Neutral Proteinases I and II of Aspergillus sojae

Epimerization of (?)-isodihydrocarvone (I) to (?)-dihydrocarvone (II) by Pseudomonas fragi IFO 3458 was studied. I was easily epimerized to II by the growing cells, the resting cells or the cell-free extracts.

An epimerase catalyzing the conversion of I to II was partially purified from the bacterial extracts about 56-fold with heat-treatment, ammonium sulfate precipitation and DEAE-Sephadex A-50 column chromatography. By the action of this epimerase, the ratio of I to II becomes about 25: 75 (K=3). It appeared that the epimerase is very stable to heat; the activity of epimerization remains 66 and 36% after treatment at 97°C for 60 and 120 min, respectively.     

Neutral Proteinases I and II of Aspergillus sojae: Some Physicochemical Properties and Amino Acid Composition

Some physicochemical properties of neutral proteinases I and II, zinc-containing metalloenzymes, from Aspergillus sojae were investigated.

Neutral proteinase I: The enzyme protein had a sedimentation coefficient of 3.90S, an intrinsic viscosity of 0.0315 dl/g, and a partial specific volume, calculated from the amino acid and carbonhydrate composition, of 0.715 cm³/g. The molecular weight was 42,200 from the Yphantis’ procedure, and was 42,500 from the calculation according to the Scheraga-Mandel-kern’s formula. The integral numbers of amino acid residues per molecule calculated on the basis of 42,200 as molecular weight were as follows; Lys₁₆, His₆, Arg₁₃, Trp₈, Asp₅₆, Thr₂₅, Ser₂₃, Glu₃₁, Pro₁₈, Gly₄₀, Ala₃₃, l/2Cys₄, Val₁₁, Met₆, Ile₁₅, Leu₂₅, Tyr₂₀, Р?е₁₀, (amide-ammonia)₂₉, in addition to mannose₆, galactose₁, hexosamine₃.

Neutral proteinase II: The enzyme protein had a sedimentation coefficient of 2.32S, an intrinsic viscosity of 0.0270 dl/g, and a calculated partial specific volume of 0.714 cm³/g. The molecular weight was 16,800 from the Yphantis’ procedure, and was 18,000 from the sedimentation and intrinsic viscosity. The following amino acid compositions was calculated on the basis of 16,800 as molecular weight; Lys₈, His₃, Arg₃, Asp₁₉, Thr₁₇, Ser₁₁, GIu₂₃, Pro₅, Gly₉, Ala₂₄, l/2Cys₄, Val₅, Ile₃, Leu₁₃, Tyr₁₀, Phe₃, (amide-ammonia)₁₅. In the enzyme preparation, neither methionine nor tryptophan was detected and carbohydrate was also absent.

In both neutral proteinases I and II, no free SH group was detected by the PCMB-titration in the presence of 8 M urea.     

Purification and Properties of Neutral Proteinase I from Aspergillus oryzaePurification and Properties of Neutral Proteinase II from Aspergillus oryzae

Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41,000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenyl-alanine amide, poly-l-lysine and poly-l,α-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.     

The Effect of Potassium Nitrate on the Reduction of Phytophthora Stem Rot Disease of Soybeans, the Growth Rate and Zoospore Release of Phytophthora sojae

The effects of potassium nitrate (KNO₃) application on Phytophthora stem rot disease reduction of Glycine max (L.) Merr. cvs. Chusei-Hikarikuro and Sachiyutaka, and mycelium growth and zoospore release of a Phytophthora sojae isolate were investigated under laboratory conditions. The application of 4–30 m m KNO₃ prior to inoculation greatly reduced incidence of disease in the two soybean cultivars. Although a concentration of 20–30 m m KNO₃ led to a slight decrease in the growth rate of the PJ-H30 isolate on PDA medium, no significant relationship was observed between inhibition of the growth rate and disease reduction on application of 0.4–10 m m KNO₃. Disease suppression recorded in laboratory experiments using pathogen mycelium was due to the response of plant tissues rather than a direct inhibition of pathogen hyphal growth by the application of KNO₃. The extent of disease reduction was related to increased potassium concentration in plants of the two cultivars (except for some cases involving cv. Sachiyutaka), suggesting that differences existed between the two cultivars in terms of the effect of KNO₃ application on disease suppression. Scanning electron microscopic observation with fresh samples indicated marked accumulation of potassium at the penetration-stopping sites of P. sojae in the cortex layer of soybean plants treated with 30 m m KNO₃, compared with the non-treated control plants. The presence of 0.4–30 m m KNO₃ decreased the release of zoospores. These results suggest the possibility of applying a solution containing 20–30 m m of KNO₃ to decrease the incidence of disease in agricultural fields by the response of plant tissues to KNO₃.     

