The Research On Soybean Storage Protein A_1aB_1b Subunit Which Was Identified As Has ?-Amylase Inhibitor Activity

Soybean storage protein is not only an important edible seed protein,but also a major component of soybean protein.Its nutritional value is rich and full,it also has excellent food and processing characteristics and has a broad application prospects.In recent years,our laboratory obtained a series of specific subunits of the missing material,which found that Guiyangzijing is a nature lack of A_1aB_1b.In this study,we chose the normal variety Nannongdahuangdou and the nature cultivar Guiyangzijindou,which is lack of A_1aB_1b.On the basis of previous studies,the follow studies were carried out.First,the soybean Gyl genes,which encode A_1aB_1b subunit,were isolated and analyzed the sequence;Second,Our study foused on subcellular localization of A_1aB_1b subunit and the laking molecular mechanisms;Third,Prokaryotic expression and purification of the soybean Gyl genesc were researched;fourth,Research on biological characteristics of recombinant proteins were on the agenda.Main results of this study are as follows.RNA was extracted from Nannongdahuangdou and Guiyangzijindou seed after 30 days flowering.Isolating and identificating the two species Gy1 genes,we analysis structural features of Gy1 gene and its encoded protein via bioinformatics and database.The full length ORF sequence of Nannongdahuangdou's Gyl gene is 1488 bp,DNA sequence is 2753 bp;the intact ORF of Guiyangzijindou's is 1161 bp,DNA sequence is 3607 bp.Compared with the normal variety Nannongdahuangdou and normal DNA sequence,cDNA sequence on NCBI,there was a long tail-to-tail repeat sequence,involving 584 bp Nucleotide of DNA sequence and 203 bp of cDNA sequence,in the Gyl gene of natural deletant Guiyangzijindou,which's A_1aB_1b subunit was null.Accordingly,the reading frame of translation of Nannongdahuangdou's Gyl gene would be changed and the mination codon advanced,which lead to the encoding amino acid sequence lack of 104 amino acids comparing with amino acid sequence of Nannongdahuangdou's A_1aB_1b subunit.In other words,Guiyangzijindou's A_1aB_1b subunit only has acidic peptide chain and part of the basic peptide chain,which is the fundamental reason for the missing subunit mechanism.A_1aB_1b protein manifested as instability,hydrophilic acidic protein whithout transmembrane.The normal A_1aB_1b protein contains 2.2%sulfur amino acids,in which methionine accounts for 1.4%;the A_1aB_1b-null protein contains 2.6%sulfur amino acids,in which methionine isl.6%.All of them are free of pyrrolysine and selenocysteine.Secondary structure prediction results show that normal A_1aB_1b protein contains alpha-helix?31.6%?,beta turn?8.29%?,extended strand?14.51%?and random coil?44.60%?;Conversely,the A_1aB_1b-null protein contains 29.7%?7.07%?19.19%?44.04%,respectively.The normal A_1aB_1b protein consists of two Integral specific "Cupin₁" domain,two nonspecific "Cupin₁" domain,a"PLN00212" conserved domains and two "ABD superfamily";the A_1aB_1b-null protein only has one specificity "Cupin₁" domain,a complete non-specific "Cupin₁" and"PLN00212" conserved domains,it also exist an incomplete "ABD superfamily".The whole A_1aB_1b protein is a non-symmetrical trimer,the A_1aB_1b-null protein only one monomer.Study on protoplasts subcellular localization shown that Nannongdahuangdou NGyl-GFP fusion protein exhibited clear signals in the chloroplast and protoplast membrane;Meanwhile,Guiyangzijindou GGy1-GFP fusion protein was mainly localized in the protoplast membrane,suggesting that the C-terminal sequence of A_1aB_1b were Significant to transportation.The deletion of C-terminal 10 amino acids of Guiyangzijindou A_1aB_1b lead to storage proteins secreted into the intercellular space;simultaneously,only a small number of 11S storage protein precursors are transported to PSV,which resulted in A_1aB_1b deficiency indirectly.The Gy1 gene of Nannongdahuangdou and Guiyangzijindou separately encoded a protein with 495 amino acids and 386 amino acids.Using prokaryotic expression methods,we obtained soluble expression of recombinant proteins in the cytoplasm of Escherichia coli.In order to obtain pure protein,HPLC was used.SDS-PAGE electrophoresis and Western blot identified that protein molecular weights were 59 kDa and 46 kDa.Protein optimal conditions:when OD₆₀₀=0.8,E.coli was induced;IPTG induction concentration within 0.5-1mmoL;induction temperature within 30-37?;The induction time is 4-6h;The shock speed of shaking is 220rpm.As for ?-amylase from fungal origin?a-amylase from aspergillus oryzae,?-amylase from bacillus subtilis?,the recombinant protein was not detected A_1aB_1b ?-amylase inhibitory activity.But,the a-amylase from mammalian sources?a-Amylase from porcine pancreas-type ?,?-Amylase from human salivary?was obviously inhibited by recombinant protein.A_1aB_1b has 58.0%inhibitory activity highly active against PPA.The effect of A_1aB_1b on HSA activities is 31.0%.The purified A_1aB_1b is without trypsin inhibitor function.But,treated by subtilisin protein,the inhibitory activity of A_1aB_1b is increased.Compared with six ?-amylase inhibitor types,which have been reported,sequence similarity of A_1aB_1b is low.And therefore presumably A_1aB_1b is a new class of storage protein inhibitors,due to storage protein have distinctive "Cupin" domain,also named "Cupin" type ?-amylase inhibitor.When the soybean storage protein A_1aB_1b a-amylase inhibitor's concentration was between 0.07 ?g/?L and 0.32 ?g/?L,its inhibitory activity against PPA and HSA is increased gradually.The inhibitory activity of the purified ?-amylase inhibitor against two?-amylase?PPA,HSA?showed optimum temperature at 37?,lower or higher than this temperature inhibition activity has declined.The inhibitory activity of the purified?-amylase inhibitor against two ?-amylase increased with the co-culture time extended,in which the highest inhibitory activity was obtained at 30 min.When co-culture time more than 30 min,the inhibition viability decreased.When the starch substrate concentration is 1.0%,the inhibitory activity of the purified ?-amylase inhibitor against PPA and HSA could reach the maximum activity.Substrate concentration over 1.0%,inhibition of viability decreased rapidly.When optimum heating temperature at 37?,soybean storage protein A_1aB_1b,which had the maximum inhibitory activity.At 65?,the inhibitory activity of A_1aB_1b would be disappear.As a health food,this imposes stringent heating requirements for Soybean protein powder.The biggest semi-inhibitory concentrations(ID₅₀)of a-amylase inhibitor against PPA and HSA were 2.03×10^-5mol/L and 4.7×10^-5mol/L,respectively.The purified soybean storage protein A_1aB_1b ?-amylase inhibitor had a significant impact on potato beetle?Leptinotarsa decemlineata?,in which obvioused diapause phenomenon.Our research provides a theoretical basis for the further development and utilization of this protein.