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Plant E2 Ubiquitin-conjugating enzymes regulate various biological pathways such as stress resistance, growth and development. Reports on its functions are more frequent in Arabidopsis thaliana, but relatively rare in soybean (Glycine max), which is one of the most important economic crops. In this study, a gene Glyma.12G161200, which may be related to the soybean cotyledon folding mutant, was cloned from soybean "Nanong 94-16". Analysis of its sequence suggested that it encodes an E2 ubiquitin binding enzyme, so it was named as GmUBC1. Its coding region is 462 bp in length, which encodes a protein of 153 amino acids with a predicted molecular mass of 17.25 kDa and an isoelectric point of 6.74. The expression pattern of GmUBC1 in different tissues of soybean and its response patterns to different stresses and hormone treatments were analyzed by real-time PCR. The results showed that the gene was expressed at the highest level in mutant seeds at 40 days after flowering. Moreover, the expression of the GmUBC1 gene was down-regulated by the treatments of PEG, cold, JA and ABA, respectively. Subcellular localization analysis of GmUBC1 revealed that the protein was expressed in the whole cell. When GmUBC1 was ectopically expressed in Arabidopsis, the 1000-grain weight and total amino acid content of some transgenic lines were found to be significantly increased. Collectively, heterologous overexpression of GmUBC1 can regulate seed weights and amino acid contents, which may provide genetic resources for soybean quality improvement.E2泛素结合酶(ubiquitin-conjugating enzyme)参与植物的抗逆、生长发育等多种生物途径的调控,其功能研究在拟南芥(Arabidopsis thaliana)中的报道较多,但在重要经济作物大豆(Glycine max)中鲜有报道。本研究从大豆“南农94-16”中克隆了1个与大豆子叶折叠突变体可能相关的基因Glyma.12G161200,序列分析结果表明该基因编码一个E2泛素结合酶,因此将其命名为GmUBC1。该基因编码区全长为462 bp,编码一个含153个氨基酸的蛋白,预测其分子量为17.25 kDa,等电点为6.74。利用qRT-PCR技术对GmUBC1在大豆不同组织中的表达模式及其对不同非生物胁迫和激素处理的响应模式进行分析,发现该基因在开花后40 d种子中表达量最高,在PEG、低温、JA和ABA处理下表达下调。亚细胞定位分析发现GmUBC1蛋白在整个细胞内都有表达。进一步在拟南芥中异源表达GmUBC1,发现转基因株系的千粒重和总氨基酸含量显著提高,表明异源过表达GmUBC1可以调控种子重量和氨基酸含量,为大豆品质改良提供了基因资源。.

Soybean (Glycine max) production is severely affected in unfavorable environments. Identification of the regulatory factors conferring stress tolerance would facilitate soybean breeding. In this study, through coexpression network analysis of salt-tolerant wild soybeans, together with molecular and genetic approaches, we revealed a previously unidentified function of a class B heat shock factor, HSFB2b, in soybean salt stress response. We showed that HSFB2b improves salt tolerance through the promotion of flavonoid accumulation by activating one subset of flavonoid biosynthesis-related genes and by inhibiting the repressor gene GmNAC2 to release another subset of genes in the flavonoid biosynthesis pathway. Moreover, four promoter haplotypes of HSFB2b were identified from wild and cultivated soybeans. Promoter haplotype II from salt-tolerant wild soybean Y20, with high promoter activity under salt stress, is probably selected for during domestication. Another promoter haplotype, III, from salt-tolerant wild soybean Y55, had the highest promoter activity under salt stress, had a low distribution frequency and may be subjected to the next wave of selection. Together, our results revealed the mechanism of HSFB2b in soybean salt stress tolerance. Its promoter variations were identified, and the haplotype with high activity may be adopted for breeding better soybean cultivars that are adapted to stress conditions.

