The extraction and characterization of soil polysaccharide by whole soil methylation

A soil of the Countesswells series was repeatedly methylated by the Hakomori procedure and a chloroform-soluble product isolated after each methylation. Ninety-two per cent of the material engendered by seven methylations was released during the first four methylations. This had a methoxyl content of about 20% and contained 2% N. Later fractions had lower methoxyl and N contents. Residual carbohydrate in the soil had reducing sugar content on hydrolysis, equivalent to about 3% of the original value. The extracted material had the infrared spectrum of a methylated soil polysaccharide and, on hydrolysis by 2 m trifluoracetic acid, released methylated sugar derivatives of which 68 were characterised by GC-MS. Derivatives corresponding to (1→4) linked sugars predominated for both hexose and pentose sugars but there was also a large proportion of (1→3) linkages, (1→3) linkages predominated for the deoxyhexose sugars. There were more sugars with only one or two methylated hydroxyl groups than could be accounted for as branching points because of the relatively small numbers of end groups. Prior reduction of the soil with sodium borohydride had no measurable effect on the nature or yield of the methylated product. This indicates that amino acid sugar linkages susceptible to β-elimination reactions can have only a very small influence on the reaction. The isolated sugar derivatives accounted for 70% of the total soil sugars. The methylated material before hydrolysis had a low nominal molecular weight on diafiltration, with 68% < 10000. Some of the sugars unaccounted for were probably lost during the dialysis stage necessary to remove dimethyl sulphoxide.     

Influence of polysaccharide structure on dextran adsorption by montmorillonite

Two dextrans of similar molecular weight (?2 × 10⁶) but containing different structural linkages (B-215F: 95% α-1→ 6 and 5% α-1→ 3 and Polytran: 75% β-1→ 3 and 25% β-1→ 6) were adsorbed on Na-montmorillonite. Adsorption isotherms showed strong, H-2-type (high-affinity. Langmuir mono-layer adsorption) clay-dextran interactions for both polymers. Maximum adsorption of the Polytran dextran (60 $\text{mg}\text{100mg}$ clay) was 33 per cent greater than that of the B-512F dextran (44.5 $\text{mg}\text{100mg}$ clay) for comparative equilibrium systems. Stable clay-Polytran complexes containing up to 47% dextran were prepared. Excess Na₂SO₄ (0.1 m) did not affect the quantity of dextran adsorption by montmorillonite at the 15% dextran concentration. Acid hydrolysis and modified Pregl method-C analyses did not quantitatively recover adsorbed dextran from complexes containing more than 13 mg of dextran adsorbed per 100 mg of clay. Loss on ignition determinations were in good agreement with the difference measurements of dextran adsorption, suggesting that part of the C was expelled as something other than CO₂ in the ignition determinations. The maximum adsorption segment for Polytran was much smaller than the individual molecule. In contrast, the maximum adsorption segment for the B-512F dextran appeared to be the same magnitude as the individual polymer molecule. Adsorption segment length was regarded as a manifestation of the relative proportions of primary and secondary alcohol groups of the molecules.     

A-type proanthocyanidins from lychee seeds and their antioxidant and antiviral activities

Two new A-type trimeric proanthocyanidins with two doubly bonded interflavanoid linkages, litchitannin A1 [epicatechin-(2β→O→7,4β→6)-epicatechin-(2β→O→7,4β→8)-catechin] (1) and litchitannin A2 [epicatechin-(2β→O→7,4β→6)-epicatechin-(2β→O→7,4β→6)-epicatechin] (2), were isolated from lychee (Litchi chinensis Sonn. cv. Heiye) seeds together with aesculitannin A (3), epicatechin-(2β→O→7,4β→8)-epiafzelechin-(4α→8)-epicatechin (4), proanthocyanidin A1 (5), proanthocyanidin A2 (6), proanthocyanidin A6 (7), epicatechin-(7,8-bc)-4β-(4-hydroxyphenyl)-dihydro-2(3H)-pyranone (8), and epicatechin (9). Their structures were elucidated on the basis of spectroscopic and chemical evidence. It is the first time that compounds 1-4, 7, and 8 have been reported in this species. Compounds 1-9 showed more potent antioxidant activity than L-ascorbic acid with ferric reducing antioxidant power (FRAP) values of 3.71-24.18 mmol/g and IC50 values of 5.25-20.07 μM toward DPPH radicals. Moreover, litchitannin A2 (2) was found to exhibit in vitro antiviral activity against coxsackie virus B3 (CVB3) and compounds 3 and 6 displayed antiherpes simplex virus 1 (HSV-1) activity.     

