Enzymatic Characteristics Of Storage-Protein-type Alpha-amylase Inhibitor CL-AI And Its Molecular Mechanism Of Expression And Regulation In Chickpea(Cicer Arietinum L.)

Alpha-amylase inhibitor and amylases are formed effectively into enzyme-inhibitor complexes,and then the carbohydrate hydrolysis and digestion are blocked by the loss and reduction of activities of amylases.Alpha-amylase inhibitor can be used as a tool for prevention and treatment of high blood glucose,diabetes and high cholesterol and other diseases;On the other hand,it can be applied to control diet and reduce obesity in daily life care;Moreover,genes and products of cc-amylase inhibitor have an important application value in agricultural insect control and reducing insecticide to protect the ecological environment.The proteinaceous ?-amylase inhibitors found in plants are classified into six types:Kunitz-like?Knottin-like?Cereal-like?Lectin-like??-Purothionin-liken and Thaumatin-like.Chickpea(Cicer arietinum L.),which belongs to the Leguminosae Cicer,is the main source of protein for southern Asia,western,Eastern and Northern Africa human,who consider cereal as the staple food.At present,the chickpea ranking the third in legume crops is one of the large edible legume crops in cultivation area.A new type a-amylase inhibitor,belonged to storage protein,was purified and identified from chickpea seeds by ammonium sulfate precipitation(ASP),ion exchange chromatography(IEC)and reversed?phase liquid chromatography(RPLC)in our previous work.To reveal the biological characteristics and the molecular mechanism of expression regulation in chickpea seed of new type ?-amylase inhibitor,based on previous work,this study is focused on selection of chickpea with high a-amylase inhibitory activity,purification of a-amylase inhibitor from chickpea seeds,gene cloning,prokaryotic expression,analysis of conserved domains with inhibitory activity,the synthesis pathway and change in inhibitory activity in vivo and biological characteristics.The main results of the research are as follows:1.The screening of chickpea germplasm resources with high a-amylase inhibitory activity(W1?W7),and compare of the a-amylase inhibitory activities in crude solution and ammonium sulfate precipitations were well investigated and the inhibitory activity in W4 was found higher than W2,The Kabuli(W2)and Desi(W4)were selected respectively as the materials for separation and purification of?-amylase inhibitor from chickpea due to the high overall inhibitory activity.Ammonium sulfate precipitated protein 60%-80%and 80%-100%with higher inhibitory activities from W2 were merge red and applied to ion exchange chromatography.Seven protein peaks were eluted peaks,of which P4 has a higher inhibitory activity(54.54%);P4 peak was used for reversed phase liquid chromatography(RPLC),and 3 protein peaks were separated,of which the P ? peak had the highest inhibition activity(80.85%).The SDS-PAGE band of P ? was hydrolyzed by trypsin and peptides were detected by mass spectrometry.The alignment of results and protein database was implemented by the MASCOT software,and 3 peptides were identified be consistent with pea(Pisum sativum)Albumin-2 protein,so the purified protein was identified as plant chickpea lectin protein CL-A2(gi| 625987).The part of 60%-80%and 80%-100%form W4 with higher inhibitory activities were used for ion exchange chromatography respectively,and 5 protein peaks were eluted from ASP(60%-80%).of which peaks P1.1 and P1.2 had high inhibitory activities(81.16%,50.72%),and 5 protein peaks were separated from ASP(80%-100%),in which peaks P2.4 and P2.5 had higher inhibitory activity(65.24%,39.43%).The parts from ion exchange chromatography with higher activities were combined and applied to reversed phase liquid chromatography(RPLC),and 4 peaks were further separated from ASP(60%-80%),of which the peak ? and IV were the higher inhibitory activity protein peak(75.93%,81,48%),moreover 60 kDa proteins were detected in peak ? and IV peak,and another with size of 25 kDa was also in protein peak IV.5 peaks of ASP(80%?100%)were further separated,of which peak ?,? and V had higher inhibitory activities(60.38%,71.70%,60.38%),and a 60 kDa protein appeared in the activity peaks.Two kinds