Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.     

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)⁺ RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I–IV. Polypeptides synthesized in vitro were labeled with (³⁵S)-cysteine and (³H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I–IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.     

Coupling diet quality and Bowman-Birk and Kunitz-type soybean proteinase inhibitor effectiveness to Diatraea saccharalis development and mortality

We used bioassays to investigate the effect of Bowman‐Birk and Kunitz‐type soybean proteinase inhibitors on two artificial diets (diets 1 and 2) which are commonly used to feed laboratory colonies of larvae of the moth Diatraea saccharalis, monitoring food intake and utilization, and larval development and mortality. Diet 1 was less nutritious, with a low protein content and reduced mineral and essential amino acid (e.g., cysteine, lysine, and methionine) content, while diet 2 was richer and more complete. When proteinase inhibitors were incorporated into the artificial diets, the effects on larval development were significantly greater for those larvae fed diet 1, with the chronic ingestion of proteinase inhibitors reducing the level of trypsin‐like activity in the midgut of larvae fed this diet. Larvae fed diet 2 also showed a reduced level of tryptic activity in the midgut, but this was less marked than for diet 1. These results indicate that despite their inhibitory effect on midgut enzymes, the effectiveness of proteinase inhibitors is directly dependent on the quality of the diet. The different effects seen on insect biology when proteinase inhibitors are added to rich or poor diets suggests that the role of anti‐nutritional proteins in the control of insects might not be adequately addressed by bioassays based on the incorporation of inhibitors into artificial diets.     

Cell type specific expression of a CaMV 35S-GUS gene in transgenic soybean plants

A number of independently derived transgenic soybean plants expressing a chimeric β-glucuronidase (GUS) gene under the control of the 355 CaMV promoter and a nopaline synthase polyadenylation signal were recovered using direct DNA transfer via electric discharge particle acceleration. Expression of GUS in R, plants was localized using thin tissue sections. Many tissue types expressed GUS at various levels. Pericycle cells in root, parenchyma cells in xylem, and phloem tissues of stem and leaf had high levels of enzyme activity. Procambium, phloem, and cortex cells in root, vascular cambium cells in stem, and the majority of cortex cells in leaf midrib, expressed low or no GUS activity. Intermediate levels of GUS activity were detected in leaf mesophyll cells, certain ground tissue cells in stem and leaf midrib, and in trichome and epidermal guard cells. Thus, we conclude that the 35S CaMV promoter is cell-type specific and is developmentally regulated in soybean.     

Development of protein bodies and accumulation of carbohydrates in a soybean (Leguminosae) shriveled seed mutant

The soybean seed mutant T311, when grown under specific environmental conditions, produces shriveled seed. This research investigated changes in development of protein bodies and accumulation of carbohydrates during seed development by comparing the mutant with P2180 seeds. The shriveled seeds contained larger protein bodies but fewer protein bodies per cell than round seeds. Protein bodies in T311 seeds included more dispersed crystals and less globoid regions than P2180 seeds. The elemental compositions of the crystals and of whole seeds in T311 were different from that in P2180 seeds. Starch breakdown was reduced with concomitant lower soluble sugar content in T311 seeds after the D11 stage (10.0-11.9 mm long seeds). The reduced starch breakdown and lowered soluble sugar content were consistent with lower a-amylase activity and earlier and greater water loss in T311 seeds. Changes in development of protein bodies and accumulation of carbohydrates were associated with the development of the shriveled seeds.     

Unstable expression of a soybean gene during seed coat development

Summary The R gene of soybean is involved in anthocyanin synthesis in the seed coat, and its r-m allele conditions a variegated distribution of black spots and/or concentric rings of pigment superimposed on an otherwise brown seed coat. We describe an unusual feature of r-m that causes expression at the R locus to switch between active and inactive phases both somatically and germinally. Non-heritable somatic changes of the allele produce single plants containing mixtures of seed with different coat colors (black+striped or brown+striped). Heritable changes of the r-m allele are manifested in progeny plants which produce all black seed or all brown seed. Surprisingly, subsequent generations from revertant sublines show continued instability of the allele such that brown revertants (r*/r*) or homozygous black seed revertants (R*/R*) can give rise to striped or striped+black-seeded plants. Thus, the revertants produced by the r-m allele are not stable but interconvert between all three forms (R*, r*, and r-m) at detectable frequencies. Mutability of the r-m allele in a different genetic background has also been found after inter-crossing various soybean genotypes.     

