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Characteristic analysis of transformants in T-DNA mutation library of Monascus ruber

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Abstract

Monascus ruber, a red mold species, has been widely used in the fields of food and medicine. In this research, we transformed Monascus ruber spores using Agrobacterium tumefaciens as a tool for random insertional mutagenesis with the hygromycin phosphotransferase gene as the selected marker. Three types of mutants including citrinin-producing mutants, mutants with abnormal aerial hyphae and pigment change mutants were screened for molecular analysis. Southern blot analysis showed that more than 83.3% of transformants contained single T-DNA insertions. The genomic DNA segments of the transformants flanking the T-DNA could be amplified from their left borders with TAIL-PCR. Homologous comparison using the Blast tool showed that none of the isolated DNA sequences had any similarity to each other, suggesting that the T-DNA was randomly integrated into the fungal genome, which provided the hypothetical reason for the variant phenotypes of the transformants. The successful creation of transformants with a single T-DNA tag insertion may help us to clone functional genes related to the metabolism and differentiation of Monascus spp., which will greatly facilitate the molecular analysis of this important fungus and the improvement of strains at the genetic level.

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Abbreviations

ATMT:

Agrobacterium tumefaciens-mediated T-DNA transformation

TAIL-PCR:

Thermal asymmetric interlaced PCR

GABA:

γ-Aminobutyric acid

SON-PCR:

Single oligonucleotide nested PCR

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Acknowledgments

Thanks are due to Dr. Daohong Jiang for his technical assistance and for kindly providing pTFCM for this experiment. This work was financially supported by the National High Technology Research and Development Program of the People’s Republic of China (863 program:No2006AA10Z1A3) and the Financial Aid Program for New Century Talents by the Ministry of Education of the People’s Republic of China (NoNCET-05-0667)

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  1. College of Food Science and Technology, Huazhong Agricultural University, Hongshan District, 430070, Wuhan, Hubei, People’s Republic of China

    Yanchun Shao, Yuedi Ding, Ying Zhao, Sha Yang, Bijun Xie & Fusheng Chen

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  1. Yanchun Shao
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  2. Yuedi Ding
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  3. Ying Zhao
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  4. Sha Yang
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Correspondence to Fusheng Chen.

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Shao, Y., Ding, Y., Zhao, Y. et al. Characteristic analysis of transformants in T-DNA mutation library of Monascus ruber . World J Microbiol Biotechnol 25, 989–995 (2009). https://doi.org/10.1007/s11274-009-9977-6

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  • DOI: https://doi.org/10.1007/s11274-009-9977-6

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