ¹⁵N values of two of the varieties of sweet potato harvested in September were significantly lower than those of pumpkin, while δ¹⁵N values of sweet potato and pumpkin harvested in August, as well as those of sweet sorghum and castor bean harvested in August and September, did not significantly differ. The lower δ¹⁵N values observed in the September-harvested sweet potato may indicate that as much as 40% of the N intake of this species is derived from dinitrogen. This species is known to have a high ability to take up N from undefined sources. Received: 23 February 1997     

甘薯抗疮痂病多胺与吲哚乙酸代谢的研究

研究不同抗性甘薯品种受疮痂病菌侵染前后多胺和吲哚乙酸代谢变化结果表明 ,感染疮痂病菌后抗病与感病品种甘薯叶片中吲哚乙酸含量和多胺氧化酶活性降低 ,多胺总量及腐胺含量增加 ,腐胺 / (精胺 亚精胺 )比值升高 ,感病品种变化幅度高于抗病品种。疮痂病菌侵入后感病品种甘薯叶片中绿原酸含量下降 ,吲哚乙酸氧化酶和过氧化物酶活性升高 ,而抗病品种则相反。     

58个不同品种甘薯茎叶营养与功能成分的研究

为了明确不同品种甘薯茎叶的营养与功能成分组成,本试验系统比较了58个不同品种甘薯茎叶中的粗蛋白、粗脂肪、膳食纤维、维生素、多酚类物质、黄酮类物质含量及抗氧化活性。结果表明,甘薯茎叶富含粗蛋白(9.35~38.45 g·100 g^-1 DW)、粗脂肪(1.36~12.30 g·100 g^-1 DW)、总膳食纤维(35.30~45.0 g·100 g^-1 DW)、维生素C(1.47~131.64 mg·100 g^-1 DW)、β-胡萝卜素(6.75~59.35 mg·100 g^-1 DW)及维生素E(0.39~23.30 mg·100 g^-1 DW);湛薯01-2品种甘薯茎叶中总酚含量(21.39 g CAE·100 g^-1 DW)及抗氧化活性(40.28 g TE·100 g^-1 DW)最高。抗氧化活性与营养成分之间的相关性分析结果表明,甘薯茎叶中主要的抗氧化活性物质为多酚类物质(R²=0.748)。本研究为甘薯茎叶的合理开发利用提供了一定的指导。     

Antioxidant activities of trypsin inhibitor, a 33 KDa root storage protein of sweet potato (Ipomoea batatas (L.) Lam cv. Tainong 57).

Trypsin inhibitors (TIs), root storage proteins, were purified from sweet potato (Ipomoea batatas[L.] Lam cv. Tainong 57) roots by trypsin affinity column according to the methods of Hou and Lin (Plant Sci. 1997, 126, 11-19 and Plant Sci. 1997, 128, 151-158). A single band of 33 kDa TI was obtained by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. This purified 33 kDa TI had scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. There was positive correlation between scavenging effects against DPPH (2 to 22%) and amounts of 33 kDa TI (1.92 to 46 pmol). The scavenging activities of 33 kDa TI against DPPH were calculated from linear regression to be about one-third of those of glutathione between 5 and 80 pmol. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detection, it was found that 33 kDa TI could capture hydroxyl radical, and the intensities of EPR signal were significantly decreased from 1.5 to 6 pmol of 33 kDa TI compared to those of the controls. It is suggested that 33 kDa TI, one of the sweet potato root storage proteins, may play a role as an antioxidant in roots and may be beneficial to health when it is consumed.     