Genome-wide identification and characterization of novel microRNAs in seed development of soybean

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in eukaryotes. However, the information about miRNAs population and their regulatory functions involving in soybean seed development remains incomplete. Base on the Dicer-like1-mediated cleavage signals during miRNA processing could be employed for novel miRNA discovery, a genome-wide search for miRNA candidates involved in seed development was carried out. As a result, 17 novel miRNAs, 14 isoforms of miRNA (isomiRs) and 31 previously validated miRNAs were discovered. These novel miRNAs and isomiRs represented tissue-specific expression and the isomiRs showed significantly higher abundance than that of their miRNA counterparts in different tissues. After target prediction and degradome sequencing data-based validation, 13 novel miRNA–target pairs were further identified. Besides, five targets of 22-nt iso-gma-miR393h were found to be triggered to produce secondary trans-acting siRNA (ta-siRNAs). Summarily, our results could expand the repertoire of miRNAs with potentially important functions in soybean.     

Inheritance of reduced linolenic acid content in soybean seed oil

 Linolenic acid is the unstable component of soybean [Glycine max (L.) Merr.] oil that is responsible for the undesirable odors and flavors commonly associated with poor oil quality. Two mutants, M-5 and KL-8, have been identified that have lower linolenic acid levels in the seed oil than the ‘Bay’ cultivar. Our objective was to determine the relationships between the genetic systems controlling linolenic acid in these mutants. Reciprocal crosses were made between the mutants and ‘Bay’, and between the two mutants. No maternal effect for linolenic acid content was observed from the analysis of F₁ seeds in any of the crosses. The data for linolenic acid content in F₂ seeds of M-5בBay’ and KL-8בBay’ crosses satisfactorily fit a 1 : 2 : 1 and 3 : 1 ratio, respectively. For the M-5×KL-8 cross, segregation observed from the analysis of F₂ seeds for linolenic acid content satisfactorily fit a ratio of 3 more than either mutant: 12 within the range of the two mutants: 1 less than either mutant. The segregation ratio of F₂ seeds and the segregation of F₃ seeds from F₂ plants indicated that M-5 and KL-8 have alleles at different loci that control linolenic acid content. The allele in KL-8 has been designated as fanx (KL-8) to distinguish it from fan (M-5). The low linolenic acid segregates with the genotype fanfanfanxfanx provide additional germplasm to reduce the linolenic acid content from the seed oil of soybean. Received: 18 December 1995 / Accepted: 12 July 1996     

QTLs associated with chlorimuron ethyl sensitivity in soybean: Effects on seed yield and related traits

 Soybean, Glycine max (L.) Merr., genotypes are known to differ in chlorimuron ethyl sensitivity (CS). Earlier we have reported two putatively independent marker loci linked to two quantitative trait loci (QTLs) controlling CS in a soybean population derived from a cross of PI97100 (sensitive to chlorimuron ethyl) and ‘Coker 237’ (tolerant to chlorimuron ethyl). The objective of the present study was to quantify the association of the two marker loci with seed yield and related traits in this soybean population following application of chlorimuron ethyl. Phenotypic data were collected for 111 F₂-derived lines of the cross grown in replicated plots at Athens, G.A., in 1994 and 1995, and at Blackville, S.C., in 1995. The two CS marker loci explained as much as 50% of the genetic variation in seed yield and seed number m^-2, but had no association with seed weight, plant height, lodging, seed protein, and seed oil. There were no epistatic interactions between the two marker loci for any of the traits. The marker locus (cr168-1 on USDA linkage group E) linked to the major CS QTL explained between 13 and 23% of the variation in seed yield. The Coker 237 allele at this locus was associated with decreased CS and increased seed yield. The marker locus (Blt015-2 on an unknown linkage group) linked to the minor CS QTL accounted for a maximum of 11% of the variation in seed yield. The Coker 237 allele at this locus was associated with an increase in CS and a decrease in seed yield. The association of the two marker loci with seed number m^-2 strongly resembled their association with seed yield. Seed yield had a strong positive correlation (r=0.74 – 0.94) with seed number m^-2, and the effect of chlorimuron ethyl on seed yield was due mainly to its effect on seed number m^-2 rather than seed weight. Received: 6 August 1996 / Accepted: 28 February 1997     