Phosphorus and Nitrogen Interactions in Field-Grown Soybean as Related to Genetic Attributes of Root Morphological and Nodular Traits

Two field experiments with different soybean (Glycine max L.) materials were conducted to investigate the interactions between phosphorus (P) and nitrogen (N) as related to the genetic attributes of root morphological and nodular traits. In experiment one, 13 cultivated soybean varieties were grown in a field with relatively low soil P and N availability. P application with 160 kg P/hm2 as triple superphosphate produced a significant simultaneous increase in the content of both P and N in shoot, demonstrating positive P and N interactions. The addition of P also increased root dry weight, root nodule number, nodule mass, nodule size, and nodulation index, but decreased root length and root surface area, indicating that P may affect N nutrition in soybean through a number of root morphological and nodular traits. Interestingly,like P content, N content appeared to be more correlated with root morphological traits (root weight, root length, and root surface area) than with root nodular traits (nodule number, nodule size, nodule mass, and nodulation index) at both P levels, implying that N taken up by the roots may contribute more to the plant N status than biological N2 fixation under the present experimental conditions. In experiment two, 57 soybean lines of a recombinant inbred line (RIL) population derived from a cross between a cultivated variety and a wild genotype were grown on another field site with moderately sufficient P and N levels to further characterize the genetic attributes of root morphological and nodular traits and their relationships with P and N interactions. The results indicated that all morphological and nodular traits measured continually segregated in the RIL population with a normal distribution of the phenotypic values, indicating that these traits are possibly controlled by quantitative trait loci (QTLs). Genetic analysis revealed that all these root traits had relatively low heritabilities (h2b=74.12, 70.65, 73.76, 56.34, 52.59, and 52.24 for root weight, root length,root surface area, nodule number, nodule mass, and nodule size, respectively), suggesting that root morphology and nodule formation are influenced greatly by environmental factors. Correlation analysis of the RILs showed that shoot N content was significantly correlated with P content, confirming positive P×N interactions. Similar to experiment one, shoot N content was only significantly correlated with root morpho logical traits, but not with root nodular traits, again denoting the fact that the N status in soybean could be attributed more to N uptake from the soil than to biological N2 fixation under the present experimental conditions.     

大豆体细胞胚胎发生与农杆菌介导的遗传转化

以55个大豆基因型未成熟子叶为外植体,用高浓度2,4-D诱导大豆体细胞胚胎发生与植株再生,并对生产上种植面积大、体细胞胚胎发生率高的大豆基因型用农杆菌介导法进行遗传转化。结果表明,东北地区主栽的大豆基因型中有14个基因型体细胞胚胎发生率超过40%。用含有pGBI121S4ABC质粒的LBA4404农杆菌侵染5个东北地区主栽大豆基因型的2147个未成熟子叶,经卡那霉素抗性筛选得到12株PCR阳性植株。 Abstract：Somatic embryogenesis was induced and the regenerated plants were obtained by higher concentrations of auxins with immature cotyledon of 55 genotypes in soybean. Bivalent insect resistant genes were transformed into immature cotyledon of soybean which have high frequency of somatic embryogenesis via Agrobacterium-mediated. The results showed that 14 genotypes possessed high frequency of somatic embryogenesis (more than 40%) among soybean genotypes from Northeast area. 2147 immature cotyledons of 5 different soybean genotypes cultured in Northeast area were inoculated with LBA4404 (including pGBI121S4ABC plasmid). 12 regenerated plants selected by Kanamicy gave positive PCR reaction.     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