Soybean mosaic virus (SMV) is widely recognized as a highly damaging pathogen of soybean, and various strains/isolates have been reported to date. However, the pathogenic differences and phylogenetic relationships of these SMV strains/isolates have not been extensively studied. In the present work, by first obtaining 18 new genomic sequences of Chinese SMV strains/isolates and further compiling these with available data, we have explored the evolution of SMV from multiple aspects. First, as in other potyviruses, recombination has occurred frequently during SMV evolution, and a total of 32 independent events were detected. Second, using a maximum-likelihood method and removing recombinant fragments, a phylogeny covering 83 SMV sequences sampled from all over the world was reconstructed and the results showed four separate SMV clades, with clade I and II recovered for the first time. Third, the population structure analysis of SMV revealed significant genetic differentiations between China and two other countries (Korea and U.S.A.). Fourth, certain SMV-encoded genes, such as P1, HC-Pro and P3, exhibited higher non-synonymous substitution rate (dN) than synonymous substitution rate (dS), indicating that positive selection has influenced these genes. Finally, four Chinese SMV strains/isolates were selected for inoculation of both USA and Chinese differential soybean cultivars, and their pathogenic phenotypes were significantly different from that of the American strains. Overall, these findings have further broadened our understanding on SMV evolution, which would assist researchers to better deal with this harmful virus.

LEAFY/FLORICAULA (LFY/FLO) is a family of plant-specific transcription factors, which plays an important role(s) in the regulation of floral organ formation and development. So far, LFY regulation on floral development in wild soybean has not been reported in the literature. In this study, the LFY gene, GsLFY, has been isolated from Glycine soja, and characterized with molecular and transgenic techniques. The cDNA for GsLFY gene is 1224 bp in length and contains an open reading frame encoding a polypeptide of 407 amino acids. Quantitative RT-PCR analysis shows that GsLFY is prominently expressed in various tissues, including roots, flowers and seeds. Among the four floral organs, GsLFY is expressed highly in sepals and stamens while weakly in the petals and carpels. Yeast two-hybrid experiments show that GsLFY possesses transactivation activity while transient expression analysis with Arabidopsis thaliana protoplasts shows that GsLFY protein is localized in the nucleus, supporting the notion that GsLFY is a transcription factor. The GsLFY transgenic tobacco plants flower about 29 days earlier than the wild-type plants, thereby providing a potential rationale for developing new soybean varieties with altered flowering time.

The insensitive response to photoperiod and temperature is an important quantitative trait for soybean in wide adaptation breeding. The natural variation in response to photoperiod and temperature was detected using 275 accessions of soybean [Glycine max (L.) Merrill] from China. Genome-wide association mapping, based on population structure analysis, was carried out using 118 SSR markers by the TASSEL GLM (general linear model) program. Nine SSR markers (P

Cytoplasmic male sterility plays an important role in utilization of crop heterosis. Screening of soybean for novel genes related to male sterility in soybean could provide a basis for studying the molecular mechanism of male sterility in plants. In this study, gene differential expressions between the cytoplasmic male-sterile line NJCMS1A and its maintainer line NJCMS1B in soybean were analyzed using cDNA-AFLP. A differentially expressed fragment, GmMF-T4A15, was isolated from large flower buds of NJCMS1B. By searching the soybean genomic library and PCR amplification, the cDNA full-length sequence of 1 311 bp was obtained and named GmMF1. The expression characteristics of GmMF1 were studied by semiquantitative real-time PCR and real-time quantitative PCR. The results showed that GmMF1 was expressed highly in flower buds of NJCMS1B. The deduced protein contains 436 amino acids and shows high similarity to members of the DUF620 protein family with unknown functions in other plant species. It is predicted that the protein encoded by GmMF1 is localized in the nucleus.

Soybean (Glycine max (L.) Merrill) is a salt-sensitive crop, and its production is severely affected by saline soils. Therefore, the response of soybean seeds to salt stress during germination was investigated at both physiological and proteomic levels. The salt-tolerant cultivar Lee68 and salt-sensitive cultivar N2899 were exposed to 100 mmol/L NaCl until radicle protrusion from the seed coat. In both cultivars, the final germination percentage was not affected by salt, but the mean germination times of Lee68 and N2899 were delayed by 0.3 and 1.0 d, respectively, compared with controls. In response to salt stress, the abscisic acid content increased, and gibberellic acid (GA₁+₃) and isopentenyladenosine decreased. Indole-3-acetic acid increased in Lee68, but remained unchanged in N2899. The proteins extracted from germinated seeds were separated using two-dimensional gel electrophoresis (2-DE), followed by Coomassie brilliant blue G-250 staining. About 350 protein spots from 2-DE gels of pH range 3 to 10 and 650 spots from gels of pH range 4 to 7 were reproducibly resolved, of which 18 protein spots showed changes in abundance as a result of salt stress in both cultivars. After matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of the differentially expressed proteins, the peptide mass fingerprint was searched against the soybean UniGene database and nine proteins were successfully identified. Ferritin and 20S proteasome subunit β-6 were up-regulated in both cultivars. Glyceraldehyde 3-phosphate dehydrogenase, glutathione S-transferase (GST) 9, GST 10, and seed maturation protein PM36 were down-regulated in Lee68 by salt, but still remained at a certain level. However, these proteins were present in lower levels in control N2899 and were up-regulated under salt stress. The results indicate that these proteins might have important roles in defense mechanisms against salt stress during soybean seed germination.