Amino acid sequence of a Bowman-Birk proteinase inhibitor from pea seeds

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified by ammonium sulfate precipitation, gel filtration, and anion and cation exchange chromatography and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Their molecular weights were determined by electrospray mass spectrometry as 6916, 6807, 7676, 7944, 7848, and 7844 D, respectively, and the sequences of the first 20 N-terminal amino acid residues of these six inhibitors were found to be identical. The complete amino acid sequence of PSTI IVa was determined. This protein comprises a total of 72 residues and has 14 cysteines, all involved in disulfide bridges. Comparison of the sequence of PSTI IVa with those of other leguminous Bowman-Birk type inhibitors revealed that PSTI could be classified as a group III inhibitor, closely related toVicia faba andVicia angustifolia inhibitors.     

种子特异表达异源DGAT1基因提高大豆种子含油量和营养品质

为提高大豆Glycine max种子含油量和营养品质,文中以二酰甘油酰基转移酶1(Diacylglycerol acyltransferase 1,DGAT1)基因为遗传修饰靶标。将来自高油植物斑鸠菊Vernonia galamensis L.编码DGAT1酶蛋白的c DNA克隆Vg DGAT1A在大豆种子特异超表达。连续选择获得高代(T7)Vg DGAT1A转基因大豆株系。转基因株系表型鉴定显示,在大豆种子发育中期(30–45 DAF),Vg DGAT1A高表达,相应地DGAT酶活性是非转基因野生型和空载体转化对照的7.8倍。转基因成熟种子含油量比对照提高了5.1%,淀粉含量比对照减少2%–3%,蛋白质含量与对照无显著差异。此外,转基因大豆种子百粒重(14.5 g)和种子萌发率(95.6%)与对照亦无明显差异。种子油脂脂肪酸成分分析显示,转基因大豆种子油中抗氧化的油酸(C18:1Δ9)含量比对照提高8.2%,相应地易氧化的亚油酸(C18:2Δ9,12)和亚麻酸(C18:3Δ9,12,15)分别减少6%和2%。这些数据表明,种子特异超表达外源Vg DGAT1A基因,打破了大豆种子含油量和蛋白质含量的负连锁,显著提高种子含油量且未导致蛋白含量降低。转基因大豆种子重量和萌发率亦未显负效应,而且种子油脂抗氧化性和营养品质得以改善。研究表明应用这一高酶活性Vg DGAT1A的基因工程是提高种子含油量和改善油脂品质的一条有效途径。     

Effects of UV-B radiation on soybean yield and seed quality: a 6-year field study

Two soybean [ Glycine max (L.) Merr.] cultivars, Essex and Williams, were grown in the field for 6 consecutive seasons under ambient and supplemental levels of ultravio-Set-B radiation to determine the potential for alterations in yield or seed quality with a reduction in the stratospheric ozone column. The supplemental UV-B fluences simulated a 16 or 25% ozone depletion. The data presented here represent the first field experiment conducted over multiple seasons which assesses the effects of increased UV-B radiation on seed yield. Overall, the cultivar Essex was found to be sensitive to UV-B radiation (yield reductions of 20%) while the cultivar Williams was tolerant. However, the effectiveness of UV-B radiation in altering yield was strongly influenced by the seasonal microclimate, and the 2 cultivars responded differently to these changing factors. Yield was reduced most in Essex during seasons in which water availability was high and was reduced in Williams only when water was severely limiting. The results of these experiments demonstrate the necessity for multiple-year experiments and the need to increase our understanding of the interaction between UV-B radiation and other environmental stresses in order to assess the potential consequences of stratospheric ozone depletion.     

Factors influencing regeneration of soybean from mature and immature cotyledons

Soybean (Glycine max L.) plantlets were e?ciently regenerated from mature and immature cotyledons of?ve different genotypes by studying various parameters affecting regeneration. Green organogenic noduleswere induced at the proximal end, which subsequently differentiated into shoot buds on modi?ed MS(Murashige T. and Skoog F. 1962. Physiol. Plant. 15: 473-497) medium. The presence of 6-benzylaminopurine(BAP) and thidiazuron (TDZ) in the medium exerted a synergistic effect, in that regenerationeffciency was higher than for either cytokin alone. The regenerated shoot buds elongated and rooted onMS medium containing 0.29 lM gibberellic acid (GA3) and 2.69 lM a-naphthaleneaceticacid (NAA),respectively. Rooted plants were established in the greenhouse with 87% success and produced viable seeds.Preliminary studies with Agrobacterium show great promise for soybean transformation based on theregeneration protocol reported here.     