大豆异黄酮合酶基因的克隆及序列分析

目的:大豆异黄酮是多酚类混合物,有防治肿瘤发生,提高机体免疫力等多种保健功能。异黄酮合酶(isoflavone synthase,IFS)是合成异黄酮的关键酶。本文为了利用异黄酮的特有生物学功能,从大豆中克隆了该基因。方法:采用PCR扩增从大豆[Glycine max(Linn.)Merr.]总RNA中分离了异黄酮合酶基因,并将其克隆到pUCm-T载体并测序。结果:得到全长1583bp的片段。以期用于构建诱导表达基因敲除系统,并用于无性繁殖植物的无标记基因转化。结论:序列分析表明,异黄酮合酶基因(IFS1)含1583个核苷酸,与已报道的序列比较,核苷酸的同源性为92%。     

增强UV-B辐射对大豆胚轴DNA损伤、修复和蛋白质含量的影响

大气平流层臭氧层减薄引起到达地表的 UV- B辐射增强。为探讨在增强 UV- B辐射下植物细胞 DNA的损伤修复和蛋白质含量的关系 ,利用 3H- Td R掺入法 ,研究了在 8.2 2 k J/(m2 d)和 12 .4 2 k J/(m2 d) U V- B辐射 (相当于兰州地区大气平流层臭氧减薄约 12 %和 2 0 % )胁迫下 ,大豆胚轴细胞 DNA合成和非按期合成 (UDS)变化 ,并测定了胚轴蛋白质含量变化 ,结果显示 ,UV- B辐射导致 DNA损伤 ,并诱导了 DNA损伤的修复 ,胚轴细胞 UDS效应增强 ,U DS指数增大。低 UV- B辐射强度下 ,胚轴蛋白质含量增加 ,可能是 U V- B诱导了一些与抗性有关的基因表达 ,导致一些新的与抗性有关的蛋白质合成 ;在高强度 UV- B辐射下 ,U DS指数与低强度辐射下无显著差异 (P=0 .0 5 ) ,但蛋白含量较低强度辐射下显著下降 (P=0 .0 5 ) ,说明高强度 UV- B辐射加重了 DNA损伤 ,而修复并未加强 ,并且高强度辐射抑制基因的正常表达和蛋白质合成。这些蛋白质的合成可能与大豆对 UV- B辐射的抗性有关。     

Effects of seismic stress on the vegetative growth of Glycine max (L.) Merr. cv. Wells II

Abstract. Vegetative plants of soybean [ Glycine max (L.) Merr. cv. Wells II] grown in a greenhouse and agitated periodically on a gyratory shaker had shorter stems, less leaf area, and lower leaf and plant dry weight than did undisturbed greenhouse-grown (GG) plants after 16 d of treatment. Outdoor-grown (OG) plants, which were subjected to additional environmental stresses including ultraviolet radiation, wind loading, and uncontrolled temperature and humidity fluctuations, were smaller and had less dry weight than GG controls, but growth was not inhibited further by gyratory shaking. Periodic shaking of GG soybeans resulted in the same plant and leaf dry weight as for OG soybeans. Response of GG plants to mechanical stress depended on light intensity, with minimum growth reduction occurring under full light (FL) level, and maximum growth reduction occurring under lower light levels (24–45% FL). Reduction in dry weight gain due to mechanical stress corresponded to a decrease in relative growth rate (RGR). Decreases in net assimilation rate and leaf area ratio contributed equally to the lower RGR of shaken plants, indicating that seismic stress inhibits dry weight accumulation by decreasing both the photosynthetic efficiency and the assimilatory surface of soybean.     

Responses of soybean to oxygen deficiency and elevated root-zone carbon dioxide concentration

Root flooding is damaging to the growth of crop plants such as soybean (Glycine max L.). Field flooding for 3 d often results in leaf chlorosis, defoliation, cessation of growth and plant death. These effects have been widely attributed solely to a lack of oxygen in the root-zone. However, an additional damaging factor may be CO(2), which attains levels of 30 % (v/v) of total dissolved gases. Accordingly, the effects of root-zone CO(2) on oxygen-deficient soybean plants were investigated in hydroponic culture. Soybean plants are shown to be very tolerant of excess water and anaerobiosis. No oxygen (100 % N(2) gas) and low oxygen (non-aerated) treatments for 14 d had no effect on soybean survival or leaf greenness, but plants became severely chlorotic and stunted when the roots were exposed to no oxygen together with CO(2) concentrations similar to those in flooded fields (equilibrium concentrations of 30 %). When root-zone CO(2) was increased to 50 %, a quarter of soybean plants died. Those plants that survived showed severe symptoms of chlorosis, necrosis and root death. In contrast, rice (Oryza sativa L.) plants were not affected by the combination of no oxygen and elevated root-zone CO(2.) A concentration of 50 % CO(2) did not affect rice plant survival or leaf colour. These results suggest that the high susceptibility of soybean to soil flooding, compared with that of rice, is an outcome of its greater sensitivity to CO(2).     