Leaf pubescence density (PD) is an important component for the adaptation of soybean [Glycine max (L.) Merr.] to drought-prone environment. Quantitative trait loci (QTL) controlling PD on the upper surface of leaf blade (PDU), PD on the lower surface of leaf blade (PDL), leaf wilting coefficient (WC) and rate of excised leaf drying (ELD) were identified using recombinant inbred lines (RILs) population from the cross between soybean cultivars kefeng1 and nannong1138-2 at the field soil drought stress stage from the mid-end of stem elongation to onset of flowering. A total of 20 QTLs were detected on molecular linkage groups (MLGs) A2, D1b, E, H, G and I with individual QTL explained 4.49–23.56% of phenotypic variation by composite interval mapping. The QTLs for PD on MLG H were mapped to near Ps locus while the QTLs on MLG D1b were located near Rsc-7. Three genome regions for PD and water status traits on MLGs A2, D1b and H were associated. This study revealed that leaf surface PD may play an important role in the soybean drought tolerance.

Trichomes (plant hairs) are present on nearly all land plants and are known to play important roles in plant protection, specifically against insect herbivory, drought, and UV radiation. The identification of quantitative trait loci (QTL) associated with trichome density should help to interpret the molecular genetic mechanism of soybean trichome density. 184 recombinant inbred lines (RILs), derived from a cross between soybean cultivars Kefeng 1 and Nannong 1138-2 were used as segregating population for evaluation of TDU (trichome density on the upper surface of leaf blade) and TDD (trichome density on the downer surface of leaf blade). A total of 15 QTL were detected on molecular linkage groups (MLG) A2, D1a, D1b, E and H by composite interval mapping (CIM) and among all the QTL, qtuA2-1, qtuD1a-1, qtuD1b-2, qtuH-2 qtuE-1, qtdD1b-2, and qtdH-2 were affirmed by multiple interval mapping (MIM). The contribution of phenotypic variance of qtuH-2 was 31.81 and 29.4% by CIM and MIM, respectively, suggesting it might be major gene Ps loci. Only 10 pairs of main QTL interactions for TDU were detected, explained a range of 0.2-5.1% of phenotypic variations for each pair for a total of 22.8%. The QTL on MLG D1b affecting trichome density were mapped near to Rsc-7 conditioning resistance to SMV (soybean mosaic virus). This study showed that the genetic mechanism of trichome density was the mixed major gene and polygene inheritance, and also suggested that the causal nature between trichome density and other agronomic traits.

From a preliminary experiment on 98 Chinese soybean varieties, 12 varieties with somatic embryogenesis frequency ranging from 0.0% to 85.7% were selected for further study in order to enhance the efficiency of somatic embryogenesis and plant regeneration. The effects of different mannitol concentrations, abscisic acid (ABA) concentrations, and embryo explant ages (sizes) were investigated. Significant differences in somatic embryogenesis were found among the 12 soybean varieties, with initiation frequencies varying from 22.1% to 89.0% under suitable mannitol concentration, and with N25281, N25263, and N06499 having the highest somatic embryogenic capacity. The results showed that all three factors were relevant for raising rates of callus initiation and somatic embryogenesis, but with differential responses among the genotypes. The treatment of 3.0% (w/v) mannitol, 5 mg l−1 ABA, and a 4- to 5-mm-sized explant was found to be optimal for somatic embryogenesis, generating the highest explant-based regeneration rate at 83.0%. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281. The above results provide a basis for efficient regeneration of soybean and are informative for the development of genetic transformation systems in Chinese soybean germplasm.

