Identification of QTLs Underlying Water-Logging Tolerance in Soybean

Soil water-logging can cause severe damage to soybean [Glycine max (L.) Merr.] and results in significant yield reduction. The objective of this study was to identify quantitative trait loci (QTL) that condition water-logging tolerance (WLT) in soybean. Two populations with 103 and 67 F_6:11 recombinant inbred lines (RILs) from A5403 × Archer (Population 1) and P9641 × Archer (Population 2), respectively, were used as the mapping populations. The populations were evaluated for WLT in manually flooded fields in 2001, 2002, and 2003. Significant variation was observed for WLT among the lines in the two populations. No transgressive tolerant segregants were observed in either population. Broad-sense heritability of WLT for populations 1 and 2 were 0.59 and 0.43, respectively. The tolerant and sensitive RILs from each population were selected to create a tolerant bulk and a sensitive bulk, respectively. The two bulks and the parents of each population were tested with 912 simple sequence repeat (SSR) markers to select candidate regions on the linkage map that were associated with WLT. Markers from the candidate regions were used to genotype the RILs in both populations. Both single marker analysis (SMA) and composite interval mapping (CIM) were used to identify QTL for WLT. Seventeen markers in Population 1 and 15 markers in Population 2 were significantly (p <0.0001) associated with WLT in SMA. Many of these markers were linked to Rps genes or QTL conferring resistance to Phytophthora sojae Kaufmann and Gerdemann. Five markers, Satt599 on linkage group (LG) A1, Satt160, Satt269, and Satt252 on LG F, and Satt485 on LG N, were significant (p <0.0001) for WLT in both populations. With CIM, a WLT QTL was found close to the marker Satt385 on LG A1 in Population 1 in 2003. This QTL explained 10% of the phenotypic variation and the allele that increased WLT came from Archer. In Population 2 in 2002, a WLT QTL was located near the marker Satt269 on LG F. This QTL explained 16% of the phenotypic variation and the allele that increased WLT also came from Archer.     

Identification of quantitative trait loci for plant height, lodging, and maturity in a soybean population segregating for growth habit

The use of molecular markers to identify quantitative trait loci (QTLs) has the potential to enhance the efficiency of trait selection in plant breeding. The purpose of the present study was to identify additional QTLs for plant height, lodging, and maturity in a soybean, Glycine max (L.) Merr., population segregating for growth habit. In this study, 153 restriction fragment length polymorphisms (RFLP) and one morphological marker (Dt1) were used to identify QTLs associated with plant height, lodging, and maturity in 111 F₂-derived lines from a cross of PI 97100 and Coker 237. The F₂-derived lines and two parents were grown at Athens, Ga., and Blackville, S.C., in 1994 and evaluated for phenotypic traits. The genetic linkage map of these 143 loci covered about 1600 cM and converged into 23 linkage groups. Eleven markers remained unlinked. Using interval-mapping analysis for linked markers and single-factor analysis of variance (ANOVA), loci were tested for association with phenotypic data taken at each location as well as mean values over the two locations. In the combined analysis over locations, the major locus associated with plant height was identified as Dt1 on linkage group (LG) L. The Dt1 locus was also associated with lodging. This locus explained 67.7% of the total variation for plant height, and 56.4% for lodging. In addition, two QTLs for plant height (K007 on LG H and A516b on LG N) and one QTL for lodging (cr517 on LG J) were identified. For maturity, two independent QTLs were identified in intervals between R051 and N100, and between B032 and CpTI, on LG K. These QTLs explained 31.2% and 26.2% of the total variation for maturity, respectively. The same QTLs were identified for all traits at each location. This consistency of QTLs may be related to a few QTLs with large effects conditioning plant height, lodging, and maturity in this population.     