Antimicrobial activity studies on a trypsin-chymotrypsin protease inhibitor obtained from potato

A 5.6 kDa trypsin-chymotrypsin protease inhibitor was isolated from the tubers of the potato (Solanum tuberosum L cv. Gogu) by extraction of the water-soluble fraction, dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This inhibitor, which we named potamin-1 (PT-1), was thermostable and possessed antimicrobial activity but lacked hemolytic activity. PT-1 strongly inhibited pathogenic microbial strains, including Candida albicans, Rhizoctonia solani, and Clavibacter michiganense subsp. michiganinse. Automated Edman degradation showed that the N-terminal sequence of PT-1 was NH2-DICTCCAGTKGCNTTSANGAFICEGQSDPKKPKACPLNCDPHIAYA-. The sequence had 62% homology with a serine protease inhibitor belonging to the Kunitz family, and the peptide inhibited chymotrypsin, trypsin, and papain. This protease inhibitor, PT-1, was composed of polypeptide chains joined by disulfide bridge(s). Reduced PT-1 almost completely lost its activity against fungi and proteases indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. These results suggest that PT-1 is an excellent candidate as a lead compound for the development of novel oral or other anti-infective agents.     

Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein

 Embryogenic soybean [Glycine max (L.) Merrill.] suspension cultures were bombarded with five different gene constructions encoding the jellyfish (Aequorea victoria) green fluorescent protein (GFP). These constructions had altered codon usage compared to the native GFP gene and mutations that increased the solubility of the protein and/or altered the native chromophore. All of the constructions produced green fluorescence in soybean cultures upon blue light excitation, although a soluble modified red-shifted GFP (smRS-GFP) was the easiest to detect based on the brightness and number of foci produced. Expression of smRS-GFP was visible as early as 1.5 h after bombardment, with peak expression at approximately 6.5 h. Large numbers of smRS-GFP-expressing areas were visible for 48 h postbombardment and declined rapidly thereafter. Stably transformed cultures and plants exhibited variation in the intensity and location of GFP expression. PCR and Southern hybridization analyses confirmed the presence of introduced GFP genes in stably transformed cultures. Received: 23 September 1998 / Revision received: 4 January 1999 / Accepted: 15 January 1999     

Entity evidence for differentiation between Tia and Tib types of soybean Kunitz trypsin inhibitor: detection of a novel transitional variant type between Tia and Tib in wild soybean (Glycine soja Sieb. & Zucc.)

Soybean Kunit trypsin inhibitor (SKTI) has several polymorphic types. Of these SKTI, there are large differences of nine amino acid substitutions between Tia and Tib. So far no transitional type between them has been found. A novel transitional intermediate variant between Tia and Tib was detected in 11 lines from 720 Japanese wild soybeans (Glycine soja Sieb. & Zucc.). This variant showed identical electrophoretic mobility to Tib in the Davis system polyacrylamide gel electrophoresis (PAGE), but higher electric points than other SKTI proteins (Tia, Tib, Tic) in isoelectric focusing PAGE. The genetic analysis of SKTI in F₂ seeds from a cross between the novel variant type and Tib showed that this variant type is inherited as codominant alleles in a multiple allelic system at an SKTI locus. This variant also showed inhibitory activity to trypsin. We propose the genetic symbol Ti b ⁱ⁵ for this novel variant. The sequence analysis of Tib ⁱ⁵ revealed that six nucleotides were different between Tib ⁱ⁵ and Tia, and the nucleotides of these mutated positions were identical to Tib. This causes substitution of five amino acids at the residue position 62 (Tyr→Phe), 74 (Ser→Arg), 114 (Met→Val), 120 (Leu→Ile) and 137 (Pro→Thr). These substitutive amino acids are completely in accord with the amino acids of Tib, showing that Tib ⁱ⁵ is an intermediate between Tia and Tib types. Tib ⁱ⁵ type is widely distributed throughout seven separate areas from northeast to southwest Japan with a 1.5% frequency of total materials examined. This indicated that Tib ⁱ⁵ type did not originate from a recent mutation event, but had spread in wild soybean from ancient times.