Construction, Expression And Antibacterial Activity Analyses Of Wheat Puroindoline A Artificial Mutants And Its Functional Analyses In Transgenic Wheat

Wheat grain hardness, one of the most important agronomic traits of wheat, directlydetermines the milling and baking quality of wheat. Ha locus, consisting of Puroindolinea (Pina) and Puroindoline b (Pinb) locating on chromosome5D short arm, controls thewheat grain hardness. Pina and Pinb are the major genes of hardness phenotype,encoding two13kDa basic proteins known as PINA and PINB. Both PINA and PINBproteins exist in the developing wheat endosperm and aleurone cells, binding to thesurface of starch granules, and of great abundance in mature seeds. Besides, Both PINAand PINB can inhibit the growth of many plant pathogens, such as gram-negativebacteria, gram-positive bacteria and fungi. PINA exhibits stronger antibacterial effectthan that of PINB.Bacterial resistance to antibiotics is mainly caused by the expression of antibioticresistance genes in bacteria. PIN proteins kill pathogens by destruction the plasmamembrane of specific lipid composition. As it is difficult for bacteria to replace the entireplasma membrane phospholipid composition, the plant antibacterial proteins are expectedto prevent the drug resistance as potential antimicrobial agents.Both PIN proteins contain a conserved cysteine backbone formed by five disulfidebonds. PIN mature proteins have a four α-helical based secondary structure. Between thefirst two α-helixes there is a tryptophan-rich domain containing several hydrophobic Trpresidues and basic Lys, Asp residues. Many evidences indicate that the tryptophan-richdomain (TRD) directly involves in grain hardness and its antibacterial function.In this study, Pina artificial mutants designated ABBC and ABBBC, with two orthree TRD, were constructed in order to obtain a more effective antibacterial protein andto explore the mechanism of PIN proteins determining grain hardness. Through thesecondary and tertiary structure prediction, the molecular structures of two PINA mutants were speculated preliminarily.Pina/ABBC/ABBBC were successfully constructed into the prokaryoticexpression vector pET43.1a and highly expressed in Rosetta-gami (DE3). RecombinantPina/ABBC/ABBBC were purified to obtain high-purity and high concentration ofrecombinant protein by means of Ni-NTA Affinity chromatography. Determination ofminimum inhibitory concentration and analyses of fluorescence microscopy were carriedout to evaluate difference in antibacterial activity of recombinant proteins. The resultsshowed that the antimicrobial activity of recombinant ABBC was significantly higherthan that of recombinant PINA, demonstrating that ABBC could be potentially effectiveanti-bacterial proteins. While the antimicrobial activity of recombinant ABBBC wassignificantly lower than that of recombinant PINA, showing no obvious application.These results indicate that this approach can improve the PINA protein antibacterialactivity by increasing the TRD, but when it contains3copies of TRD in one protein, theantibacterial activity decreased.Pina, ABBC and ABBBC were successfully constructed into the eukaryoticexpression vector pLRPT. Through the gold powder package and gene gun bombardment,the vectors were transformed into tetraploid wheat cultivar Ofanto. Several transgenicdescendants were survived. Using PCR analysis, Southern Blot test, SDS-PAGE andWestern Blot test, four transgenic T0generation plants were identified.