利用PCR-RFLP方法鉴别黄曲霉毒素产毒菌株

doi:10.3864/j.issn.0578-1752.2014.18.015

摘要/Abstract

摘要： 【目的】开发一种精准度高、灵敏性好、简便快捷的鉴别黄曲霉毒素产毒菌株的方法，为粮食储藏中黄曲霉毒素污染提供早期预警，为食品工业的质量安全控制提供技术保障，为黄曲霉毒素污染的生物防治提供无毒微生物来源。【方法】利用生物信息学方法分别以黄曲霉（Aspergillus flavus）、寄生曲霉（Aspergillus parasitieus）、米曲霉（Aspergillus oryzae）、酱油曲霉（Aspergillus sojae）的黄曲霉毒素合成基因簇中的关键调控基因aflR及其启动子序列为研究对象，采用PCR-RFLP方法对黄曲霉毒素可能产生菌株进行鉴别分析研究；为进一步验证PCR-RFLP方法对产毒菌株区分结果，利用大豆培养基对上述菌株进行发酵培养，并利用HPLC方法测定了菌株产毒能力。【结果】生物信息学分析结果表明黄曲霉毒素可能产生菌株的aflR高度同源, 但启动子和结构基因的部分碱基出现了规律性变异，并导致了限制性酶切图谱中的NheⅠ、PvuⅡ和HincⅡ等限制性酶切位点的改变；通过提取各菌株基因组DNA，PCR扩增得到aflR和启动子序列片段，测序得知片段长度分别为798 bp和 515bp；PCR-RFLP鉴定结果表明产生黄曲霉毒素菌株的aflR启动子片段经NheⅠ酶切后得到176 bp和339 bp 2个片段，不产毒菌株无该限制性酶切位点；试验进一步对黄曲霉菌和寄生曲霉菌aflR结构基因的分析发现，利用HincⅡ分别处理黄曲霉和寄生曲霉的aflR的结构基因，可以分别得到3个片段（387、250和161 bp）和2个片段（548、250 bp）；利用PvuⅡ分别处理黄曲霉和寄生曲霉的aflR结构基因，可以分别得到2个片段（652、146 bp）和3个片段（413、239和146 bp）；产毒试验表明，米曲霉菌和酱油曲霉菌不产生黄曲霉毒素，PCR-RFLP分析表明不产毒的黄曲霉菌的确丧失了产生黄曲霉毒素的能力；产生黄曲霉毒素各曲霉菌株之间产毒能力差异显著，不但产生的黄曲霉毒素总量高低相差几十倍，其产生的黄曲霉毒素组分也不尽相同。【结论】本研究阐明了在可能产生黄曲霉毒素关键基因aflR序列中存在的差异，通过PCR-RFLP方法快速鉴别黄曲霉菌株产毒与否，并且对黄曲霉和寄生曲霉两种曲霉进行了区分。黄曲霉毒素产生菌株鉴定方法的开发，将为粮油食品安全检测和质量控制，粮食食品生产过程的质量安全控制与监测提供基于分子水平的新型技术。

关键词: 黄曲霉毒素, aflR基因, PCR-RFLP, 产毒菌株鉴别

Abstract: 【Objective】The objective of this study is to develop an identification method of aflatoxin-producing strains with the advantages of high accuracy, good sensitivity, and convenience, which provide early warning to aflatoxin contamination of stored grain, provide technical support for the safety control in food industry and provide the non-toxic microbial sources for biological control of aflatoxin contamination.【Method】The key gene of aflatoxin synthesis, aflR, which belongs to Aspergillus flavus, Aspergillus parasitieus, Aspergillus oryzae and Aspergillus sojae, was analyzed by bioinformatics method. The potential strains producing aflatoxin were identified by PCR-RFLP technology. To verify these experimental results above, their toxin-producing ability was tested and analyzed by HPLC after these strains were cultured and fermented with soybean medium.【Results】The sequences of their aflR gene were analyzed in this research and the results showed high homology among their sequences. On the other hand, the partial bases of their aflR and promoter mutated regularly to induce variation of restriction sites NheⅠ, PvuⅡ, and HincⅡ. The genomic DNA were extracted from all these strains, the sequences of their aflR and promoter were amplified by PCR which includes 798 bp and 515 bp respectively. These promoters of aflR gene were digested by NheⅠand produced 176 bp and 339 bp two short fragments from toxin-producing strains. However, there was no restriction site of NheⅠin those of the non-toxin-producing strains. The results showed thatthe aflR of A. flavus and A. parasitieus were digested into three fragments (387 bp, 250 bp, 161 bp) and two fragments (548 bp, 250 bp) by HincⅡ, and those ofA. flavus andA. parasitieus were digested into two fragments (652 bp, 146 bp) and three fragments (413 bp, 239 bp, 146 bp) by PvuⅡ, respectively. The analysis of aflatoxin-producing demonstrated that both A. oryzae and A. sojae could not produce aflatoxin under the same cultural condition. It showed the same results that no aflatoxin produced by the strains in PCR-RFLP analysis. Among toxin-producing Aspergillus strains, the differences of their toxin-producing ability were remarkable. Not only their toxin yields rose a great many times in variation but their compositions were different.【Conclusion】This study illustrated the differences existed in the key gene aflR sequence among the strains. The active toxin-producing A. flavus and non-toxin-producing ones as well as toxin-producing A. flavus and A. parasitieus were identified by PCR-RFLP rapidly. The development of the method for identifing toxin-producing Aspergillus strains is probably useful for safety check and quality control not only in productive process of cereal and oil products but during food storage.

Key words: Aflatoxin, aflR gene, PCR-RFLP, identification of produced strains

孙长坡，常晓娇，伍松陵，沈晗. 利用PCR-RFLP方法鉴别黄曲霉毒素产毒菌株[J]. 中国农业科学, 2014, 47(18): 3675-3683.

SUN Chang-po, CHANG Xiao-jiao, WU Song-ling, SHEN Han. Identification of Aflatoxin Producing Strains by Using PCR-RFLP Method[J]. Scientia Agricultura Sinica, 2014, 47(18): 3675-3683.

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