of protein with high inhibitory activities obtained and purified from ASP(60%-80%)and ASP(80%-100%)by RPLC separation(Protein A:25 kDa and Protein B:60 kDa)were hydrolyzed and detected by mass spectrometry.By searching and comparing database,protein A was indentified as chickpea lectin-like protein CL-A2(gi|625987),and the identification result is consistent with the W2 result.Two peptides,R.DFLEDALNVNR.R and K.SEGGLIET?rNPSNK.Q were identified from protein B and consistent with chickpea legumin protein Q9SMJ4,which was also according with preliminary laboratory results.Comparing of protein molecular size,the a-amylase inhibitor protein CL-Al we purified from chickpea was precursor of chickpea legunin Q9SMJ4 protein presumably.2.The gene sequences ofa-amylase inhibitor proteins CL-AI and CL-A2 in chickpea were cloned and the full-length ORF of CL-AIgene was 1491 bp,encoding 497 amino acids,ORF of CL-A2 gene was 693 bp and it encoded 231 amino acids.Analysis of comparison with the DNA sequence showed that the CL-AI gene contained 4 exons and no intron was contained in CL-A2 gene.Bioinformatics analysis indicated that,CL-AI gene sequence showed a high similarity with peas,vetch,alfalfa,soybean and other crops.CL-AI protein contained of a-helix(28.97%),p-tum(11.07%),extended strand(21.53%),and random coil(38.43%)in the structure.An N terminal signal peptide with length of 21 amino acids was existed,among which the signal peptide enzyme cutting sites was located in the No.21 and No.22 amino acids and the CL-AI was predicted mainly in the ion of secretory pathway protein(SP).CL-AI protein,which was classified as seed storage protein 11S,contained two Rm1C-like cupin domains.CL-A2 gene showed sequence similarity with pea,alfalfa,beans and other crops,and CL-A2 protein structure contained a-helix(27.83%),?-turn(16.52%),extended strand(26.09%),random coil(29.57%);There is no signal peptide in CL-A2 of which subcellular localization was in cytoplasm;supersecondary structure mainly consists of a Hemopexin-like domain,and was classified as Lectin-like protein.3.Sequence of CL-AI ORF without signal peptide and CL-A2,encoded the CL-AI protein subunits were inserted into the prokaryotic expression vector respectively,and then transformed into E.coli expression strain BL21(DE3).CL-A2,CL-AI,CL-AI-? solube-fusion protein and CL-AI-? inclusion fusion protein were obtained from induced protein expression and using Ni column affinity chromatography and CL-AI-? inclusion body protein was taken renaturation in vitro.Determination of inhibitory activities of prokaryotic expression fusion protein against human salivary amylase(HSA)respectively showed that CL-A2,CL-AI,CL-AI-a,CL-AI-? had inhibitory against HSA,with inhibitory activities were 56.47%,67.06%,70.59%,67.65%respectively,but there was no significant difference between them.4.The experiments of deletion conserved Cupin domain of CL-AI protein and prokaryotic expression showed that the inhibitory activities of CL-AI,with Cupin-deletion in ?-subunit??subunit respectively,against HSA decreased(36.90%,44.51%)and the difference was significant.Moreover,CL-AI protein with complete deletion of Cupin domain had a lost inhibitory activity(1.42%).These results indicated that Cupin structure of the CL-AI protein plays an important role in inhibiting activity.CL-AI-? protein with amino acid point mutation results showed that the Gly75 mutation affected inhibitory activity of CL-AI-? protein with a significantly decrease,of which inhibitory activity retained only 0.90-7.69%.The inhibitory activity against HSA of CL-AI-? with Pro123 mutation of protein was significantly decreased when content was low(0.08 ?g/?L)with the activity(38.91%),But with the increase of protein content,its inhibitory activity rose and the inhibitory activity of mutant protein was significantly higher than the control(47.96%)at high content(0.24 ?g/?L).The effects of mutation of Asn132,Pro123 and Asn132 influenced inhibitory activity of CL-AI-? were similar mutation of Pro123.These results showed the conserved amino acid Gly75 in the first conservative region of Cupin domain plays an important role in the inhibitory activity of CL-AI-?,and the Pro123 and Asn132 mutations in the second conserved region would