To investigate the genetic mechanism of Al-tolerance in soybean, a recombinant inbred line population (RIL) with 184 F(2:7:11) lines derived from the cross of Kefeng No.1 x Nannong 1138-2 (Al-tolerant x Al-sensitive) were tested in pot experiment with sand culture medium in net room in Nanjing. Four traits, i.e. plant height, number of leaves, shoot dry weight and root dry weight at seedling stage, were evaluated and used to calculate the average membership index (FAi) as the indicator of Al-tolerance. The composite interval mapping (CIM) under WinQTL Cartographer v. 2.5 detected five QTLs (i.e. qFAi-1, qFAi-2, qFAi-3, qFAi-4 and qFAi-5), explaining 5.20%-9.07% of the total phenotypic variation individually. While with the multiple interval mapping (MIM) of the same software, five QTLs (qFAi-1, qFAi-5, qFAi-6, qFAi-7, and qFAi-8) explaining 5.7%-24.60% of the total phenotypic variation individually were mapped. Here qFAi-1 and qFAi-5 were detected by both CIM and MIM with the locations in a same flanking marker region, GMKF046-GMKF080 on B1 and satt278-sat_95 on L, respectively. While qFAi-2 under CIM and qFAi-6 under MIM both on D1b2 were located in neighboring regions with their confidence intervals overlapped and might be the same locus. Segregation analysis under major gene plus polygene inheritance model showed that Al-tolerance was controlled by two major genes (h(2) (mg) = 33.05%) plus polygenes (h(2) (pg) = 52.73%). Both QTL mapping and segregation analysis confirmed two QTLs responsible for Al-tolerance with relatively low heritability, and there might be a third QTL, confounded with the polygenes in segregation analysis.

Pollen development is disturbed in the early tetrad stage of the YX-1 male sterile mutant of wolfberry (Lycium barbarum L.). The present study aimed to identify differentially expressed anther proteins and to reveal their possible roles in pollen development and male sterility. To address this question, the proteomes of the wild-type (WT) and YX-1 mutant were compared. Approximately 1760 protein spots on two-dimensional differential gel electrophoresis (2D-DIGE) gels were detected. A number of proteins whose accumulation levels were altered in YX-1 compared with WT were identified by mass spectrometry and the NCBInr and Viridiplantae EST databases. Proteins down-regulated in YX-1 anthers include ascorbate peroxidase (APX), putative glutamine synthetase (GS), ATP synthase subunits, chalcone synthase (CHS), CHS-like, putative callose synthase catalytic subunit, cysteine protease, 5B protein, enoyl-ACP reductase, 14-3-3 protein and basic transcription factor 3 (BTF3). Meanwhile, activities of APX and GS, RNA expression levels of apx and atp synthase beta subunit were low in YX-1 anthers which correlated with the expression of male sterility. In addition, several carbohydrate metabolism-related and photosynthesis-related enzymes were also present at lower levels in the mutant anthers. In contrast, 26S proteasome regulatory subunits, cysteine protease inhibitor, putative S-phase Kinase association Protein 1(SKP1), and aspartic protease, were expressed at higher levels in YX-1 anthers relative to WT anthers. Regulation of wolfberry pollen development involves a complex network of differentially expressed genes. The present study lays the foundation for future investigations of gene function linked with wolfberry pollen development and male sterility.

Soybean is an important source of edible oil, protein and protein diet. The breeding process of high quality soybean can be accelerated via employment of transgenic technology, by which the key genes for soybean quality traits could be directly manipulated. Thus, various soybean varieties could be bred to fulfill different needs for specific consumers. Here, we reviewed the contribution of transgenic technology to improvement of soybean qualities in recent years. We also introduce some newly developed safe transgenic technologies and hope this information could relieve some concerns on the GM food.