Expression of two cloned mRNA sequences during development and germination of spores of the sensitive fern,Onoclea sensibilis L.

A library of complementary DNA (cDNA) clones has been prepared from poly(A)⁺RNA of spores of the sensitive fern, Onoclea sensibilis L. By differential hybridization with labeled probes made to poly(A)⁺ RNA of spores, gametophytes and leaves, two spore-specific clones (pOSS68 and pOSS194) were selected and characterized. Northern blot analysis showed that RNA sequences homologous to the two cDNA clones first appear in the post-meiotic spore and increase in abundance during spore maturity. Both RNA sequences decay during photoinduced germination of the spores and do not reappear in the gametophytes. In spores imbibed in the dark under conditions which do not favor germination, no significant decrease in pOSS194-mRNA abundance is noted. In contrast, the decrease in pOSS68 mRNA in dark-imbibed spores parallels that observed in photoinduced spores. The predicted amino-acid sequence of pOSS194 has a striking similarity to the early light-inducible proteins expressed during the greening of etiolated pea and barley seedlings, whereas that of pOSS68 shows some homology to proteins encoded by late-embryogenesis-abundant mRNAs of angiosperm embryos.Abbreviations bp base pairs - cDNA complementary DNA - ds double-stranded - ELIP early light-inducible proteins - LEA late embryogenesis abundant - nt nucleotide - ss single stranded This work was partially supported by a NASA grant (NAGW-901) and by an allocation from the Research Challenge Investigators' Fund of the Ohio State University to V.R. Thanks are due to Mr. Clayton L. Rugh for sequencing our clones and to Dr. Paul A. Fuerst for help in the computer search of sequence alignments.     

大豆籽粒发育过程中异黄酮合成相关酶基因的表达模式与异黄酮积累的相关分析

利用高效液相色谱法和实时定量PCR方法,分别测定了2个异黄酮含量显著差异的大豆品种鲁黑豆2号(LHD2)和南汇早黑豆(NHZ)在子粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化,试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆子粒发育过程中,异黄酮含量逐渐升高,而不同异黄酮合成相关酶基因的表达趋势不同,CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致,而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在2个大豆品种中存在相反的趋势,在LHD2中与异黄酮组分积累趋势相反,而在NHZ中与异黄酮组分积累趋势相同。结果还表明,同一基因家族中不同基因在子粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高,异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量,预示这些基因家族在大豆子粒异黄酮积累过程中存在功能分化。此外,各基因表达模式与异黄酮积累的相关分析结果表明,不同基因表达模式与异黄酮积累的相关性在2个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在子粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关,CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中,IFR在子粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明,在2个品种中,苯丙氨酸水解酶PAL1与4CL,4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为今后利用基因工程提高大豆异黄酮含量奠定了基础。     

Purification and Some Physical Properties of Acid-cellulase from Aspergillus niger

A cellulase preparation which exhibits the highest activity at a lower pH range, 2.3 to 2.5, was purified from a commercial cellulase preparation from a culture filtrate of Asp. niger and referred to as acid-cellulase.

The purification involves ammonium sulfate fractionation, gel filtration and ion-exchange and adsorption chromatographies. The purified enzyme was revealed to be homogenous in ultracentrifugation and disc as well as ampholine electrophoreses and to be an acidic protein of which isoelectric point lied at pH 3.3. The sedimentation coefficient and molecular weight were determined to be 3.27 S and 46,000, respectively. The optical properties were also studied.     

