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作者	单位
李添梦,胡小平，杜光源，范三红	(1.西北农林科技大学生命科学学院，陕西杨凌 712100； 2.西北农林科技大学理学院，陕西杨凌 712100； 3.西北农林科技大学植物保护学院，陕西杨凌 712100)

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中文摘要:

禾谷镰刀菌（F.graminearum）产生的单端孢霉烯族毒素会污染小麦籽粒，给人类及家畜健康带来严重威胁。 Tri8基因编码一种分泌型乙酰转移酶，催化毒素前体双乙酰DON（3,15-diAcDON）脱去一个乙酰基，形成3-乙酰DON（3-AcDON）或15-乙酰DON（15-AcDON）。本研究利用重叠PCR及无缝克隆技术构建了 Tri8基因敲除载体pOSCAR-△ Tri8，通过农杆菌介导的遗传转化方法筛选获得 Tri8基因敲除菌株PH-1-△ Tri8，最后利用液质联用仪对原始菌株和PH-1-△ Tri8菌株中的毒素类型进行分析。结果表明，对照菌株毒素以DON为主，未检测到3,15-diAcDON，而突变菌种毒素以3-AcDON和15-AcDON为主，并出现一定数量的3,15-diAcDON。本研究为后续3,15-diAcDON标准品的制备及利用，以及Tri8蛋白结构和体外功能研究奠定了基础。

英文摘要:

Trichothecene mycotoxins produced by Fusarium graminearum can cause wheat grain pollution and pose a serious threat to human and livestock health. Tri8 encodes an secretory acetyltransferase that catalyzes the removal of an acetyl group from the mycotoxin precursor 3,15-diacetyldeoxynivalenol(3,15-diAcDON) to form 3-acetyldeoxynivalenol(3-AcDON) or 15- acetyldeoxynivalenol(15- AcDON). In this study,the Tri8 knock-out vector pOSCAR-△ Tri8 was constructed by overlapping PCR and seamless cloning,and the Tri8 knock-out strain PH-1-△ Tri8 was obtained by Agrobacterium mediated genetic transformation. Finally,the chemotypes of mycotoxinproduced by the original strain and PH-1-△ Tri8 strain were analyzed by liquid chromatography-mass spectrometry. The results showed that DON was the main chemotype in the control strain,and 3,15-diAcDON was not detected,while 3-AcDON and 15-AcDON were the main chemotypes in the knock-out strain,and a certain amount of 3,15-diAcDON appeared. This study laid a foundation for the preparation and utilization of 3,15-diAcDON standards,as well as the study of Tri8 protein structure and its activity in vitro.

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