盐度对哈氏仿对虾肌肉一般营养成分和氨基酸组成及含量的影响

通过调查不同盐度(12～36)环境下养殖的哈氏仿对虾(Parapenaeopsis hardwickii)肌肉一般营养成分和氨基酸组成及含量,研究了盐度对该虾肌肉营养品质的影响。结果显示,肌肉的水分随环境盐度升高出现显著的直线下降,而粗蛋白含量随环境盐度升高而直线升高;各盐度组的粗脂肪含量虽然没有明显差异,但其含量随盐度升高有一定的线性下降趋势;36盐度组的粗灰分含量比12、16、20和28盐度组的明显高,32盐度组的灰分含量比20和24盐度组的也明显高;所检测的肌肉干样16种氨基酸中,只有3种氨基酸的含量随盐度升高而升高,而其他13种氨基酸的含量随盐度升高明显降低,其中有10种氨基酸的含量在盐度12～24条件下的比盐度28～36条件下的明显高,而这些氨基酸含量在盐度12～24之间没有明显差异。各盐度组间氨基酸总量和鲜味氨基酸含量均没有明显差异;必需氨基酸和半必需氨基酸含量在盐度12～24条件下的均比盐度28～36条件下的明显高,而在12～24盐度组之间以及28～36盐度组之间均没有明显差异。必需氨基酸/氨基酸总量的比值和必需氨基酸/非必需氨基酸比值随盐度升高均明显线性降低;盐度12～24组的必需氨基酸指数(66.13～67.42)高于盐度28～36组的(62.56～64.46)。综上所述,盐度12～24环境下养殖的哈氏仿对虾肌肉营养价值相对较高,表现为低盐趋向,考虑到肌肉的水分含量和生长性能,哈氏仿对虾在养殖期间选择16～24盐度范围比较合适,同时也说明哈氏仿对虾适合大多数沿海地区环境的盐度条件。     

Stability of Soybean Recombinant Plastome Over Six Generations

The stability of a plastid transgene has been evaluated in soybean transformants over six generations. These transformants had integrated the aadA selection cassette in the intergenic region between the rps12/7 and trnV genes. Three independent homoplasmic T0 transformation events were selected and ten plants from each event propagated to generation T5 in the absence of selection pressure. No transgene rearrangement nor wild-type plastome were detected in generation T5 by Southern blot analysis. All tested progenies were uniformly resistant to spectinomycin. Therefore, soybean transformants of generations T0 and T5 appear to be genetically and phenotypically identical.     

Molecular markers associated with seed weight in two soybean populations

Seed weight (SW) is a component of soybean, Glycine max (L.) Merr., seed yield, as well as an important trait for food-type soybeans. Two soybean populations, 120 F₄-derived lines of YoungxPI416937 (Pop1) and 111 F₂-derived lines of PI97100xCoker 237 (Pop2), were mapped with RFLP makers to identify quantitative trait loci (QTLs) conditioning SW across environments and populations. The genetic map of Pop1 consisted of 155 loci covering 973 cM, whereas Pop2 involved 153 loci and covered 1600 cM of map distance. For Pop1, the phenotypic data were collected from Plains, GA., Windblow, N.C., and Plymouth, N.C., in 1994. For Pop2, data were collected from Athens, GA., in 1994 and 1995, and Blackville, S.C., in 1995. Based on single-factor analysis of variance (ANOVA), seven and nine independent loci were associated with SW in Pop1 and Pop2, respectively. Together the loci explained 73% of the variability in SW in Pop1 and 74% in Pop2. Transgressive segregation occurred among the progeny in both populations. The marker loci associated with SW were highly consistent across environments and years. Two QTLs on linkage group (LG) F and K were located at similar genomic regions in both populations. The high consistency of QTLs across environments indicates that effective marker-assisted selection is feasible for soybean SW.     

