Construction And Study Of △ku70 Strain In Aspergillus Chevalieri Var.Intermedius

The "Golden Flower Fungus" Aspergillus chevalieri var. intermedius is the dominant fungus during the fermentation of Fuzhuan Tea, and associated with the quality, healthcare mechanism, and security mechanism of Fuzhuan Tea. The previous researches reveal that, unlike other fungi, the sexual and asexual reproduction of Aspergillus chevalieri var. intermedius are obviously regulated by osmotic pressure, so Aspergillus chevalieri var. intermedius can be used as a type strain in the studies of sexual and asexual generation mechanism of filamentous fungi. Based on those distinctive features, the molecular biology researches of Aspergillus chevalieri var. intermedius have both industrial values and theoretical values. Our team is the originator of the genomic researches of Aspergillus chevalieri var. intermedius. Agrobacterium tumefaciens mediated transformation has been successfully constructed, and several genes are cloned and researched through gene knockout by our team. However, the singleness selectable marker hph and the lower efficiency of gene knockout hinder the genomic researches of Aspergillus chevalieri var. intermedius. Therefore, it is necessary to find a new selectable maker and construct a genetically engineered strain which has an higher efficiency of gene disruption.In this paper, promoter PgpdA, G418-resistance gene nptⅡ, and terminator TtrpC were amplified from the plasmid pUR5750 firstly. Then the npt II gene expression cassette —PgpdA-nptⅡ-TtrpC, which could lessen virulence of G418 in filamentous fungi, was constructed in the helper vector pcDNA3.1(+). The binary vector pDHt/sknt was constructed after the npt II gene expression cassette replaced bar gene expression cassette in the binary vector pDHt/sk-bar. Finaly, the nptⅡ gene was successfully transformed Aspergillus chevalieri var. intermedius through Agrobacterium tumefaciens mediated transformation. PCR alysis confirmed that nptⅡ gene had been integrated into genome of transformants, and southern analysis showed that nptⅡ gene was single copy in transformants. The experiment of hereditary stability revealed that transformants of nptⅡ gene had well genetic stability. All of those results indicated that the binary vector pDHt/sknt was suitable for Agrobacterium tumefaciens mediated transformation, and npt Ⅱ gene could be used as a selectable maker in Aspergillus chevalieri var. intermedius.On the basis of previous researches, this study had constructed a ku70 gene disruption strain Δ ku70. Firstly, cDNA fragment of ku70 gene was cloned by the degenerate primers which was designed based on the conserved sequences of Ku70 proteins; and the sequence of ku70 gene and its flanking sequence were cloned by RACE or chromosome walking PCR. Then the ku70 gene disruption vector pDHt/sknt-D-U was constructed in binary vector pDHt/sknt, and the ku70 gene disruption strain A ku70 was isolated though Agrobacterium tumefaciens mediated transformation. Finally, phenotype and efficiency of gene disruption were compared betweenΔku70 strain and wild strain, veA gene and flbA gene were used for analysis the efficiency of gene knockout, and veA gene disruption vectors were constructed in this paper. Compared with wild strain, Δ ku70 showed slightly lower in growth rate, and displayed similar sensitivity to temperature, osmotic pressure, and chemical agents of ethyl methane sulfonate and H2O2. Otherwise, no apparent defect were observed. Therefore, we concluded thatΔku70 was suitable for investigating the function of new gene with high efficiency in Aspergillus chevalieri var. intermedius.