钾肥基施利于甘薯块根产量的形成

【目的】 研究不同施钾方法对甘薯块根产量形成的影响,可为甘薯高产稳产寻找合理的钾肥施用方法,并阐明其增产机理。【方法】 选取典型的食用型甘薯品种红香蕉和北京553为试材,于2013~2014年两个生长季在山东农业大学农学试验站进行田间试验。设4个施钾处理:不施钾肥（CK）、全部钾肥基施（JS）、全部钾肥封垄期追施（FS）、基施钾肥和封垄期追施钾肥各占50%（1/₂ JS+1/₂ FS）。在主茎第4、5片展开叶上标记13CO₂,24、48和96 h后取样,用质谱仪（Isoprime 100）测定不同部位的13C含量。从甘薯栽秧后50 d开始,每隔20 d取样直到收获。按根、茎、叶、柄四部分分别称重,将主茎自功能叶所在位置至基部等分为三段,取上、下两端用于测定蔗糖含量。收获期测定生物产量和块根产量,计算经济系数。【结果】 甘薯施用钾肥能显著提高生物产量、经济系数和块根产量,增产21.33%~34.38%。各施钾处理之间比较,生物产量差异不显著,经济系数和块根产量差异显著,其中基施钾肥处理的经济系数和块根产量最高。栽秧后50 d,基施钾肥处理植株干物质积累量和功能叶光合产物在块根中的分配率都显著高于其他施钾处理。栽秧后50 d和110 d,基施钾肥处理光合产物源端装载和库端卸载的效率显著高于其他处理,栽秧后150 d,所有施钾肥处理趋于一致。栽秧后50 d,基施钾肥处理光合产物转运效率显著高于其他施钾处理,栽秧后100 d和150 d,施钾肥处理间效果趋于一致。在栽秧后50~130 d,基施钾肥处理的块根膨大速率一直高于其他施钾处理。【结论】 在相同供试土壤和气候条件下,钾肥施用时间越早,越有利于甘薯早发、快长,促进块根早形成、快膨大;光合产物由叶片向块根运转效率的高值持续期越长,越有利于提高光合产物在块根中的分配。因此,全部钾肥基施甘薯经济系数和块根产量最高,是最经济有效的施肥方法。     

A pectinmethylesterase gene associated with a heat-stable extract from citrus

A putative thermostable pectinmethylesterase (TSPME) protein of 36 kDa was isolated from heat-treated citrus finisher pulp. After purification and partial sequencing of the protein, a reverse genetic approach was used to obtain the complete genomic sequence of a new pectinmethylesterase (PME) gene, CsPME4, from Citrus sinensis (L.) Osb. cv. Valencia. The CsPME4 gene contained two exons of 1203 and 690 bp interrupted by a single positionally conserved intron of 1230 bp. A full-length CsPME4 cDNA clone amplified from Valencia orange juice vesicles shared 98% identity with the genomic clone. The encoded protein of the full-length CsPME4 cDNA shared 66 and 39% amino acid identity with the full-length encoded proteins of the citrus PME, CsPME1, and CsPME3, respectively, whereas the predicted mature protein of CsPME4 shared 80 and 61% identity with the predicted mature proteins of CsPME1 and CsPME3, respectively. Southern analysis demonstrated that CsPME4 was present in at least two copies in the Valencia orange genome. Northern analysis revealed that CsPME4 mRNA was accumulated mainly in young and developing tissues of Valencia orange. Several approaches to express recombinant CsPME4 in different systems failed to obtain active protein. Further research will be necessary to successfully express the putative TSPME gene CsPME4 for biochemical characterization.     

Quantitation of methylxanthinic alkaloids and phenolic compounds in mate (Ilex paraguariensis) and their effects on blood vessel formation in chick embryos

Methylxanthinic alkaloids and phenolic compounds are related to the therapeutic properties of Ilex paraguariensis infusions. Considering the known vascular tropism of xanthines, an aqueous extract (mate) and caffeine were evaluated on blood vessel formation, in connection with the analysis of those secondary metabolites, which was performed in young and mature leaf samples collected in three cultivation systems located in the southern region in Brazil (Santa Catarina State). Samples of young and mature leaves from a monoculture cultivation system (MC) showed the highest content of phenolic compounds (149.68 microg/mL, young leaves; 135.50 microg/mL, mature leaves) and caffeine (young leaves, 148.07 microg/mL; mature leaves, 244.63 microg/mL) as compared to samples from agroforesty (AF) and shaded-native (NT) cultures. Theophylline was not detected in samples by reverse phase-high performance liquid chromatography, and mature leaves showed lower theobromine amounts (11.46 microg/mL). Treatments performed with mate aqueous extract and caffeine (1.03-4.12 microM/disk) in the yolk sac vascular membranes of 2-day-old chick embryos revealed pro-vasculo- and angiogenic properties as well as embryonic growth enhancement. These findings, uncoupled from any detectable embryotoxic effect, suggest a potential therapeutic and/or prophylactic use in cardiovascular disorders for caffeine and related constituents of mate plant extracts, an issue that waits further studies.     

