Monitoring the Production of Aflatoxin B(1) in Wheat by Measuring the Concentration of nor-1 mRNA

A real-time reverse transcription-PCR system has been used to monitor the expression of an aflatoxin biosynthetic gene of Aspergillus flavus in wheat. Therefore, total RNA was isolated from infected wheat samples, reverse transcribed and subjected to real-time PCR. In parallel all samples were analyzed by high-pressure liquid chromatography for aflatoxin B(1) production. The primer-probe system of the real-time PCR was targeted against nor-1, a gene of the aflatoxin biosynthetic pathway. By application of this method the nor-1 transcription was quantified during the course of incubation. After 4 days of incubation nor-1 mRNA could be detected for the first time. The amount of nor-1 mRNA increased rapidly, and the maximum was achieved after 6 days. Then, starting very slowly, the mRNA was degraded until day 8, and this was followed by a very fast degradation, reaching nondetectable levels at days 9 and 10. First traces of aflatoxin B(1)could be detected between the 5th and 6th day of incubation. The aflatoxin concentration reached its maximum after 9 days of incubation and remained constant for the whole period of observation. To ensure that differences in the nor-1 mRNA concentration were due to different expression levels, the expression of the constitutively expressed β-tubulin gene (benA56) has also been monitored. The expression of benA56 remained constant during the whole incubation time. As a parameter for fungal growth, the number of nor-1 gene copies was determined during the course of incubation. The numbers of nor-1 gene copies increased at the beginning of the incubation and reached a plateau at day 5. They correlate well with the viable counts albeit at a higher level.本研究采用实时逆转录聚合酶链式反应（real-time reverse transcription-PCR）体系，监测受黄曲霉（Aspergillus flavus）侵染的小麦中黄曲霉毒素生物合成基因的表达情况。为此，我们从受感染的小麦样本中提取总RNA（total RNA），经逆转录后开展实时PCR扩增。同时，通过高效液相色谱（high-pressure liquid chromatography）对所有样本的黄曲霉毒素B₁（aflatoxin B₁）产量进行平行分析。 该实时PCR的引物探针系统靶向黄曲霉毒素生物合成途径中的nor-1基因。依托该方法，我们对培养过程中nor-1的转录水平进行了定量检测。培养4天后，首次检测到nor-1信使RNA（mRNA）；其丰度迅速上升，并于培养第6天达到峰值。随后，mRNA以极慢的速率开始降解，直至培养第8天，此后降解速率显著加快，至第9天和第10天时已无法被检出。 黄曲霉毒素B₁的首批痕量产物可在培养第5至6天之间被检出，其浓度在培养9天后达到峰值，并在整个观测周期内保持稳定。 为确保nor-1 mRNA浓度的差异源于真实的表达水平差异，本研究同时监测了组成型表达的β微管蛋白基因（β-tubulin gene）benA56的表达情况。结果显示，benA56的表达在整个培养周期内始终保持恒定。 作为真菌生长的表征参数，本研究对培养过程中的nor-1基因拷贝数进行了定量。该基因拷贝数在培养初期有所上升，并于第5天达到平台期；尽管其数值高于活菌计数（viable counts）结果，但二者的变化趋势具有良好的相关性。