An intron in an AtGUS genomic sequence was removed via three-step polymerase chain reaction (PCR) to express the gene in E. coli BL21 (Figure 2A ). According to the database of A. terreus NIH2624, a primer set containing P1(59-CCGTACgTAATGCT-GAAGCCCCGACAAACACCTT-39) and P2(59-CATGCGG-CCGCTTAAGCGCCAAATAGGAAGTATAGT-39) was designed to obtain the sequence with an intron from the A. terreus Li-20 genome under the following conditions: 94uC for 10 min, 30 cycles of 94uC for 1 min, 58uC for 1 min, 72uC for 2 min, and a final extension at 72uC for 10 min with Ex Tag (TaKaRa, Japan). After ligated into PMD19-T, a primer set containing P3(59-CACTCCACCGTGTTTTCAATGTATGAGCTGCAGC-39) and P4(59-CCGGCTTCGCAGCTATGTGTCTTGAGCATC-39) was used for the second PCR, and the reaction was performed by Pfu polymerase (Shenggong, China) under the following conditions: 94uC for 10 min, 30 cycles of 94uC for 1 min, 55uC for 1 min, and 72uC for 5 min. The fragment amplified in the second PCR was ligated by T4 DNA ligase after a terminal phosphation with T4 polynucleotide kinase (Takara, Japan), and the positive clones were screened in an LB plate with 100 mg/mL ampicillin. The primer set containing P1 and P5(CATGCG-GCCGCTTAACTCCACCGTGTTTTCAATGTATG-39) was used for AtGUS(-3t) under the same conditions as those of P3 and P4.