Isolation, structure determination, synthesis, and sensory activity of N-phenylpropenoyl-L-amino acids from cocoa (Theobroma cacao)

Application of chromatographic separation and taste dilution analyses recently revealed besides procyanidins a series of N-phenylpropenoyl amino acids as the key contributors to the astringent taste of nonfermented cocoa beans as well as roasted cocoa nibs. Because these amides have as yet not been reported as key taste compounds, this paper presents the isolation, structure determination, and sensory activity of these amino acid amides. Besides the previously reported (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (clovamide), (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine (deoxyclovamide), and (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, seven additional amides, namely, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine, (+)-N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-N-[(E)-cinnamoyl]-L-aspartic acid, were identified for the first time in cocoa products by means of LC-MS/MS, 1D/2D-NMR, UV-vis, CD spectroscopy, and polarimetry, as well as independent enantiopure synthesis. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying oral sensation were determined to be between 26 and 220 micromol/L (water) depending on the amino acid moiety. In addition, exposure to light rapidly converted these [E]-configured N-phenylpropenoyl amino acids into the corresponding [Z]-isomers, thus indicating that analysis of these compounds in food and plant materials needs to be performed very carefully in the absence of light to prevent artifact formation.     

Structural characterization and hypoglycemic effects of arabinogalactan-protein from the tuberous cortex of the white-skinned sweet potato (Ipomoea batatas L.)

An arabinogalactan-protein (WSSP-AGP) was isolated from the tuberous cortex of the white-skinned sweet potato (WSSP; Ipomoea batatas L.). It consists of 95% (w/w) carbohydrate and 5% (w/w) protein with high contents of hydroxyproline, alanine, and serine. Its sugar composition is α-L-Rha:α-L-Ara:β-D-Gal:β-D-GlcA in a molar ratio of 1.0:4.1:7.6:1.3. Its weight-average molecular weight was estimated to be 126,800 g/mol by high-performance size exclusion chromatography coupled with multiangle laser light scattering. Structural analysis indicated that WSSP-AGP is a (1→3)-β-D-galactan highly branched at O-6 with (1→6)-β-D-galactan, in which the branched chains are substituted at the O-3 position with α-L-Araf-(1→ and α-L-Araf-(1→5)-α-L-Araf-(1→ and at the O-6 position typically with α-L-Rhap-(1→4)-β-D-GlcAp-(1→ as terminating groups. Continuous administration of WSSP-AGP to KKAy mice significantly lowered fasting plasma glucose levels. This indicates that WSSP-AGP plays an important role in the hypoglycemic effects of WSSP.     

Spiro cross-links: representatives of a new class of glycoxidation products

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates is largely unknown. Investigations are reported on the isolation of 6-[2-[[(4S)-4-amino-4-carboxybutyl]amino]-6,7-dihydroxy-6,7-dihydroimidazo[4,5-b]azepin-4(5H)-yl]-L-norleucine (10) and N-acetyl-6-[(6R,7R)-2-[[4-(acetylamino)-4-carboxybutyl]amino]-6,7,8a-trihydroxy-6,7,8,8a-tetrahydroimidazo[4,5-b]azepin-4(5H)-yl]-L-norleucine (12) formed by oxidation of the major Maillard cross-link glucosepane 1. Independent synthesis and unequivocal structural characterization are given for 10 and 12. Spiro cross-links, representing a new class of glycoxidation products, were obtained by dehydrogenation of the amino imidazolinimine compounds N6-[2-[[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-5-[(2S,3R)-2,3,4-trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysinate (DOGDIC 2) and N6-[2-[[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysinate (DOPDIC 3). These new oxidation products were synthesized, and their unambiguous structural elucidation proved the formation of the spiro imidazolimine structures N6-[(7R,8S)-2-[[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-8-hydroxy-7-(hydroxymethyl)-6-oxa-1,3-diazaspiro[4.4]non-1-en-4-ylidene]-L-lysinate (16), N6-(8R,9S)-2-[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-8,9-dihydroxy-6-oxa-1,3-diazaspiro[4.5]dec-1-en-4-ylidene)-L-lysinate (19), and N6-[(8S)-2-[(4-amino-4-carboxybutyl)amino]-8-hydroxy-6-oxa-1,3-diazaspiro[4.4]non-1-en-4-ylidene]-L-lysinate (18), respectively. It was shown that reaction of the imidazolinone 15 led to the formation of spiro imidazolones, structurally analogous to 16 and 19.     

