## 3 Results
### GC-MS Analysis  
A GC-MS analysis was performed to determine the function of the _tps1_ gene. In this analysis, volatiles extracted from the wild-type, deletion strains, and overexpression strains were examined using the HS-SPME method, and three biological replicates of each strain were performed to ensure the accuracy of the experimental results. Under conditions where the wild-type was used as a control group, the HS-SPME-GCMS analyses showed that the deletion or overexpression of the _tps1_ gene did not affect the production of dimethyl sulfide,furan, 2-ethenyl-, n-octane, and other non-terpene VOCs (Electronic  
Figure 3: qRT-PCR analysis and confirmation of _tps1_ transformants. (**a**) The principle of homologous recombination was utilized to delete the terpene synthase gene _tps1_ by inserting the screening marker _hph_ gene. Following amplification with oligonucleotide TPSF/TPSR, a 1645 bp fragment of the _tps1_ gene was produced in the wild-type strain, while a 3600 bp recombinant fragment was generated in the deletion strain (M—GL 5000 Maker; 16, 17—amplification of recombinant fragments of picked transformants; 29—pMD18T-\(\Delta\)_tps1_-_tps_ plasmid; 30—wild-type strain HB20111 genome). (**b**) qRT-PCR analysis of _T. atroviride_ wild-type (HB20111) strain, _tps1_-deletion mutants (16, 17), and _tps1_-overexpression mutants.  
Supplementary Material), but the deletion or overexpression of the _tps1_ gene affected the production of terpene VOCs.  
Two major classes of terpene volatile compounds were produced in the wild-type strain: eight monoterepenes and two sesquiterpenes (Table S2), with the major compounds being 4-methyl-2(5H)-furanone, \(\beta\)-phellandrene, geranylacetone, \(\alpha\)-phellandrene, \(\alpha\)-angelica lactone, and acorenone b, which comprised 88.61% of the total terpene volatiles (electronic Supplementary Material). The presence of (5e,9e)-farnesyl acetone and beta-cedrene was not detected in the deletion mutant compared to the control WT (Table S2). Interestingly, both the deletion mutant and the overexpression mutant produced new sesquiterpene compounds, trans-neralidol and meridold, respectively, at RT 31.5329 (Table S2). This suggests that the _tps1_ gene may be involved in terpenoid synthesis. Additionally, a single volatile compound, 1,3,6,10-cyclotetradecatetraene, 3,7,11-trimethyl-14-(1-methylethyl)-, (s-(e,z,e,e))-, exhibited a unique presence in the _tps1_-gene-deletion or -overexpression strains, despite its low relative abundance. Concurrently, we observed that the terpene spectra of overexpressed inverters exhibited an increase in content for only four out of the nine sesquiterpenes when compared with those of the wild-type, indicating that the overexpression of the _tps1_ gene did not result in a complete enhancement in volatile organic compound production. Conversely, monoterepene species were reduced in all the overexpressed strains, with only one monoterepene (2, 3-dihydro-3, 5-dihydroxy-6-methyl-4h-pyrano-4-one) being comparable to the wild-type (Table S2). Furthermore, this study also revealed that four sesquiterpenes (neralidol, Epizonarene, \(\beta\)-acorenol, guaiene) showed a significant increase in abundance of the _Otps1_ strain with _tps1_ gene overexpression compared to _tps1_ gene deletion (Table S2).