acidum[20]. A full-length cDNA clone of MaAC was amplified using Pyrobest DNA polymerase (TaKaRa, Japan) from a cDNA library of M. acridum established in our laboratory [21] with gene-specific learn more primers MaAC-F (5′- TTCCACGCCAAACCTCAA -3′) and MaAC-R (5′- AGCCAAGTTGTTTCGGTA -3′). The resulting PCR product was subcloned into the pMD19-T vector, and transformed into E. coli XL-Blue for determination by GenScript (Nanjing, China). To study the function of MaAC, an RNA interference (RNAi) vector was constructed. The click here partial sequence of MaAC (500 bp) was amplified

by MaAC-F (5′- GCGATACACGCCACAAGGACAAAGA-3′) and MaAC-R (5′-CCCAAGCTTACTACCAATCTCATCCACCTC-3′) from M. acridum MaAC cDNA. The resulting PCR product was cloned into pMD19-T (Takara, China) to form pMD19-MaAC. A fragment of MaAC was recovered from pMD19-MaAC by digestion with EcoRI and EcoRV and inserted into the vector pDPB [22]. The fragment PgpdA-MaAC-PtrpC

from pDPB was inserted at the site between HindIII and XbaI of pPK2-pB [23, 24] to form pPK2-pB-MaAC-RNAi. Transformation of M. acridum was mediated by Agrobacterium tumefaciens according to the procedure described previously [25]. Transformants were screened on Czapek-dox medium containing 80 μg/mL phosphinothricin (PPT) and incubated at 27°C for 8 d. Transformants were confirmed by PCR amplification of the RNAi cassette. Real-time quantitative reverse transcript (qRT-PCR) analysis To confirm the expression levels of MaAC, the wild type and MaAC-RNAi XAV-939 clinical trial transformants were grown in PD liquid culture for 2 d and the mycelia were collected and washed with sterile water. Total RNA was isolated using the SV Total RNA Isolation System (Promega, USA). The synthesis of cDNA and real-time RT-PCR were performed using the method described by Leng et al. [26]. Primers of MaAC-F (5′- GGACGAAGGACTTGACAGACC-3′) and MaAC-R (5′-CACAGCATCTCCAGACGAGG-3′) were used to detect MaAC expression levels. Determination of fungal growth To characterize the role of MaAC in vegetative growth,

the growth rate of the wild type and Thalidomide the RNAi mutants were analyzed using CellTiter 96® AQueous One Solution Assay (Promega, USA). In this study, the wild type or RNAi mutants were inoculated in PD liquid culture for 24, 30, 36, 48, 54, 60 and 72 h, respectively. CellTiter 96® AQueous One Solution Reagent (20 μL) and 100 μL culture fluid were directly added to the culture wells, the mixture was incubated for 2 h at 37°C, and then the absorbance was recorded at 490 nm with a 96-well plate reader. cAMP assay The MaAC mutant and the wild type were cultured in PD liquid culture for 36 h. After harvesting, 20 mg mycelia were collected and washed three times with sterile water, followed by treatment with 2 mL 0.01 M PBS. Samples were then lyophilized and dissolved in the mixture.