Reactivity of lysine moieties toward an epoxyhydroxylinoleic acid derivative: aminolysis versus hydrolysis.

Epoxyols are generally accepted as crucial intermediates in lipid oxidation. The reactivity of tert-butyl (9R,10S,11E,13S)-9, 10-epoxy-13-hydroxy-11-octadecenoate (11a,b) toward lysine moieties is investigated, employing N(2)-acetyllysine 4-methylcoumar-7-ylamide (12) as a model for protein-bound lysine. The prefixes R and S denote the relative configuration at the respective stereogenic centers. Independent synthesis and unequivocal structural characterization are reported for 11a,b, its precursors, and tert-butyl (9R,10R,11E, 13S)-10-(?5-(acetylamino)-6-[(4-methyl-2-oxo-2H-chromen-7-yl)amino ]-6 -oxohexyl?amino)-9,13-dihydroxy-11-octadecenoate (13a-d). Reactions of 11a,b and 12 in 1-methyl-2-pyrrolidone (MP) and MP/water mixtures at pH 7.4 and 37 degrees C for 56 days show formation of the aminols 13a-d to be favored by an increased water content. The same trend is observed for hydrolytic cleavage of 11a,b to tert-butyl (E)-9,10, 13-trihydroxy-11-octadecenoate (14) and tert-butyl (E)-9,12, 13-trihydroxy-10-octadecenoate (15). Under the given conditions, aminolysis proceeds via an S(N)2 substitution, in contrast with the S(N)1 process for hydrolysis. In the MP/water (8:2) incubation, 15. 8% of 12 has been transformed to 13a-d and 10.5% of 11a,b hydrolyzed to the regioisomers 14 and 15 after 8 weeks, respectively. Aminolysis of alpha,beta-unsaturated epoxides by lysine moieties therefore is expected to be an important mode of interaction between proteins and lipid oxidation products.     

Structural identification of novel oligosaccharides produced by Lactobacillus bulgaricus and Lactobacillus plantarum

β-Galactosidases (β-Gal) of lactic acid bacteria produce oligosaccharides from lactose when suitable acceptor carbohydrates are present. This study aimed to elucidate the structure of oligosaccharides formed by galactosylation of N-acetylglucosamine (GlcNAc) and fucose. Crude cellular extract of Lactobacillus bulgaricus and LacLM of Lactobacillus plantarum were used as sources of β-Gal activity. Disaccharides obtained by galactosylation of GlcNAc were identified as Gal-β-(1→4)-GlcNAc or Gal-β-(1→6)-GlcNAc by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and comparison with external standards. Trisaccharides were identified as Gal-β-(1→6)-Gal-β-(1→[4 or 6])-GlcNAc by LC-MS, analysis of the MS/MS spectra of selected in-source fragment ions, and their relative retention times. LC-MS analysis revealed the presence of five galactosylated fucosides, but their linkage type could not be identified, partly due to the lack of reference compounds. β-Gal of lactic acid bacteria may serve as suitable tools for the chemoenzymatic synthesis of therapeutic oligosaccharides.     

Studies on the starch and hemicelluloses fractionated by graded ethanol precipitation from bamboo Phyllostachys bambusoides f. shouzhu Yi

Starch from bamboo Phyllostachys bambusoides f. shouzhu Yi evaluated by means of solid-state 13C CP/MAS NMR and X-ray diffraction showed a typical B-type pattern with a very low degree of crystallinity (10.9%). In addition to starch, alkali-soluble hemicelluloses were further fractionated by graded precipitation at ethanol concentrations of 0 (HA), 15, 30, 45, 60, and 75% (v/v). Chemical composition and structural features of the six hemicellulosic subfractions were investigated by a combination of sugar analysis, GPC, FT-IR, GC-MS, 1D (1H and 13C) and 2D (HSQC) NMR spectra, and thermal analysis. The results showed that the bamboo hemicelluloses were O-acetylated 4-O-methyl-glucuronoarabinoxylans (GAX) consisting of a linear (1→4)-β-D-xylopyranosyl backbone decorated with branches at O-3 of α-L-arabinofuranosyl (5-12 mol%) or at O-2 of 4-O-methylglucuronic acid units and acetyl groups (0.8-11 mol%). The molecular weights of these polysaccharides ranged between 13400 and 67500 g/mol, and the molar ratios of A/X and G/X increased with ascending ethanol concentrations. Moreover, xylo-oligosaccharides (XOS) with DP 1-6 were produced by enzymatic hydrolysis of hemicelluloses and the total yields of XOS were range of 21.5 to 40.6%. The structure-property relationships were also established in order to improve enzyme accessibility.     

