Combination of serum phosphorylated neurofilament heavy subunit and hyperintensity of intramedullary T2W on magnetic resonance imaging provides better prognostic value of canine thoracolumbar intervertebral disc herniation

The aim of this study was to evaluate the prognostic value of concurrent measurement of serum phosphorylated neurofilament heavy subunit (pNF-H) concentration and intramedullary T2W hyperintensity in paraplegic to paraplegic dogs. Our hypothesis was that concurrent measurement of these would provide a more accurate prediction of functional outcome in dogs with thoracolumbar intervertebral disc herniation (IVDH). A prospective case-control clinical study was designed using 94 dogs with acute onset of thoracolumbar IVDH. The association of serum pNF-H concentration, T2W hyperintensity on sagittal MRI (T2H/L2), deep pain perception and surgical outcome were evaluated with logistic regression analysis after three months for all 94 surgically treated dogs. Sensitivity to predict non-ambulatory outcome was compared among pNF-H and T2H/L2 and combination of both. Logistic regression analysis indicated that serum pNF-H concentration and T2H/L2 were significantly correlated with surgical outcome (P<0.05); however, deep pain perception was not (P=0.41). The results of logistic regression analysis indicated that the odds ratios of unsuccessful long-term outcome were 2.6 for serum pNF-H concentration, 1.9 for T2H/L2 and 2.3 for deep pain sensation. The sensitivity and specificity to predict non-ambulatory outcome for using serum parameter pNF-H>2.6 ng/ml, using T2H/L2 value of>0.84 and using both serum pNF-H and T2H/L2, were 95% and 75.7%, 65% and 86.5%, and 90.0% and 97.5%, respectively. Therefore, combined measurements of serum pNF-H and T2H/L2 might be useful for predicting long-term outcome in dogs with thoracolumbar IVDH.     

东方粘虫中肠V-ATPase H亚基原核表达及纯化

对东方粘虫Mythimna separate(Walker)中肠V-ATPase H亚基基因(VATPH)进行克隆、原核表达及纯化。首先采用RT-PCR技术克隆基因,再构建原核表达载体(pET15b-VATPH)并转入E.coil BL21(DE3),经异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导表达,用Ni-NTA柱及S-200分子筛纯化,最后进行SDS-PAGE分析。结果表明,pET15b-VATPH可高效表达V-ATPase H亚基,纯化后可获得单一条带且分子质量约为55ku的重组蛋白。该结果为进一步研究V-ATPase H亚基的晶体结构,药物小分子与H亚基大分子的相互作用,尤其是以H亚基为筛选模型创制新农药奠定基础。     

Cloning of vacuolar H<Superscript>+</Superscript>-ATPase subunit c genes from Japanese iris,and functional characterization in yeast

Five different isoforms (IrlVHA-c1-c5) of V-ATPase subunit c (VHA-c) were cloned from a Japanese iris (Iris lactea Pall. var. chinensis Fisch. Koidz) cDNA library using degenerate primers PCR and the 5'-RACE technique. The sequence analysis showed the open reading frame (ORF) of the IrlVHA-c1 c5 to be 495 bp, corresponding to a protein of 164 amino acids. Among the five isoforms, IrlVHA-c1 and IrlVHA-c2 are completely homologous. The IrlVHA-c protein is localized at the vacuolar membrane as indicated by a g...     

拟南芥VHA-c基因在非生物胁迫响应中的作用

利用RT-PCR技术检测VHA-c基因对高盐、水和ABA胁迫的响应,结果表明所有的VHA-c基因都参与了V-ATPase的抗盐胁迫反应;在响应水胁迫的反应中VHA-c2、VHA-c4和VHA-c5基因的表达量增加;在ABA胁迫反应中VHA-c1,VHA-c2,VHA-c3和VHA-c4基因的表达水平均提高,但是VHA-c1和VHA-c3基因只在早期的ABA胁迫中增加其表达量。     

Development of two multiplex PCR assays targeting improvement of bread-making and noodle qualities in common wheat

Wheat quality properties are genetically determined by the compositions of high and low molecular weight glutenin subunits, grain hardness, polyphenol oxidase (PPO) activity and starch viscosity. Two multiplex PCR assays were developed and validated using 70 cultivars and advanced lines from Chinese autumn‐sown wheat regions. Multiplex PCR I includes molecular markers for genes/loci ω‐secalin, Glu‐B1‐2a (By8), Glu‐D1‐1d (Dx5), Glu‐A3d, Glu‐B3 (for non‐1B·1R type) and Pinb‐D1b targeting improved gluten parameters and pan bread quality. Multiplex PCR II comprises markers for genes/loci Ppo‐A1, Ppo‐D1 and Wx‐B1b targeting improved noodle quality. The results were consistent with those achieved by SDS‐PAGE and RP‐HPLC, indicating that the two multiplex assays were highly effective, with good repeatability and low costs enabling their use in wheat breeding programmes. In total, nine alleles (subunits) at locus Glu‐B1, four at Glu‐D1 and five at Glu‐A3 locus were identified, and the alleles (subunits) Glu‐B1b (7 + 8), Glu‐B1c (7 + 9), Glu‐D1a (2 + 12), Glu‐D1d (5 + 10), Glu‐A3a, Glu‐A3c and Glu‐A3d were most frequently present in the cultivars and lines tested. The 1B·1R translocation was present in 28 (40.0%) lines, whereas the Wx‐B1 null allele for better noodle quality was present in only seven (10.0%) cultivars and advanced lines, and 37 (52.9%) lines had Pinb‐D1b associated with hard grains. The allele Ppo‐A1b on chromosome 2AL associated with lower PPO activity was present in 38 (54.3%) genotypes, whereas the less effective allele Ppo‐D1a on chromosome 2DL, also associated with low PPO activity was present in 45 (64.3%) of genotypes. These two multiplex PCR assays should be effective in marker assisted selection targeting improved pan bread‐making and noodle qualities.     

