## 2 Materials and Methods
### Construction of a Homologous Integrated Expression Vector  
#### 2.3.1. Cloning of the _lip_ Gene  
_P. chrysosporium_ total RNA was extracted using Trizol reagent following the manufacturer's instructions. Based on the full _lip_ cDNA sequence of the _lip_ gene (GenBank-K accession Number: M74229.1), primers Lip-R1+1 and Lip-F1+1 were designed. The cDNA was generated from the total RNA extracted from _P. chrysosporium_ using the BcaBEST\({}^{\text{TM}}\) RNA PCR kit Ver.1.1 (RR023A, TAKARA) with oligo dT primers. The _lip_ cDNA was amplified via PCR with the upstream primer Lip-R1+1 and the downstream primer Lip-F1+1. The amplification was carried out under the following conditions: the first step was at 94 \({}^{\circ}\)C for 1 min, followed by 30 cycles of 94 \({}^{\circ}\)C for 30 s, 51 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 2 min, and the final extension was carried out at 72 \({}^{\circ}\)C for 5 min. The purified PCR products were cloned into the pMD\({}^{\text{TM}}\)19-T vector Cloning Kit (6013, TAKARA), and transformed into _Trans_1-T1 Phage Resistant Chemically Competent Cell (CD501-02, TransGen Biotech, Beijing, China) to construct the recombinant clone. The PCR-positive clones were sequenced, and the cloned sequence was confirmed through comparing the sequence within the recombinant construct with the sequence published in GenBank. 3.2. Cloning of _C. jadinii_ GAP Promoter Sequence, GAP Terminator Sequence, Cycloheximide Resistance Gene (_CYH_) and 188 rDNA Fragment  
Primers were designed based on the _C. jadinii_ GAP gene promoter sequence-_GAP-p_ (ID: FJ664342.1), _GAP_ gene terminator sequence-_GAP-t_ (ID: FJ664343.1) and _185 rDNA_ gene sequence (ID: AB054569.1) published in GenBank and the sequence characteristics of the pBR322 plasmid. Appropriate restriction sites (GAP-p1/GAP-p2 (_Eco_RV/_Xbal_), GAP-t1/GAP-t2 (_Xbal_/_Bam_HI), 185 rDNA-P3/185 rDNA-R3 (_Eco_RV and _Eco_RV)) were also introduced (Table 1). The _C. jadinii_ genome was used as the template. Yeast GAP-p, GAP-t and 188 rDNA fragments were obtained via PCR amplification with the corresponding \begin{table}
\begin{tabular}{c c c c c} \hline  & **Primers** & **Primers Sequence (5\({}^{\prime}\) to 3\({}^{\prime}\))** & **Size (bp)** & **Source** \\ \hline \multirow{4}{*}{1} & GAP-p1 & GGATATCTTACAGCGAGCACTCAA & & \\  & GAP-p1 & _Eco_RV & 975 & C. _jadiii_ \\  & GAP-p2 & GCICTAGAGTTGTTTGTGT & & \\  & _Xbal_ & & & \\  & GAP-t1 & CTAGCTATAGCTTTTTAT & & \\
2 & _Nh_eI & 462 & C. _jadiii_ \\  & GAP-t2 & GGATCCTTCATTCATCCCTCACTACG & & \\  & _Bam_HI & & & \\  & L41-P4 & CGTCGACAGTAGTAGAAAAGAG & & \\  & _Sall_ & & & \\
3 & L41-RM4 & GGATTCCG GTTGTCTATGTGT & & \\  & _Bam_HI & & & \\  & mL41-P5 & AACCAAGCTTTCCCAC & & \\  & mL41-R5 & GTGGAAAACTTGCTTGGTT & CAA code Gln & C. _jadiii_ \\  & 185 rDNA-P3 & CGATATCTGCCAGTGTCATGT & & \\
4 & & _Eco_RV & & \\  & & CGATATCTGACTTGCCTTACTAG & & \\  & 185 rDNA-R3 & EcoRV & & \\  & Lip-R1+1 & GCCTTAGAATGGCCTTCAAGCAGCTCTCTC & & \\
5 & & _Xbal_ & & \\  & Lip-F1+1 & CTAGCTTAGCCTTGTGCCGGG & & \\  & & _Nh_eI & & \\  & M1-1S & GGCCCTTCTGGGTCTTGAATT & & \\
6 & M1-1 AS & GCATATGACTACTGGCAAGTAGTAAAAAGAGCCAAT & & \\  & M2-1S & ATTGGCCTTTTCATACTACTGTCC & & pGMLR \\  & M2-1 AS & GCCCTGTATCGTTATTATTATTATTGTGTCACACTCCCTG & & \\ \hline \end{tabular}
\end{table} 
Table 1: PCR primers used in this study. primers. These PCR-amplified fragments were ligated into the pMD19-T vector to obtain ligation products, named pT-GP, pT-GT and pT-rD plasmid, respectively. All three plasmids were transformed into _Trans_-5\(\alpha\) chemically competent cells (CD201-01, TransGen Biotech), and the positive clones were identified via PCR. Primers L41-P4 and L41-RM4 were designed, and suitable restriction sites (_Sal_I and _Bam_HI) were introduced using the GenBank published sequence of the _C. jadini_ CYH sensitive gene (_L41_) (ID: D67040.1), combined with the sequence characteristics of the pBR322 plasmid. At the same time, a pair of reverse complementary primers, mL41-P5 and mL41-R5, was also designed.