Structure characterization and antioxidant activity of a novel polysaccharide isolated from pulp tissues of Litchi chinensis

A novel polysaccharide (LCP50S-2) with antioxidant activity was isolated from Litchi chinensis Sonn. The structure of LCP50S-2 was elucidated on the basis of physicochemical and instrumental analyses, and its average molecular weight was determined by gel permeation chromatography to be 2.19 × 10(2) kDa. The backbone of LCP50S-2 was composed of (1→3)-linked β-L-rhamnopyranosyl residues, (1→4)-linked α-D-xylopyranosyl residues, (1→4)-linked β-D-glucopyranosyl residues, and (1→4)-linked α-D-glucopyranosyl residues which branched at O-6. The two branches consisted of α-L-arabinopyranosyl residues and (1→6)-linked β-D-galactopyranosyl residues terminated with α-L-arabinopyranosyl residues, respectively. In the in vitro antioxidant assay, LCP50S-2 was found to possess DPPH radical-scavenging activity and hydroxyl radical-scavenging activity with IC(50) values of 220 and 266 μg/mL, respectively.     

Synthesis and characterization of a potential prebiotic trisaccharide from cheese whey permeate and sucrose by Leuconostoc mesenteroides dextransucrase

The production of new bioactive oligosaccharides is currently garnering much attention for their potential use as functional ingredients. This work addresses the enzymatic synthesis and NMR structural characterization of 2-α-D-glucopyranosyl-lactose derived from sucrose:lactose and sucrose:cheese whey permeate mixtures by using a Leuconostoc mesenteroides B-512F dextransucrase. The effect of synthesis conditions, including concentration of substrates, molar ratio of donor/acceptor, enzyme concentration, reaction time, and temperature, on the formation of transfer products is evaluated. Results indicated that cheese whey permeate is a suitable material for the synthesis of 2-α-D-glucopyranosyl-lactose, giving rise to yields around 50% (in weight respect to the initial amount of lactose) under the optimum reaction conditions. According to its structure, this trisaccharide is an excellent candidate for a new prebiotic ingredient, due to the reported high resistance of α-(1→2) linkages to the digestive enzymes in humans and animals, as well as to its potential selective stimulation of beneficial bacteria in the large intestine mainly attributed to the two linked glucose units located at the reducing end that reflects the disaccharide kojibiose (2-α-D-glucopyranosyl-D-glucose). These findings could contribute to broadening the use of important agricultural raw materials, such as sucrose or cheese whey permeates, as renewable substrates for enzymatic synthesis of oligosaccharides of nutritional interest.     

Large-Scale Purification and Characterization of Barley Limit Dextrinase,a Member of the α-Amylase Structural Family

Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol consists of precipitation from the extract at 20–70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on β-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mMβ-cyclodextrin and contains a single polypeptide chain of 105 kDa (SDS-PAGE) and pI 4.3. Sequence analysis of tryptic fragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPLC, identified short motifs recognized in β-strand 2, 3, and 5 characteristic of a catalytic (β/α)₈-barrel domain of the α-amylase family of amylolytic enzymes. Barley LD has ≈50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using ¹H-NMR spectroscopy, LD hydrolyzes specifically α-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 6²-O-α-maltotriosyl-maltotriose, with retention of the α-anomeric configuration. β-Cyclodextrin competitively inhibits the LD activity with K_i of 40 μM, while K_i is 1.9 mM and 2.4 mM for α-cyclodextrin and γ-cyclodextrin, respectively.     

Tetrahydro-beta-carbolines, potential neuroactive alkaloids, in chocolate and cocoa

Tetrahydro-beta-carbolines (THbetaCs), potential neuroactive alkaloids, were found in chocolate and cocoa. 6-Hydroxy-1-methyl-1,2, 3,4-tetrahydro-beta-carboline (6OHMTHbetaC), 1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (THCA), 1-methyl-1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) in both diastereoisomers (1S,3S and 1R,3S), and 1-methyl-1,2,3, 4-tetrahydro-beta-carboline (MTHbetaC), besides serotonin and tryptamine biogenic amines, were identified and quantified in dark chocolate, milk chocolate, cocoa, and chocolate-containing cereals by RP-HPLC-fluorescence and HPLC-MS. For each THbetaC, the concentration ranges were determined: 6OHMTHbetaC (0.16-3.92 microg/g), THCA (0.01-0.85 microg/g), 1S,3S-MTCA (0.35-2 microg/g), 1R,3S-MTCA (0.14-0.88 microg/g), and MTHbetaC (nd-0.21 microg/g). The highest content was generally found in chocolates and cocoas, but cereals containing chocolate also showed an appreciable amount of THbetaCs. The possible biological implications of this novel group of alkaloids in chocolate are discussed.     