To research the mechanism of soybean reproductive development, we identified a number of flower development related genes in soybean by microarray hybridization. A gene predominately expressed in soybean flowers was chosen for further analysis. Through bioinformatic and RT-PCR approaches, the full-length gene was cloned from soybean flowers. The results of BLAST searching indicated that this gene encoded for an aldehyde dehydrogenase and was named as GmALDH3-1. GmALDH3-1 contains a complete open reading frame of 1485 bp in length, which encodes for a peptide of 494 amino acids. The product encoded by GmALDH3-1 shows 83% similarity and 68% identity with Populus tomentosa PtALDH3, respectively, and 39% and 59% with human ALDH3B. Phylogenetic analysis shows that GmALDH3-1 and other ALDH3 subfamily members are grouped into the same branch and GmALDH3-1 is close to PtALDH3 and Arabidopsis AtALDH3F1. Real-time RT-PCR analysis demonstrated that the highest expression level of GmALDH3-1 occurred in flowers, but the expression of this gene was almost undetectable in leaves and roots. We further analyzed GmALDH3-1 expression during the course of seed development based on publicly available microarray data and found that GmALDH3-1 was highly expressed in the seed endothelium, epidermis, outer integument, and hilum.

To investigate the genetic basis of drought tolerance in soybean (Glycine max L. Merr.) a recombinant inbred population with 184 F(2:7:11) lines developed from a cross between Kefeng1 (drought tolerant) and Nannong1138-2 (drought sensitive) were tested under water-stressed and well-watered conditions in field and greenhouse trials. Traits measured included leaf wilting coefficient, excised leaf water loss and relative water content as indicators of plant water status and seed yield. A total of 40 quantitative trait loci (QTLs) were identified: 17 for leaf water status traits under drought stress and 23 for seed yield under well-watered and drought-stressed conditions in both field and greenhouse trials. Two seed yield QTLs were detected under both well-watered and drought-stressed conditions in the field on molecular linkage group H and D1b, while two seed yield QTLs on molecular linkage group C2 were found under greenhouse conditions. Several QTLs for traits associated with plant water status were identified in both field and greenhouse trials, including two leaf wilting coefficient QTLs on molecular linkage group A2 and one excised leaf water loss QTL on molecular linkage group H. Phenotypic correlations of traits suggested several QTLs had pleiotropic or location-linked associations. These results will help to elucidate the genetic basis of drought tolerance in soybean, and could be incorporated into a marker-assisted selection breeding program to develop high-yielding soybean cultivars with improved tolerance to drought stress.

We cultured soybean immature cotyledons to induce somatic embryos and set up soybean somatic embryo lines by culturing the induced somatic embryos in liquid medium on shaker. Regenerated plants of normal fertility were easily obtained with the cultures of various ages by culturing the somatic embryos on differentiation media. DNAs were isolated from the embryogenic cultures after 5, 9, 15 or 17 months' suspension and from 42 plants regenerated from somatic embryos of various culturing ages. 102 SSR markers covering soybean genome almost evenly were chosen to check variation of microsatellite DNA during suspension culture and differentiation. Among the eight DNA samples of soybean somatic embryos of various ages and 42 DNA samples of regenerated plants, there was no any variation of the major bands of the 102 SSR markers during one year's subculturing and differentiation. There were only six weaker subsidiary bands of five SSR markers among the 102 SSR markers added in four of the fifty DNA samples. Two of them happened to the same regenerated plant differentiated from the 9-month embryogenic cultures. Three happened to the two DNA samples from somatic embryos irregular in morphology of the 5-month embryogenic cultures. The last subsidiary band variation happened to a DNA sample of the 17-month embryogenic cultures. The results show that stable microsatellites were maintained during the suspension culture and differentiation while we made the cultures highly embryogenic potential and easy to regenerate.

Three crosses between NG94-156 (brachytic stem) and three varieties (normal stem) were made, and F2 segregative population and two recombined inbred line populations(F(7:8)) were obtained. Genetic analysis indicated that the brachytic stem of NG94-156 was controlled by two duplicate recessive genes. In searching for RAPD marker linked to the genes controlling brachytic stem, 260 RAPD primers were applied to screen four parents of three combinations and RIL. Polymorphic bands revealed by the primer S-506 exhibited the best repeatability among all primers. Linkage analysis indicated the genetic distance between S-506(1600) and brachytic stem gene was 6.94 cM.