Use of the SDS-sedimentation test and SDS-polyacrylamidegel electrophoresis for screening breeder's samples of wheat for bread-making quality

Summary Gelprotein or SDS-insoluble gel-forming glutenin was isolated from wheat flour by extraction with an aqueous 1.5% SDS solution. Remarkable intervarietal differences were observed both in amount and subunit composition of these proteins.The amount of gelprotein and the SDS-sedimentation volume both proved to be good parameters for the bread-making quality of wheat cultivars. A high correlation was observed between amount of gelprotein and SDS-sedimentation volume. The amount of gelprotein was therefore tentatively assumed to be the essential basis of the SDS-sedimentation test.The subunit composition of the gelprotein was studied by SDS-PAGE after reduction of SS bonds by mercaptoethanol. It was found that the average bread-making quality of wheat cultivars and progeny of the cross Atlas 66 x Atys which possessed subunits 3 and 10, coded for by chromosome 1D, was significantly higher than that of wheat samples possessing subunit 2 and 11, their allelic counterparts.     

Limited but specific variations of seed storage proteins in Japanese common wheat (Triticum aestivum L.)

The electrophoretic banding patterns ofgliadin in common wheat lines derived fromJapan were determined byacid-polyacrylamide gel electrophoresis. For the 107 wheat lines used in our study,27 different patterns were identified, 13corresponding to the -gliadin, 8 tothe , -gliadin and 6 to the-gliadin. The gliadin patterns ofJapanese wheat cultivars and landracesgreatly differed from the patterns of wheatlines from other countries, and thevariation seen in wheat lines from Japanwas limited to 46 patterns. Sevencollection or breeding areas in Japanshowed different frequencies in theirgliadin patterns. Combining the gliadinpatterns with high molecular weightglutenin subunit compositions, 67combinations were observed. One gliadinpattern consisting of -gliadinpattern F, , -gliadinpattern H and -gliadin pattern Dwas frequently found in many Japanese wheatlines, though the other patterns werelimited to only one or two wheat lines.     

小麦远缘杂交后代节燕98-2分离类型贮藏蛋白的研究

采用A-PAGE和SDS-PAGE聚丙烯酰胺凝胶电泳方法,对远缘组合分离出来的节燕98-2类型入选11个遗传性基本稳定的具有高产、多抗的小麦株系的醇溶蛋白和高分子量谷蛋白亚基进行了分析.结果表明,在A-PAGE电泳分析中,11个供试株系具有11种不同的醇溶蛋白带型.在SDS-FAGE电泳分析中,出现了7种不同的高分子量谷蛋白亚基(HMW-GS)及6种亚基组合类型,优质亚基及亚基组合所占的比例较少,品质评分偏低,其变幅为5～8分,平均为6.36分.但在所分析的材料中,出现了一个少见的特殊亚基:2 10 12.并研究了这些HMW-GS和组合频率及特点.11个株系中7个具有45 10优质亚基和2个具有2*亚基,它们可供小麦优质育种利用.研究表明,通过远缘杂交能够选育出具有高产、抗病和优质的小麦新材料.     

Breeding of tetra null soybean (Glycine max) for lipoxygenase,kunitz trypsin inhibitor,lectin, and 7S α' subunit proteins

Soybean seed includes various bioactive substances. Also, they contain a variety of antinutritional factors including lipoxygenase, Kunitz trypsin inhibitor (KTI), lectin, and 7S α' subunit proteins. The genetic removal of these proteins will improve the nutritional value of soybean seed. The objective of this research was to breed new soybean with tetra recessive alleles (lox1lox2lox3/lox1lox2lox3‐ti/ti‐le/le‐cgy1/cgy1) for lipoxygenase, KTI, lectin, and 7S α' subunit proteins. Seven parents were used to breed tetra null strain. SDS‐PAGE and Western blot analysis were used to determine the presence or absence of lipoxygenase, 7S α' subunit, KTI, and lectin proteins in mature seed. Tetra null soybean line has a purple flower, determinate growth habit, tan pod, and yellow seed coat colour. Stem height of the breeding line was 62.3 cm. The 100‐seed weight of the breeding line was 27.1 g and yield (t/ha) was 2.84. This is the first soybean strain with lox1lox2lox3/lox1lox2lox3‐ti/ti‐le/le‐cgy1/cgy1 genotype (absence of lipoxygenase, KTI, lectin, and 7S α' subunit proteins).