Differential Effect of Cd2+ and Ni2+ on Amino Acid Metabolism in Soybean Seedlings

In 10-d-old soybean seedlings, the growth of roots and shoots was significantly inhibited at 50 and 100 M and more Cd²⁺, respectively, and by 50 M or more Ni²⁺. Although total protein content of roots exposed to 200 M Cd²⁺ or Ni²⁺ was similarly decreased compared to the control, the activity of nitrate reductase was much more inhibited by Cd²⁺. Ni²⁺-treatment (200 M) induced an accumulation of all free amino acids in roots associated with a decrease in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities reflecting the accumulation of both alanine and aspartic acid, respectively. Cd²⁺-treatment (200 M) decreased the amount of all free amino acids. In addition, cysteine which is the main amino acid consisting the phytochelatin complexes constituted about 17.5 % of total free amino acids. The activities of both ALT and AST in Cd²⁺-treated roots were higher than in Ni²⁺-treated roots suggesting higher conversion of alanine and aspartate to pyruvate and oxaloacetate. Primary leaves excised from either Cd²⁺ or Ni²⁺-treated seedlings showed similar pattern of enzyme activities as roots.     

Differential Expression of Peroxidase Isoenzymes in Soybean Roots Treated with the Benzothiadiazole

The protection compound benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) was applied to soybean roots or leaves in a dose of 20 cm³ of a solution containing 25 μg g⁻¹ of the active ingredient. Electrophoretic profiles of chitinase and superoxide dismutase were not altered by the product. Increased activity of two root anionic peroxidases and three differential isoforms of these enzymes were observed in plants with roots treated by BTH, which can be used as biochemical markers of the BTH effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Evolution of Amino Acid Repeat Arrays in Plasmodium and Other Organisms

Repeat arrays in protein-coding sequences were analyzed by a novel approach, based on analyzing the distribution of the pairwise proportion of nucleotide differences among units within a repeat array. The results showed that evidence of recent repeat array expansion was particularly characteristic of the repeat arrays of the malaria parasites (genus Plasmodium), supporting the hypothesis that Plasmodium is particularly prone to repeat array expansion by slipped-strand mispairing or a similar mechanism. Repeat arrays in Plasmodium asexual-stage antigens (which are exposed to the immune system of the vertebrate host) had unique characteristics with respect to the number of repeat units, as well as nucleotide and amino acid composition, suggesting that natural selection exerted by the host immune system has shaped features of these arrays.[Reviewing Editor: Dr. Manyuan Long]     

Soybean aphid resistance genes in the soybean cultivars Dowling and Jackson map to linkage group M

The soybean aphid [Aphis glycines Matsumura] is an important pest of soybean [Glycine max (L.) Merr.] in North America. Single dominant genes in the cultivars ‘Dowling’ and ‘Jackson’ control resistance to the soybean aphid. The gene in Dowling was named Rag1, and the genetic relationship between Rag1 and the gene in Jackson is not known. The objectives of this study were to map the locations of Rag1 and the Jackson gene onto the soybean genetic map. Segregation of aphid resistance and simple sequence repeat (SSR) markers in F _2:3 populations developed from crosses between Dowling and the two susceptible soybean cultivars ‘Loda’ and ‘Williams 82’, and between Jackson and Loda, were analyzed. Both Rag1 and the Jackson gene segregated 1:2:1 in the F _2:3 populations and mapped to soybean linkage group M between the markers Satt435 and Satt463. Rag1 mapped 4.2 cM from Satt435 and 7.9 cM from Satt463. The Jackson gene mapped 2.1 cM from Satt435 and 8.2 cM from Satt463. Further tests to determine genetic allelism between Rag1 and the Jackson gene are in progress. The SSR markers flanking these resistance genes are being used in marker-assisted selection for aphid resistance in soybean breeding programs. Trade and manufacturers’ names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Dynamics of Seed Protein Biosynthesis in Two Soybean Genotypes Differing in Drought Susceptibility

The dynamics of seed storage protein biosynthesis was studied under field conditions during two vegetative seasons. Two soybean (Glycine max L. Merr.) genotypes were examined: BOSA (drought tolerant) and L 121 (drought susceptible). Seed samples were taken from plants at three stages of seed maturation (50 and 70 d after flowering, and at full maturity). The earlier synthesis of the -subunit of the 7S protein occurred in the drought susceptible cultivar. We have not found such differences in the synthesis of the - and -subunits of the 7S protein. Our results did not confirm significant genotypic differences in protein composition of the mature seeds between the cultivars studied, but have pointed out to the differences in the dynamics of protein biosynthesis during seed maturation and desiccation.     