接种根内球囊霉提高氮素向甘薯块根转移和再分配的机理

【目的】研究接种丛枝菌根真菌 (arbuscular mycorrhiza, AM) 对甘薯 (Ipomoea batatas L.) 的侵染率及叶片氮代谢酶活性的影响,探索甘薯氮素吸收后在植株体内的转移和分配规律,以期为全面了解菌根真菌促进氮代谢的过程提供理论依据。【方法】采用盆栽试验方法,供试菌种为一种根内球囊霉Glomus intraradices BEG141。土壤灭菌后,以不接种菌根 (–AM) 为对照,在8 kg土中接种100 g菌剂 (+AM)。于甘薯幼苗移栽后30天、60天和90天,从甘薯茎蔓顶部往下数第5片完全展开叶的叶柄与茎蔓交叉处定量注射99% (15NH₄)₂SO₄溶液,15N总施用量为199.5 μg/plant。每次注射后三天取植株样,分为茎、叶、纤维根和块根4部分,测定生物量干重、根系菌根侵染率、15N丰度、氮代谢酶活性。【结果】接种AM处理显著增加了甘薯根部真菌侵染率及泡囊丰度、根内菌丝丰度和丛枝丰度。随着移栽天数的增加,侵染率显著增加,最高达到67%。移栽后30天接种和不接种菌根真菌处理间甘薯生物量和氮素吸收量差异不显著,移栽后60天和90天,接种AM真菌处理的甘薯生物量和氮素吸收量显著高于不接种AM处理 (P < 0.05)。与CK相比,同一生育期接种AM处理显著提高了甘薯叶片谷氨酸脱氢酶 (GDH)、谷氨酰胺合成酶 (GS) 和谷氨酸合成酶 (GOGAT) 的活性,对硝酸还原酶 (NR) 活性无显著影响。双因素分析表明,接种菌根与接种后时间对提高甘薯生物量干重、氮素累积量及GDH和GS活性的正交互效应显著 (P < 0.05)。移栽后30天,接种AM处理显著提高了甘薯茎蔓和叶片15N积累量和分配率;移栽后60天,叶片中15N积累量较前一时期显著增加。接种AM处理的叶片和茎蔓中15N积累量在30 d和60 d显著高于不接种AM处理 (P < 0.05),而在移栽后90天显著低于不接种AM处理,说明接种AM处理显著促进15N向块根的转移和分配。【结论】接种AM真菌可提高GDH、GS和GOGAT的代谢活性,促进无机氮向有机氮的转化。接种AM菌剂可促进生育前期氮素在叶片中的分配,有利于地上部的生长,而后期促进地上部积累氮素向地下部转运,进而增加甘薯块根中的干物质积累,提高甘薯的经济产量。     

Dehydroascorbate reductase cDNA from sweet potato (Ipomoea batatas [L.] Lam): expression, enzyme properties, and kinetic studies

A cDNA encoding a putative dehydroascorbate reductase (DHAR) was cloned from sweet potato. The deduced protein showed a high level of sequence homology with DHARs from other plants (67 to approximately 81%). Functional sweet potato DHAR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 12% native PAGE. The protein's half-life of deactivation at 50 degrees C was 10.1 min, and its thermal inactivation rate constant K(d) was 6.4 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 6.0-11.0 and in the presence of 0.8 M imidazole. The K(m) values for DHA and GSH were 0.19 and 2.38 mM, respectively.