*2.3. Promoter Isolation and Analysis*

The promoter region of the *Cppsy* gene was cloned from *C. protothecoides* CS-41 genomic DNA. The cloned *Cppsy* promoter region was determined to be 1980 bp in length, and the sequence is shown in Figure 4. Furthermore, the cloned *Cppsy* promoter region was analyzed using the PLACE and PlantCARE databases. Several core fragments were identified, which are homologous to the *cis*-acting elements of higher plants and of great importance for the promoter functions (Figure 4). Three types of elements, which have been found to be regulated by hormones in the upstream region of some 

plant genes, are present in the *Cppsy* promoter: the ABRE type (CCTGCGTGGC, CACGTG, and GCCTCGTGGC) involved in abscisic acid responsiveness; the TGACG-motif (TGACG), the *cis*-acting regulatory element involved in methyl jasmonate (MeJA) responsiveness; and the Sp1 (CCCCCGCCA and ACCCGCCATG), MNF (GTGCCCCATGCAGGTT) and Box I (TTTCAAA) types involved in light responsiveness. 

The transcriptional start site (TSS) of the *Cppsy* promoter was determined by 5<sup>ȝ</sup>-RACE using total RNA extracted from *C. protothecoides*. The TSS is an adenine (A) at 34 bp upstream of the translation initiation codon. The distance between the putative  TATA-box and TSS is approximately ƺ24 to ƺ28 bp, which is consistent with the distance of 32 bp ± 7 bp from previous data [30]. 

A previous study showed that the *psy* expression level is affected by light [31] and other biotic and abiotic stresses [16] in higher plants. Gene expression response to environmental stress is related to the regulation mechanism. To understand more about the regulation mechanism, we need to know more information about the gene, including the gene structure and regulatory domains. Here, many elements were found in the *Cppsy* promoter that belong to light-responsive elements, such as Sp1, MNF1, G-box, and chs-CMA2a. There were also some *cis*-acting elements involved in abscisic acid (ABRE) and MeJA (TGACG-motif) responsiveness. Light is one of the most important environmental factors for algae. To determine whether these motif sequences from the *Cppsy* promoter are involved in light, abscisic acid, and MeJA signaling, loss-of-function analysis needs to be carried out in future studies. 


**Figure 4.** Promoter sequence of *Cppsy* from *C. protothecoides* CS-41. Numbers indicate the positions relative to transcriptional start site (TSS). The TSS is indicated as +1 and in bold; important *cis*-elements are underlined. 