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F₂ segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Decrease of Extracted Apoplast Protein in Soybean Root Tip by Aluminium Treatment

Aluminium effect on the mobility of apoplast protein in root tips was studied. Two-day seedlings of soybean (Glycine max. (L.) Merr. cv. Tsurunoko) were treated with 50 μM AlCl₃ for 2 h. Using infiltration method, the apoplast protein in root tips was extracted with 20 or 100 mM MgCl₂. When 20 mM MgCl₂ was used to collect weakly bound protein to apoplast, the amount of protein extracted was reduced to be about 20 % compared with that of control and the band of 97 kDa disappeared in SDS-PAGE gel. However, the 97 kDa protein could be extracted by 100 mM MgCl₂, which were used for extraction of more tightly bound protein to apoplast, and the amount was estimated to be the same as that of control. When the protein was further developed in two-dimensional electrophoresis, three spots were found between pI 6.4 and 6.5. This is the first report of an Al effect on the mobility of apoplast protein. This revised version was published online in July 2006 with corrections to the Cover Date.     

RFLP loci associated with soybean seed protein and oil content across populations and locations

Molecular markers provide the opportunity to identify marker-quantitative trait locus (QTL) associations in different environments and populations. Two soybean [Glycine max (L.) Merr.] populations, Young x PI 416 937 and PI 97100 x Coker 237, were evaluated with restriction fragment length polymorphism (RFLP) markers to identify additional QTLs related to seed protein and oil. For the Young x PI 416937 population, 120 F₄-derived lines were secored for segregation at 155 RFLP loci. The F₄-derived lines and two parents were grown at Plains, G.a., and Windblow and Plymouth, N.C. in 1994, and evaluated for seed protein and oil. For the PI 97100 x Coker 237 population, 111 F₂-derived lines were evaluated for segregation at 153 RFLP loci. Phenotypic data for seed protein and oil were obtained in two different locations (Athens, G.a., and Blackville, S.C.) in 1994. Based on single-factor analysis of variance (ANOVA) for the Young x PI 416937 population, five of seven independent markers associated with seed protein, and all four independent markers associated with seed oil in the combined analysis over locations were detected at all three locations. For the PI 97 100 x Coker 237 population, both single-factor ANOVA and interval mapping were used to detect QTLs. Using single-factor ANOVA, three of four independent markers for seed protein and two of three independent markers for seed oil were detected at both locations. In both populations, singlefactor ANOVA, revealed the consistency of QTLs across locations, which might be due to the high heritability and the relatively few QTLs with large effects conditioning these traits. However, interval mapping of the PI 97100 x Coker 237 population indicated that QTLs identified at Athens for seed protein and oil were different from those at Blackville. This might result from the power of QTL mapping being dependent on the level of saturation of the genetic map. Increased seed protein was associated with decreased seed oil in the PI 97100 x Coker 237 population (r = –0.61). There were various common markers (P0.05) on linkage groups (LG) E, G,H,K, and UNK2 identified for both seed protein and oil. One QTL on LG E was associated with seed protein in both populations. The other QTLs for protein and oil were population specific.     

