*3.7. Promoter Isolation and Analysis*

The Genomic Walking Kit (TaKaRa, Dalian, China) was used to obtain promoter regions of the *Cppsy* gene from *C. protothecoides* CS-41. Based on the *Cppsy* genomic sequences, gene-specific primers were designed and are listed in Table 1. Primary and nested PCRs were performed with the *Cppsy* gene-specific primers and Genome Walking adapter primers (AP1) in the kit according to the manufacturer's instruction. The primary nested PCR products were diluted to 1:50 with distilled water for subsequent nested PCR. The nested PCR products were purified from 1.2% (w/v) agarose gel and sub-cloned into the pMD18T vector (TaKaRa, Dalian, China). The cloned vectors were then sequenced and the putative *cis*-regulatory elements were analyzed using the PLACE [40] and PlantCARE databases [41].

## *3.8. Gene Expression Response to Light and MeJA*

To analyze the light regulation pattern of the *Cppsy* gene in *C. protothecoides*, algal cells in the late log phase were cultivated in the dark for more than 2 days, then collected by centrifugation at 5000 rpm for 15 min in the darkness. The pellet was resuspended in fresh medium without glucose, and then subjected to light treatment under a light intensity of 120 μmol mƺ2·sƺ1 for different induction times 

(0, 0.5, 1.0, 2.0, and 4.0 h). Each treatment was carried out with three parallel repetitions. 

To analyze the MeJA regulation pattern of the *Cppsy* gene in *C. protothecoides*, algal cells in the log phase were treated with 100 μM MeJA (Sigma, St. Louis, MO, USA)  diluted in dimethyl sulfoxide (DMSO) for 0, 2, 4, 6, 8 10, and 12 h. Control cultures were treated with DMSO only. 

The effects of light and MeJA on the *Cppsy* gene transcripts in *C. protothecoides* were quantified 

by reverse transcriptase quantitative PCR (RT-qPCR). The RT-qPCR experiment was performed in 

two steps: the cDNA templates were synthesized from RNA samples using Prime Script™ Reverse Transcriptase Reagent according to the manufacturer's instructions (TaKaRa, Dalian, China) using oligo (dT) as the primer; then qPCR was conducted on an iQ Cycler (Bio-Rad, Watford, UK) using the specific primers (Table 1) and the SYBR ExScript RT-PCR kit (TaKaRa, Dalian, China). The specific primers for the corresponding genes included psyRT-F and psyRT-R for the *Cppsy* gene, and 16SRT-F and 16SRT-R for the 16S gene (Table 1). Before the qPCR expression analysis, we checked the amplification efficiency of each primer pairs, and all were well controlled between 99.83% and 101.25%. 

Each qPCR measurement was carried out independently at least three times, and the mean value was used for quantification. The 2ƺ̇̇CT method was used to analyze the relative changes in gene expression, the expression of the 16S gene was used as a normalized control, and the expression of the untreated samples was used as a negative control. 