The recombinant inbred line (RIL) population, NJRIKY with 184 families derived from a cross Kefeng No.1 x Nannong1138-2 was used in mapping QTLs of root weight of soybean. Based on the linkage map constructed by Wang, with the software Cartographer V. 1.21 of the composite interval mapping procedure, three QTLs of root weight were mapped on N3-B1 and N6-C2 linkage group. The left telomere distance of rw1 was 66.31cM on N3-B1 linkage group, and those of rw2 and rw3 were 169.91cM and 179.71 cM, respectively, on N6-C2 linkage group. The former was located in A520T approximately ACCCAGO5 and the latter were overlapped with OPW13 and ACGCATO6, respectively. Their LOD values were 10.34,4.01 and 3.15, respectively. The QTLs of the root weight explained 26.3%,9.2% and 6.8% of the total variation, and their additive effects were -0.514,-0.303 and -0.260, respectively.

While there is linkage between molecular marker and trait controlled by two duplicate genes in the DH or RIL population, the recombination rate (RR) between molecular marker and one gene controlling the above trait may be estimated by the maximum likelihood method. Moreover, the standard deviation of RR was also obtained in this paper. Finally, the results from Monte Carlo simulation with 3000 replications showed that the unbiasedness of RR for various sample size and RR was good, and the variation of the estimated value of RR decreased with the increase of sample size or RR.

Soybean mosaic virus (SMV) is one of the most prevalent pathogens that impact the soybean world widely. Previous reports showed that most of the resistances were controlled by one pair of dominant genes. In this study, a soybean RIL population NJRIKY derived from Kefeng 1 x Nannong 1138-2 was used to study the inheritance of resistance to five SMV strains (Sa,Sc-8, Sc-9, N1, and N3) and mapping of resistant genes. Kefeng 1 is resistant to all five SMV strains while Nannong 1138-2 is susceptible to all five SMV strains. The parents and RIL populations were planted in green house and five SMV strains were inoculated on different populations. The results showed that each ratio of the number of resistant families to that of susceptible families was consistent with 1:1 for the five strains. This indicated that the resistance to each of the five strains was controlled by one dominant gene, respectively. RFLP and SSR markers were used to analyze the RIL population, Mapmaker/Exp 3.0b was used to study the linkage between markers and the resistant genes. Through linkage analysis, Rsa was found linked Rn1, Rn3 and Rsc9 with 21.4 cM, 23.5 cM and 35.3 cM, Rsc8 was found to be linked only Rn1 with 35.8 cM. Multi locus analysis showed that the order and intervals of the five resistance genes were Rsc8-35.8 cM-Rn1-10.3 cM-Rn3-21.5 cM- Rsa-35.8 cM-Rsc9. According to the result of RFLP and SSR analysis, a genetic map was constructed which consisted of 256 markers that covered 3050.9 cM and converged into 22 linkage groups. The five resistance genes were mapped on the linkage group N8-D1b + W. The RFLP markers A691T, K4771, LC5T were found linked to the resistant genes Rn1 and Rn3 with distances of 15.04 cM, 17.82 cM, 15.37 cM, 16.14 cM, 17.82 cM, 16.58 cM.

A recombinant inbred line (RIL) population, NJRIKY, which was derived from the cross Kefeng 1 xNannong 1138-2, was used to constructed the genetic linkage map. Larval weight and pupae weight of cotton worm [Prodenia litura (L.) Fabricius] were examined and used as indicators of resistance. Based on the linkage map constructed with SSR markers of this RIL population, one QTL responsible for larval weight was mapped on linkage group G20-O and the position was 31.91 cM. The QTL's additive effect was 0.0408 and explained 11.74 of the total variation of the larval weight. Two QTLs associated with pupae weight were mapped on linkage group G8-D1b+W and G17-L and the positions were 14.71 cM and 0.01 cM, respectively. The QTLs' additive effects were -0.0139 and 0.0103 ,which explained 11.30 and 6.36 of the total variation of larval weight, respectively.

CEL I, extracted from celery, is the first known eukaryotic nuclease that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. It is a key enzyme for TILLING research. Here we reported a crude extraction method and activity assay of CEL I. Incision at mismatches of single nucleotide suggested that CEL I can effectively detect DNA at G-->A base substitution and the result can be obtained from an ABI377 Sequencer. Therefore, the extracted enzyme can be used in TILLING.