甘蔗ACC氧化酶全长cDNA的克隆及序列分析

ACC氧化酶是高等植物乙烯生物合成途径中的限速酶。根据报道的植物ACC氧化酶基因序列设计特异引物,从甘蔗cDNA文库中克隆到一ACC氧化酶基因片段,命名为GZ-ACO,该基因长792bp,与甘蔗基因组文库中克隆的ACO基因片段仅18个碱基之差。根据该cDNA片段序列,设计两对末端扩增的特异引物,利用RACE-PCR技术,获得GZ-ACO片段的5′端和3′端序列。用VectorNTI7.0软件对三个序列进行拼接和分析,结果得到全长的甘蔗GZ-ACO氧化酶基因。GZ-ACOcDNA核苷酸序列长1307bp,具有一个972bp完整的读码框,启动子ATG位于126bp,终止子TAA位于1097bp,推导编码323个氨基酸。系统进化分析表明,GZ-ACO基因氨基酸序列与其它植物已报道的ACC氧化酶基因具有65%~86%的同源率,且与单子叶禾本科植物首先聚类,其次与单子叶芭蕉科、兰科植物聚类,最后与双子叶植物聚类,与植物形态的系统进化结果一致。该基因已在DDBJ/EMBL/GenBank基因数据库注册,注册号为AY521566。     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶(P450aromB)在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型P450arom的同源性较高(48.3%-66.1%),与性腺型P450arom的同源性较低(34.2%-49.9%),与自身的性腺型芳香化酶同源性为45.1%。RT-PCR分析表明:P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

Study on the Superoxide Dismutase of Soybean Seeds from Different Species in Subgenus

The SOD isoenzymes of different soybeans were studied in this experiment and the results showed that: 1. there were 7 SOD isoenzyme bands in all soybean genotypes used in this experiment, 2. the SOD activity (unit/g fr. wt.) in different soybean seeds decreased as the evolutionary process advanced, 3 the SOD activity decreased as the wt./100 seeds increased, and the color of seed coat changed from black to yellow. The relationship between SOD activity and the evolutionary process was discussed.     

长效油菜素内酯TS303和二氢茉莉酸丙酯协同提高大豆光合能力

研究了长效油菜素内酯TS303、二氢茉莉酸丙酯(PDJ)及二者复配对大豆光合作用的影响及作用机理。试验结果表明：(1)0.01~1 mg·L^-1 TS303浸种促进大豆干物质积累, 以0.1 mg·L^-1的浓度效果最好, TS303对干物质在地上部和根之间的分配没有明显的影响, 1～10 mg·L^-1 PDJ浸种促进干物质积累, 以5 mg·L^-1增幅最大,50和100 mg·L^-1则抑制干物质积累,1～100 mg·L^-1 PDJ均促进同化物质向根系分配;(2)0.1 mg·L^-1 TS303和5 mg·L^-1 PDJ能增加大豆光合叶面积及净光合速率, 增强光合能力, 二者混合使用表现出协同效应;(3)0.1 mg·L^-1 TS303和5 mg·L^-1 PDJ及二者复配增加叶绿素含量和提高PSⅡ的实际光转化效率 (ФPSⅡ), 二者对ФPSⅡ的提高途径不同, TS303增加光合淬灭(qP)而对有效光转化效率(Fv′/Fm′)影响不大, PDJ增加Fv′/Fm而对qP影响不大;(4)0.1 mg·L^-1 TS303和5 mg·L^-1 PDJ及二者复配增加大豆气孔导度、碳酸酐酶活性、RuBPCase含量和活性, 增强CO₂转运和固定能力;(5)0.1 mg·L^-1 TS303和5 mg·L^-1 PDJ及二者复配增加叶片中蔗糖的含量, 提高蔗糖/淀粉比率, 加快同化物质的转运, 增加根中淀粉含量。总体上, TS303在光能转化和CO₂固定方面效果好于PDJ, 而PDJ促进同化物质运出效果好于TS303, 这可能是二者协同提高大豆光合能力的原因。     

Relative growth rates of leaves from soybean grown under drought-stressed and irrigated field conditions

Abstract. The relative growth rates and leaf area were graphed against leaf area, normalized with respect to final leaf area, to assess the applicability of the Lockhart cell wall expansion equation to soybean, Glycine max (L.) Merr., leaf development under field conditions. For leaves that had completed more than 20% of their growth, relative growth rates decreased linearly with an increase in the normalized leaf area, indicating that these leaves were undergoing strictly expansive growth. Drought stress significantly decreased the relative growth rate of these larger leaves. Small leaves which had completed less than 20% of their growth, were found to have highly variable relative growth rates. The large variability in relative growth rates indicated that the Lockhart cell wall expansion equation was inadequate to evaluate the growth of these young leaves. Drought stress had virtually no influence on the relative growth rates observed in the small leaves.