A Kelch Motif-Containing Serine/Threonine Protein Phosphatase Determines the Large Grain QTL Trait in Rice

A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice (Oryza sativa L.) varieties. In this study, we generated an F₂ population by crossing the large‐grain japonica cultivar CW23 with Peiai 64 (PA64), an elite indica small‐grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly‐identified in this study. In particular, qGL3‐1, a newly‐identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine‐mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution (C→A) at position 1092 in exon 10, resulting in replacement of Asp (D) in PA64 with Glu (E) in CW23 for the 364^th amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near‐isogenic line (NIL) for qGL3‐1 revealed that the allele qGL3‐1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker‐assisted selection.     

Synthetic esters recognized by glucuronoyl esterase from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-d-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-d-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-d-glucuronic and d-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-d-glucuronic acid than with that of d-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-d-glucopyranuronosyl residues α-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-d-glucuronic acid.     

Effects of straw mulching on soil temperature, evaporation and yield of winter wheat: field experiments on the North China Plain

Straw mulching is an effective measure to conserve soil moisture. However, the existence of straw on the soil surface also affects soil temperature, which in turn influences crop growth, especially of winter crops. Five‐year field experiments (2000–2005) investigated the effects of straw mulching and straw mass on soil temperature, soil evaporation, crop growth and development, yield and water use efficiency (WUE) of winter wheat (Triticum aestivum L.) at Luancheng Station on the North China Plain. Soil is a moderately well‐drained loamy soil with a deep profile at the station. Two quantities of mulch were used: 3000 kg ha^?1 [less mulching (LM)] and 6000 kg ha^?1 [more mulching (MM)], representing half and all of the straw from the previous crop (maize). In the control (CK), the full quantity of mulch was ploughed into the top 20 cm of soil. The results showed that the existence of straw on the soil surface reduced the maximum, but increased the minimum diurnal soil temperature. When soil temperature was decreasing (from November to early February the next year), soil temperature (0–10 cm) under straw mulching was on average 0.3°C higher for LM and 0.58°C higher for MM than that without mulching (CK). During the period when soil temperature increased (from February to early April, the recovery and jointing stages of winter wheat), average daily soil temperature of 0–10 cm was 0.42°C lower for LM and 0.65°C lower for MM than that of CK. With the increase in leaf area index, the effect of mulching on soil temperature gradually disappeared. The lower soil temperature under mulch in spring delayed the development of winter wheat up to 7 days, which on average reduced the final grain yield by 5% for LM and 7% for MM compared with CK over the five seasons. Mulch reduced soil evaporation by 21% under LM and 40% under MM compared with CK, based on daily measuring of microlysimeters. However, because yield was reduced, the overall WUE was not improved by mulch.     

Development of a small-scale bioreactor: application to in vivo NMR measurement

A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta). Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH.     

脂肪酶和酯酶的定向进化及其应用

简单介绍了脂肪酶和酯酶的定向进化的方法和策略。重点介绍了脂肪酶和酯酶的高通量筛选方法及定向进化研究所取得的主要进展。     

SDS-PAGE、同工酶等电聚焦和RAPD标记鉴定两系杂交水稻F1种子纯度对比研究

目的：探讨利用种子贮藏蛋白SDS—PAGE电泳、酯酶同工酶超薄等电聚焦电泳和RAPD分子标记鉴定五个两系杂交水稻组合F_1种子纯度的可行性。方法和结果：利用SDS—PAGE电泳技术未能找到所试五个组合各自的父本特征蛋白质带;利用酯酶同工酶超薄等电聚焦电泳技术和RAPD分子标记可找到所试五个组合的父母本特征酶带和RAPD标记,但酯酶同工酶的多态性同时也受种子萌发时间的影响。酯酶同工酶超薄等电聚焦电泳和RAPD分子标记可用于所试五个两系杂交水稻组合F_1种子纯度的鉴定。     

A versatile library of activity-based probes for fluorescence detection and/or affinity isolation of lipolytic enzymes

This work describes the synthesis of a library of fluorescent and/or biotinylated alkylphosphonate inhibitors being reactive towards serine hydrolases, especially lipases and esterases. Fluorescent inhibitors can be used for sensitive and rapid detection of active proteins by gel electrophoresis. Biotinylated inhibitors are applicable for the enrichment and isolation of active enzymes. Functionality as well as the different detection methods of the synthesized inhibitors were successfully tested with an enzyme preparation, namely cholesterol esterase from porcine pancreas (ppCE). Moreover, a biotinylated inhibitor was employed to enrich ppCE on avidin beads. Hence, our set of phosphonate inhibitors can be used for the detection and/or isolation of active serine hydrolases.     

Isoenzymes of lipolytic acyl hydrolase and esterase in potato tuber

Five varieties of potato (Solanum tuberosum) were shown by gel- and free-flow-electrophoresis to exhibit multiple forms of lipolytic acyl hydrolase (LAH) and esterase enzymes. The electrophoretic patterns of LAH and esterase activities and protein differed with the variety and were characteristic for a given variety. In the variety (Golden Wonder) with the highest LAH activity (p-nitrophenylpalmitate as substrate), this was 200-fold greater than the esterase activity (p-nitrophenylacetate as substrate) and isoenzyme patterns for both enzymes were the most complex. In the variety with a very low LAH activity (Désirée), the LAH and esterase activities were similar and more simple isoenzyme patterns for these enzymes were observed.     

Effects of faba bean,blue lupin and rapeseed meal supplementation on nitrogen digestion and utilization of dairy cows fed grass silage-based diets

There is increasing interest in using locally produced protein supplements in dairy cow feeding. The objective of this experiment was to compare rapeseed meal (RSM), faba beans (FBs) and blue lupin seeds (BL) at isonitrogenous amounts as supplements of grass silage and cereal based diets. A control diet (CON) without protein supplement was included in the experiment. Four lactating Nordic Red cows were used in a 4 × 4 Latin Square design with four 21 d periods. The milk production increased with protein supplementation but when expressed as energy corrected milk, the response disappeared due to substantially higher milk fat concentration with CON compared to protein supplemented diets. Milk protein output increased by 8.5, 4.4 and 2.7% when RSM, FB and BL were compared to CON. The main changes in rumen fermentation were the higher propionate and lower butyrate proportion of total rumen volatile fatty acids when the protein supplemented diets were compared to CON. Protein supplementation also clearly increased the ruminal ammonia N concentration. Protein supplementation improved diet organic matter and NDF digestibility but efficiency of microbial protein synthesis per kg organic matter truly digested was not affected. Flow of microbial N was greater when FB compared to BL was fed. All protein supplements decreased the efficiency of nitrogen use in milk production. The marginal efficiency (amount of additional feed protein captured in milk protein) was 0.110, 0.062 and 0.045 for RSM, FB and BL, respectively. The current study supports the evidence that RSM is a good protein supplement for dairy cows, and this effect was at least partly mediated by the lower rumen degradability of RSM protein compared to FB and BL. The relatively small production responses to protein supplementation with simultaneous decrease in nitrogen use efficiency in milk production suggest that economic and environmental consequences of protein feeding need to be carefully considered.