Identification of antioxidative flavonols and anthocyanins in Sicana odorifera fruit peel

Ten flavonols and three anthocyanins were identified in the fruit peel of melo?n de olor (Sicana odorifera), and their structures were established by spectrometric and spectroscopic (ESI-MS and NMR) techniques. One of the identified flavonols, quercetin 3-O-(6'-O-malonyl)-β-D-glucopyranoside 4'-O-β-D-glucopyranoside, has not been reported before in the plant kingdom. Although quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-d-glucopyranoside-4'-O-β-D-glucopyranoside had been reported before in literature and structure elucidation was done by comparison of NMR data with published data, to the best of our knowledge complete 1D and 2D NMR data have not been not delineated so far. Moreover, the antioxidant activity of pure compounds was measured by ABTS assay. It was established that quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside, quercetin-3-O-(6'-malonyl)-glucopyranoside, quercetin-3-O-β-D-glucopyranoside, and quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside-4'-O-β-D-glucopyranoside contribute significantly to the antioxidant activity exhibited by the fruit peel methanolic extract.     

Biotransformation of the neonicotinoid insecticide thiacloprid by the bacterium Variovorax boronicumulans strain J1 and mediation of the major metabolic pathway by nitrile hydratase

A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a β-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.     

口蹄疫病毒持续感染黄牛分离毒株VP1和3ABC基因变异特征分析

摘要：为明确口蹄疫病毒持续感染黄牛期间病毒基因变异情况,本试验主要研究口蹄疫病毒持续感染分离株结构蛋白VP1和非结构蛋白3ABC基因在牛体内的动态变化规律。用O/Akesu/58毒株以104ID50/ml剂量舌面穿刺接种5头中国黄牛,出现临床或亚临床症状之后痊愈动物会成为可能的口蹄疫病毒带毒牛。用探杯定期采集实验牛咽喉部黏性液体（O/P液）,接种BHK-21细胞增殖病毒后共分离到12株毒株。RT-PCR方法扩增持续感染分离毒株VP1和3ABC基因,克隆测序后分析发现所有持续感染分离毒株的VP1基因有16个核苷酸位点发生一致的突变,但只有两个位点造成氨基酸突变(I56→T、A210→E),而持续感染分离毒株之间有4个核苷酸位点和3个氨基酸位点发生了颠换。非结构蛋白3ABC基因较为稳定,仅有13个核苷酸位点和5个氨基酸位点的颠换,没有缺失或突变。推测口蹄疫病毒持续感染的形成与主要的抗原基因VP1变异没有显著关系,宿主嗜性相关的3ABC基因也没有明显变化。     

猪TNNC2基因的克隆及其融合蛋白在大肠杆菌中的表达

根据GenBank上猪TNNC2（Fast skeletal muscle troponin C2）基因序列（GenBank accession No. DQ629177）设计一对引物,采用RT-PCR方法克隆得到617 bp TNNC2 cDNA片段（GenBank accession No. EF673726）,包括完整的开放阅读框（ORF）,与GenBank上公布的猪TNNC2基因（GenBank accession No. AY575058）的ORF核苷酸序列同源性达99 %,并发现开放阅读框内的5个点突变,319位点T→C,320 位点G→A,321位点C→T,导致氨基酸107位Ala（丙氨酸）→Met（蛋氨酸）,322位点A→G为同义突变,433位点A→T,导致氨基酸144位Glu（谷氨酸）→Asp（天冬氨酸）。根据已获得的TNNC2基因开放阅读框序列,重新设计引物扩增得到包含BamH I和EcoR I酶切位点的完整阅读框,将其首先克隆到pMD18-T载体中,经菌液PCR筛选和酶切鉴定后,用BamH I和EcoR I将目的片段切下,再克隆到原核表达载体pRSET A中构建重组表达质粒pRSET A-TNNC2。将重组质粒转化大肠杆菌BL21（DE3）,IPTG不同诱导条件诱导表达,经SDS-PAGE电泳和Western blot分析证实重组表达质粒pRSET A-TNNC2表达出24 kD左右的融合蛋白,最佳诱导时间为4 h,最佳的IPTG诱导浓度为0.6 mmol/L,表达产物以可溶性蛋白的形式存在。     

