Meta-analysis of soybean amino acid QTLs and candidate gene mining

The composition and quantity of amino acids influence the protein content and nutritional value of soybeans and also have an important impact upon soybean quality. After integrating and proofreading 140 original QTLs associated with amino acid contentfrom soybase（http：//www.soybase.org/）, 138 QTLs were further analyzed to determine high-confidence QTL regions. Meta-analysis was first carried out using the Bio Mercator ver. 2.1 software, yielding 33 consensus QTLs. The consensus QTL confidence intervals（CIs） ranged from 0.07 to 19.85 Mb. Next, the overview method was used to optimize the CIs, and 57 ＂real＂ QTLs were mapped. Candidate genes in the consensus QTL regions were obtained from Phytozome and were annotated using the Gene Ontology（GO）, Kyoto Encyclopedia of Genes and Genomes（KEGG）, Swissprot, and gene annotation databases. Finally, 16 unpublished candidate genes controlling the content of five types of amino acids were identified with Blast. These results laid the foundation for fine mapping of soybean amino acid-related QTLs and marker-assisted selection.

Determination of basal ileal endogenous losses and standardized ileal digestibility of amino acids in barley fed to growing pigs

Background： Basal ileal endogenous amino acid（AA） losses（IAAend） and standardized ileal digestibility（SID） values of cereal grains, such as barley, are apparently underestimated when determined according to the nitrogen（N）-free method. Regression analysis between the dietary apparent ileal digestible content（c AID） and total crude protein（CP） and AA can be considered as alternative approach to obtain more accurate values for IAAendand SID of AA in cereal grains.Methods： Eight hulled barley genotypes were used, with barley being the only source of CP and AA in the assay diets. The diets contained 95 % as-fed of these eight barley genotypes each, ranging in CP content between 109.1 and 123.8 g/kg dry matter（DM）. Nine ileally T-cannulated barrows, average body weight（BW） 30 ± 2 kg, were allotted to a row-column design comprising eight periods with 6 d each and nine pigs. On d 5 and the night of d 6 of every period, ileal digesta were collected for a total of 12 h. The IAAend and the SID were determined by linear regression analysis between c AID and total dietary CP and AA.Results： There exist linear relationships between cA ID and total CP and AA（P 〈 0.001）. The IAAend of CP, Lys, Met, Thr and Trp amounted to 35.34, 1.08, 0.25, 1.02 and 0.38 g/kg DM intake（DMI）, respectively, which are greater compared to average IAAend determined previously under N-free feeding conditions. The SID of CP, Lys, Met, Thr and Trp was 90,79, 85, 79 and 86 %, respectively, and was greater when compared to tabulated values. Moreover, these SID values were greater than those reported in literature, based on correction of apparent ileal digestibility（AID） of CP and AA for their IAAendvalues. Summarized, the results of the present regression analysis indicate greater IAAendin barley-based diets compared to those obtained by N-free feeding.Conclusions： For low-protein feed ingredients like barley the regression method may be preferred over correction of AID values for their IAAenddetermined under N-free feeding conditions, as intercepts and slopes of the linear regression equations between cA ID and total dietary CP and AA provide direct estimates of IAAendand SID of CP and AA in the presence of the assay feed ingredient.

A Novel Method for the Quantitative Detection of Sericin Content in Silk Fiber Based on the Ratio of Aspartate to Alanine

The development of a method for the quantitative determination of the sericin content ratio(SCR)is urgently needed for silk refining and the purification of silk fibroin for biomedical applications.In this work,a series of sericin/fibroin mixed samples with known SCRs were prepared by mixing initial samples of extracted sericin and fibroin from Bombyx mori silk.Significant differences were found in the contents of characteristic hydrophilic amino acids abundant in sericin and hydrophobic amino acids abundant in fibroin,and several linear relation-ships of SCR associated with the content ratios of Ser/Ala,Asp/Ala,Lys/Ala,Asp/Gly and Ser/Gly were estab-lished by amino acid analysis.Subsequently,the linear equation expressing SCR(%)as a function of the Asp to Ala content ratio X(%)was established as SCR=2.5634X−12.5587(R 2=0.9972).The results indicated that the SCR of degummed silks calculated by the equation is more objective and effective than the results obtained by the traditional weight loss method.Our study provides a novel approach for the sensitive and quantitative detection of the sericin content within the detection limit in unknown silks,which can contribute to quality control in the silk production process.

Separation and Identification of Glutamine Peptides from Defatted Soybean Meal by Enzymatic Hydrolysis

Glutamine peptides were obtained from defatted soybean meal by enzymatic hydrolysis using a combination of Protamex~? and trypsinase. The results showed that the extent of hydrolysis and the concentration of Gln peptides in the hydrolysate were 22.02%and 6.05 mmol/L, respectively. The hydrolysates were fractionated by size-exclusion chromatography on a Sephadex G-15 into five major fractions(GelF1–GelF5). The peptide(GelF1) fraction with the highest glutamine peptide content(51.8%) was further evaluated to determine its molecular weight distribution. Most(92.37%) peptides were less than 1 000 Da. Glutamic acid and glutamine were the most abundant amino acids, accounting for up to 12.98% of the total amino acid content. In addition, the total amino acid content in GelF1 was higher than that in GelF2 and GelF3.

Identification in Lupin Seed of a Serine- Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Selection and determination of specific and protective antigens of Mycobacterium tuberculosis

Objective To select and identify the specific and protective antigens of Mycobacterium tuberculosis (M. tuberculosis) for searching a new approach to diagnose and treat tuberculosis Methods Extract and culture filtrates of M tuberculosis were obtained by ultrasonic treatment and millipore membrane filtration, respectively The protein samples were tested with monoclonal antibody (MAb) and patient sera The proteins showing positive reaction were sequenced on Beckman /LF 3200/peptide amino acid sequencer Results Proteins of M tuberculosis with molecular weight of 31?ku and 30?ku showed positive results when reacted with anti M tuberculosis MAb and sera of tuberculosis patients, but not with normal mouse serum and healthy human sera N terminal sequences of the 31?ku and 30?ku antigen were Ala Glu Val Asp Trp Leu Val Phe Ala Val and Phe Ser Arg Pro Gly Leu Pro Val Glu Try respectively Conclusion 31?ku and 30?ku proteins of M tuberculosis are immune protective proteins They play important or dominant roles in determination of immunorecognization