木豆不同品种和叶龄对叶片氨基酸形成的影响和聚类分析

为了解木豆(Cajanus cajan)叶片的氨基酸含量的变化,对19份种质资源和1～3叶龄的木豆叶片氨基酸含量进行研究。结果表明,不同种质资源和叶龄的木豆叶片中均含有17种氨基酸,必需氨基酸以苏氨酸、异亮氨酸和赖氨酸的含量最高,分别为0.67%、0.70%和0.48%,非必需氨基酸以丝氨酸、酪氨酸和组氨酸的含量最高,分别为0.69%、0.66%和0.44%。除赖氨酸和丙氨酸外,其余15种氨基酸含量在木豆叶片中总体上随着叶龄的增加呈现上升趋势。不同种质资源的木豆叶片中总氨基酸含量差异明显。聚类分析可将19个木豆种质资源划分为低氨基酸型(I)、中氨基酸型(II)、中高氨基酸型(III)、高氨基酸型(IV)等4大类型。这为高品质木豆叶茶的加工与高氨基酸木豆品种选育提供了理论依据。     

大豆GmABI4基因克隆及表达分析

该研究基于大豆基因组数据库,根据拟南芥ABI4蛋白的氨基酸序列,经比对分析,获得了大豆中的2个GmABI4基因,分别命名为GmABI4-1(GenBank登录号为XM_014766551.1)和GmABI4-2(GenBank登录号为NM_001249003)。TMHMM软件和系统进化转录分析表明,这2个基因编码的蛋白均不具有信号肽,二级结构主要以无规则卷曲和延伸链为主;进化树分析表明,大豆GmABI4和野生大豆亲缘关系较近。荧光定量PCR分析表明,GmABI4-1与GmABI4-2基因在大豆种子与豆荚中的表达量均高于根、茎、叶、花等其他组织,推测可能与调控大豆种子生命活动相关。     

Influence of Media Components and pH on Somatic Embryo Induction in Three Genotypes of Soybean

The influence of media components on the initiation of somatic embryogenesis in three genotypes of soybean was investigated. The following genotypes were used: Iroquois, Macon, and Savoy. Media modifications included sucrose concentration, type and concentration of auxin at two pH levels, and pH level independently. Immature cotyledons were used as the source of explant. Cotyledons were placed on a medium containing MS salts, B5 vitamins, sucrose, and auxin. Gelrite (0.2%) was used as the solidifying agent. Sucrose concentrations of 1, 2, 3, 4.5, or 6% were used. The auxins used included 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) with each at concentrations of 45.2, 90.4, 135.7, 180.9, and 226.2 M. The pH of each the media was adjusted to either 5.7 or 7.0 with 1 N NaOH. In an additional experiment, the effect of the two pH levels, 5.7 and 7.0, was investigated independently. Overall, the frequency of somatic embryogenesis significantly varied among the different genotypes used in this study, with Iroquois showing the highest response. Frequency of somatic embryogenesis also varied in response to the different treatments used, including sucrose and auxin. The highest initiation (91.7%) and mean number of somatic embryos per responding explant (14.9) of Iroquois was observed in a medium containing 2% sucrose. The highest initiation (97.1%) and mean number of somatic embryos per responding explant (19.5) was observed in Iroquois on 135.7 M 2,4-D and Savoy on 135.7 M 2,4-D, respectively, for the auxin by pH level experiment. No significant differences were observed among the two pH treatments used.     

衰老对大鼠脑区氨基酸水平的影响

本文测定了正常青龄组（３月龄）和老龄组（２０月龄）大鼠不同脑区（皮层、小脑海马、纹状体和下丘脑）谷氨酸、天门冬氨酸、甘氨酸、ｒ－氨基丁酸和牛磺酸的含量。结果表明：在衰老过程中大鼠某些脑区谷氨酸、天门冬氨酸、甘氨酸和牛磺酸水平显著降低;而纹状体γ－氨基丁酸含量则显著升高。     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Relating Trypsin Enzymatic Properties with Amino Acid Composition

In this paper, the DNA fragment of trypsin genes from eight crustaceans were cloned and sequenced. The amino acid composition of the 24 deduced and 42 selected trypsin sequences were compared. Low arginine, methionine, and proline content and high aspartic acid, glutamic acid, and isoleucine content attributed to the distinct catalytic efficiency of crustacean trypsins.