| Puroindoline protein(PINA and PINB protein),is the main protein governing the wheat hardness,which is abundant in the aleurone layer and endosperm cells of mature seeds.PINA protein,the major component of PIN proteins,can affect the formation of wheat hardness during seed development and effectively inhibit the growth of many plant pathogens,bacteria and fungi.In this research,mature wheat Pina gene sequence was amplified by PCR and heterologous expressed and purified in Escherichia coli using GST and HIS tag.Flow cytometry,fluorescence microscopy,scanning electron microscopy,transmission electron microscopy,laser confocal microscopy and western Blot were employed to investigate the inhibitory effects of PINA protein on A.flavus.The main results and contents of this study were as follows:(1)The genomic DNA of wheat(Yangmai 15)was extracted and the mature Pina gene sequence of 363 bp was amplified by PCR.Pina and pGEX4T-3 plasmid were digested by restriction enzyme Not I and EcoR I.After ligation with T4 DNA ligase,the recombinant plasmid pGEX4T-3+Pina was transformed into E.coli DH5?competent cells.The sequence of GST-Pina gene with length of 1063 bp was amplified with the template recombinant plasmid pGEX4T-3+Pina by PCR.Recombinant plasmid pET28A(+)+GST-Pina was acquired by double digestion with Xho I and Hind III and conjugation and then it was transformed into E.coli BL21(DE3)competent cells for expression.The PINA protein was expressed mainly in the form of insoluble inclusion body and was obtained by denaturation,renaturation and purification on Ni column.The antifungal activity against A.flavus was carried out.128 ?g/mL PINA protein almost completely inhibited spore germination in liquid medium of DPY and PDB.When the protein concentration was 42.42 ?g/mL,the antifungal effect was remarkable on the PDA medium.The antifungal effect against A.flavus was notable when 960 ?g PINA protein applied in the 10 g corn meal medium.(2)The mechanism of the antifungal effect against A.flavus spores by PINA protein was investigated in this study.Spores treated with 128 ?g/mL PINA protein were uneven,deformed and atrophied at 12 h.Additionally,the cell wall dissolved and blurred,cytoplasmic wall separated and the organelles were destroyed after 24 h treatment.Propidium iodide(PI)staining illustrated that the intracellular spore structure changed significantly,indicating cell membrane damage.5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide(JC-1)staining indicated decreased mitochondrial membrane potential.Large nuclear condensation and DNA fragmentation were also detected by 4'6-diamidino-2-phenylindole(DAPI)staining.A.flavus spores were cultured in PDA medium containing PINA protein(the final concentration was 22 ?g/mL and 40 ?g/mL).After 48 hours,the total RNA of mycelia was extracted and the cDNA was retrieved.RT-PCR was utilized to analyze the expression level of FKS1,ROT1,GEF1,TRK1,brlA,FPS1.The results indicated that FKS1,ROT1,GEF1,TRK1 were up-regulated while brlA,FPS1 were down-regulated.Most of the up-regulated genes were related to cell wall synthesis(FKS1,ROT1)and the cell membrane interrelated proteins(GEF1,TRK1)monitoring.The antifungal effect of PINA protein on mycelia was also carried out.The surface of mycelia without protein treatment was smooth,round and non-atrophic.Mycelia treated with 32 ?g/mL and 64 ?g/mL PINA protein was rough,wrinkled and atrophic.When the protein concentration was 96 ?g/mL,holes were found on the surface of mycelia.PI,JC-1 and DAPI staining showed that the fluorescence was continuously enhanced,with the increased protein concentration from 32 ?g/mL,64 ?g/mL 96 ?g/mL,and 128 ?g/mL,indicating that cell membrane was damaged,mitochondrial membrane potential was decreased and DNA was fragmented and condensed.In this study,the effects of PINA protein on A.flavus was preliminarily studied,which laid a foundation for molecular breeding of storage-tolerant wheat and the development of new storage fungicides. |
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