小麦病程相关蛋白wheatwin 1的表达及其对黄曲霉生长及产毒的影响

    EXPRESSION OF A PATHOGENESIS-RELATED PROTEIN,WHEATWIN 1,FROM WHEAT AND ITS EFFECTS ON GROWTH AND MYCOTOXIN PRODUCTION OF ASPERGILLUS FLAVUS

    • 摘要: 黄曲霉在储粮中的大量生长可严重危害粮食的储藏品质甚至产生真菌毒素。研究表明,小麦籽粒中含有多种病程相关蛋白,具有较强储藏霉变抗性的小麦品种胚部含有较多的病程相关蛋白,但其对储粮霉菌的生长和产毒的影响尚不清楚。通过PCR手段扩增从小麦基因组DNA中扩增出了病程相关蛋白wheatwin 1基因并将其在大肠杆菌中进行了表达,对wheatwin 1蛋白进行复性和纯化后分析其对黄曲霉生长及产生毒素的影响。结果表明编码成熟wheatwin 1蛋白的DNA序列长度为378 bp,将其在大肠杆菌中与谷胱甘肽转移酶融合表达可获得包涵体蛋白。将包涵体蛋白复性并利用Thrombin酶切后获得了具有核酸酶活性的wheatwin 1,其可降解黄曲霉RNA;进一步研究表明100 g/mL wheatwin蛋白可显著抑制黄曲霉孢子的萌发和黄曲霉毒素的积累。为进一步揭示wheatwin蛋白对黄曲霉生长和产毒的机理研究奠定了基础。

       

      Abstract: Aspergillus flavus,a common pathogenic fungus found in tropical and subtropical climates,contaminates crops (corn,wheat and peanuts) post-harvest in favorable environmental conditions and can produce carcinogenic mycotoxins known as aflatoxin,which poses a serious threat to food quality and human health.It has been reported that several pathogenesis-related proteins exist in wheat kernel and the wheat cultivars that have higher resistance to storage fungi contain more pathogenesis-related proteins in their embryos.However,the effects of these pathogenesis-related proteins on the growth of storage fungi and mycotoxin production still need to be further demonstrated.In present study,the gene of wheatwin 1,encoding a pathogenesis-related protein,was cloned from wheat genomic DNA using PCR technique,inserted in to plasmid pGEX-4T-3 and expressed in Escherichia coli Origami (DE3) strain.After renaturation and purification of wheatwin 1,the inhibitory effects on growth of A.flavus and mycotoxin production were investigated.The results showed that the gene sequence encoding mature protein of wheatwin 1 is 378 bp and the inclusion body was obtained through the fusion expression of wheatwin 1 and glutathione S-transferase (GST) in E.coli.The inclusion body was refolded and then was digested by thrombin to obtain biologically active wheatwin 1,which exhibited ribonulcease activity and could hydrolyze RNA of A.flavus.The effects of wheatwin 1 on the growth of A.flavus and aflatoxin B1 production indicated that 100 μg/mL wheatwin 1 could significantly inhibit the germination of spore as well as accumulation of aflatoxin B1,which provided experimental basis for further study of inhibitory mechanisms of wheatwin 1 on the growth of A.flavus and mycotoxin production.

       

    /

    返回文章
    返回