Effect of Chronic Nitrogen Additions on Soil Nitrogen Fractions in Red Spruce Stands

The responses of temperate and boreal forest ecosystems to increased nitrogen (N) inputs have been varied, and the responses of soil N pools have been difficult to measure. In this study, fractions and pool sizes of N were determined in the forest floor of red spruce stands at four sites in the northeastern U.S. to evaluate the effect of increased N inputs on forest floor N. Two of the stands received 100 kg N ha^-1 yr^-1 for three years, one stand received 34 kg N ha^-1 yr^-1 for six years, and the remaining stand received only ambient N inputs. No differences in total N content or N fractions were measured in samples of the Oie and Oa horizons between treated and control plots in the three sites that received N amendments. The predominant N fraction in these samples was amino acid N (31-45% of total N), followed by hydrolyzable unidentified N (16-31% of total N), acid-soluble N (18-22% of total N), and NH₄ ⁺ (9^-13% of total N). Rates of atmospheric deposition varied greatly among the four stands. Ammonium N and amino acid N concentrations in the Oie horizon were positively related to wet N deposition, with respective r2 values of 0.92 and 0.94 (n = 4, p < 0.05). These relationships were somewhat stronger than that observed between atmospheric wet N deposition and total N content of the forest floor, suggesting that these pools retain atmospherically deposited N. The NH₄ ⁺ pool may represent atmospherically deposited N that is incorporated into organic matter, whereas the amino acid N pool could result from microbial immobilization of atmospheric N inputs. The response of forest floor N pools to applications of N may be masked, possibly by the large soil N pool, which has been increased by the long-term input of N from atmospheric deposition, thereby overwhelming the short-term treatments.     

Sodium azide induced high-oleic peanut (Arachis hypogaea L.) mutant of Virginia type

Less prone to oxidation than its conventional counterpart, high-oleic peanut (Arachis hypogaea L.) containing?≥?72% oleate and?<?8% linoleate is preferred by processors, seed sellers and consumers. Development of high-oleic peanut cultivars with suitable pod/seed size and shape may satisfy the need from food processors manufacturing whole kernel or halve products. In this study, a high-oleic Virginia type peanut mutant was identified through screening of a 15 mmol/L sodium azide mutagenized M3 population with near infrared spectroscopy. Sequencing of the mutated and wild type FAD2A/FAD2B genes detected 2 point mutations. The G448A mutation in FAD2A was the same as in previous reports, causing an amino acid change of D150N. G558A in FAD2B was a novel mutation, resulting in a stop codon and premature termination of protein synthesis. 16 promising lines with acceptable productivity and pod/seed characters have been tentatively bred, which will be evaluated further in yield tests with replications after seed increase.

     

Identification of an allele attributable to formation of cucumber-like flavor in wild tomato species (Solanum pennellii) that was inactivated during domestication

Carbon 6 (C6)-aldehydes formed by fatty acid 13-hydroperoxide lyase (13HPL) specific to fatty acid 13-hydroperoxides (13-HPO) are important flavor constituents in fresh tomato fruits. C9-aldehydes are usually formed by 9/13HPL showing dual specificity to 9- and 13-HPOs and are scarcely found in tomato fruits. Mature red fruits of one of the introgression lines, IL1-4, generated by hybridization of a cultivated tomato (Solanum lycopersicon) to its wild relative Solanum pennellii, form high amounts of C9-aldehydes upon homogenization. The IL1-4 fruits showed high 9/13HPL activity. One of the genes isolated from IL1-4 showed a high similarity to plant 9/13HPLs. Recombinant proteins expressed in Escherichia coli showed 9/13HPL activity. Cleaved amplified polymorphic sequence analyses indicated that the gene was specific to IL1-4 and S. pennellii. S. lycopersicon had a gene having high similarity to the S. pennellii gene. It was absent in IL1-4. Among the differences of amino acid residues found between the two genes, a Cys to Ser exchange may be responsible for the inactivation of resultant protein product of S. lycopersicon gene because the Cys is an essential amino acid residue for HPL activity. From these observations, it could be assumed that a tomato gene corresponding to S. pennellii 9/13HPL gene had been inactivated through domestication of tomatoes.