Molecular Cloning and Expression Analysis of the Cryptochrome Gene CiPlant-CRY1 in Antarctic Ice Alga Chlamydomonas sp. ICE-L

Abstract

Cryptochrome (CRY) is a kind of flavin-binding protein that can sense blue light and near-ultraviolet light, and participates in the light response of organisms and the regulation of the circadian clock. The complete open reading frame (ORF) of CiPlant-CRY1 (GenBank ID OM389130.1), encoding one kind of CRY, was cloned from the Antarctic ice alga Chlamydomonas sp. ICE-L. The quantitative real-time PCR study showed that the expression level of the CiPlant-CRY1 gene was the highest at 5 °C and salinity of 32‱. CiPlant-CRY1 was positively regulated by blue or yellow light, suggesting that it is involved in the establishment of photomorphology. The CiPlant-CRY1 gene can respond to polar day and polar night, indicating its expression is regulated by circadian rhythm. The expression level of CiPlant-CRY1 was most affected by UVB irradiation, which may be related to the adaptation of ice algae to a strong ultraviolet radiation environment. Moreover, the recombinant protein of CiPlant-CRY1 was expressed by prokaryotic expression. This study may be important for exploring the light-induced rhythm regulation of Antarctic ice algae in the polar marine environment.

Keywords: Chlamydomonas sp. ICE-L; CiPlant-CRY1; cryptochrome; extreme Antarctic environments; heterologous expression.

Figure 1

Alignment comparison of the deduced amino acid sequences of CiPlant-CRY1 with other species. Chl-ICE-L: Chlamydomonas sp. ICE-L, Chl-rei: C. reinhardtii, Coc-sub: C. subellipsoidea C-169, Rap-sub: Raphidocelis subcapitata, Mon-neg: Monoraphidium neglectum, Gon-pec: Gonium pectoral. The frame represents the conservative domain of Plant-CRY protein.

Figure 2

Phylogenetic tree for CiPlant-CRY1 and related cryptochrome/photolyase family based on amino acid sequences. Numbers at nodes indicate levels of bootstrap support (%) based on a neighbor-joining analysis. The bootstrap test of the tree was performed with 1000 replications.

Figure 3

Relative expression of the CiPlant-CRY1 at different temperatures for different times. Standard error bars are shown, n = 3. Different capital letters indicate significant differences between different treatments (p < 0.01).

Figure 4

Relative expression of the CiPlant-CRY1 in salinity of 16‰, 32‰, 64‰, and 96‰ for different times. Standard error bars are shown, n = 3. Different capital letters indicate significant differences between different treatments (p < 0.01).

Figure 5

Relative expression of CiPlant-CRY1 in different lights for different times. W: white light, B: blue light, G: green light, Y: yellow light, R: red light. Standard error bars are shown, n = 3. Different capital letters indicate significant differences between different treatments (p < 0.01).

Figure 6

Relative expression of the CiPlant-CRY1 in different photoperiods for different times. C: normal light (12 h light:12 h dark), D: polar day (continuous light), N: polar night (continuous dark). Standard error bars are shown, n = 3. Different capital letters indicate significant differences between different treatments (p < 0.01).

Figure 7

Relative expression of the CiPlant-CRY1 in different ultraviolet lights for different times. W: white light, UVA: UVA irradiation, UVB: UVB irradiation, V: violet light. Standard error bars are shown, n = 3. Different capital letters indicate significant differences between different treatments (p < 0.01).

Figure 8

SDS-PAGE of the recombinant protein CiPlant-CRY1 induced by IPTG. M: Marker, 1: Uninduced CiPlant-CRY1 supernatant, 2: Uninduced CiPlant-CRY1 precipitate, 3: CiPlant-CRY1 supernatant induced by 0.2 mM IPTG, 4: CiPlant-CRY1 precipitate induced by 0.2 mM IPTG, 5: CiPlant-CRY1 supernatant induced by 0.5 mM IPTG, 6: CiPlant-CRY1 precipitate induced by 0.5 mM IPTG, 7: No-load bacterial supernatant induced by 0.5 mM IPTG, 8: No-load bacterial precipitate induced by 0.5 mM IPTG.