change the enzymatic properties of CL-AI-?.Glycinin G1 and Glutelin,from soybean and rice respectively,were selected to clone sequence and prokaryotic expression,due to sequence differences and structure similarity.Soluble fusion protein Glycinin GI and inclusion body Glutelin were purified and obtained,and the renaturation inclusion body was taken place subquently.Determination of inhibitory activity against HSA showed that Glycinin GI and Glutelin have inhibitory effect on HSA,so we speculated that the biological activity similarity of Glutelin,Glycinin GI and CL-A1 was caused by the similarity in Cupin domain in the 3D structure.5.In vitro,two sulfide bonds reconstruction showed that the CL-AI and CL-Al-p protein can itself,but not easy to,form two disulfide bonds,and the inhibitory activities could be increased due to formation of two disulfide bonds.Compared with the CL-AI and CL-AI-?,CL-AI-? itself was more easily to form two sulfide bonds,which reduced inhibitory activity.CL-AI(a+p)tended to form two disulfide bonds with CL-AI-?,CL-AI-?,which caused decrease in inhibitory activity obviously.CL-AI and CL-AI-? had the strongest ability in two disulfide bonds formation and also resulted in the decreased inhibitory activity.Two disulfide bonds formation among CL-AI(?+?),CL-AI-? and CL?AI-? had a negative effect on protein a-amylase inhibitory activity.6.Study on CL-AI gene at transcription level indicated that,during seed development,CL-AI gene are transcribed as a single mRNA,which encoded CL-AI precursor protein at the transcriptional level and other pathway encoding other proteins didn't exist;the mRNA was not sheared into mRNAs,which encoded the ? and ? chains.Study on CL-AI gene at translation level showed that CL?AI protein precursor was cut off the signal peptide and sheared into CL-AI-? and CL-AI-? simultaneously,then these two peptide chains formed heterologous dimer by two disulfide bonds and stored in the body.The formation of two disulfide bonds occured after CL-AI precursor was sheared,instead of before shearing.The subcellular localization results showed that the CL-AI protein was mainly synthesized in the cytoplasm,with the gradual protein synthesis accumulation,Which was transported to protein storage type vacuoles.Results indicated that precursor of CL-AI was sheared into ? and ?subunits,which remained inhibitory activities in protein synthesis process,but the inhibitory activity was reduced or even dispeared,due to the dimers of two subunits linked by two disulfide bonds.7.CL-AI-? protein derived from Prokaryotic expression and purification had a certain inhibition against amylases from human saliva(HSA),porcine pancreas(PPA),corn(MA),Bacillus subtilis(BA),Aspergillus oryzae(OA),with the inhibitory activities of 71.58%,74.32%,67.74%,53.45%and 71.11%,respectively.Inhibitory activity of CL-AI-? was mainly affected by temperature,and had certain heat stability.The most suitable pH was range of 7.0-9.0,incubation time and substrate concentration of starch also could affect inhibitory activity.With the increase of protein concentration,the inhibitory activity of CL-AI-? against various ?-amylase of decreased gradually.The CL-AI-? protein had strong inhibitory activities against amylases from the midgut and blood of Helicoverpa armigera Hubner,with inhibitory activity of 68.42%and 96.21%respectively,also had inhibitory activity of 39.09%against saliva amylase.The inhibitory acitvities of CL-AI-? against amylase from the midgut and salivary of Leptinotarsa decemlineata larvae were 55.26%and 28.71%respectively,but there was basically no inhibition against amylaseof blood.Leptinotarsa decemlineata larvae feeding test showed CL-AI-?,CL-AI and CL-AI(R)larvae made a lost weight of Leptinotarsa decemlineata larvae(4.38%,0.83%and 7.77%respectively),and larvae feeding with CL-AI-? protein had a slight increased with rate of only 0.91%,compared with the control whose body weight increased by 50.62%.The effect of CL-AI-? on seed germination of maize,wheat and rice showed that there was no obvious effect on plant seeds germination rate,dry weight and fresh weight after germination,but CL-AI-? could slow down the growth rates of root and shoot length,indicating delayed growth after seeds germination.