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

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.


Introduction
Light is an important environmental factor that affects life systems on Earth. It is not only an energy source for photosynthesis, but also a signal source for the growth and development of photosynthetic organisms. The light-dark cycle is one of the most regular periodic signals of organisms, controlling the metabolism of most species, rhythmic changes in physiology, and behavior. In addition to the beneficial effects of light, its ultraviolet rays can also cause DNA damage. As an important photoreceptor and transcriptional regulator, cryptochrome (CRY) is a kind of photochrome that affects plant growth and development, metabolism, and its own phototropism by absorbing blue light (400-500 nm) and nearultraviolet light (320-400 nm). It is a photoreceptor that participates in the process of phototransduction and the formation of the biological clock in vivo [1]. The CRY-mediated light signaling pathway is essential for plant growth and development and adaptation to changes in the external environment, and the research on the mechanism of CRY action is a hot spot and frontier in the field of international plant physiology research.
In plants, CRYs are mainly involved in the regulation of photomorphogenesis, including the regulation of plant circadian rhythms, stomatal opening, root growth, osmotic stress response, shade avoidance, leaf senescence, and other circadian rhythms and life cycle processes [2]. CRY generally has two domains, including an amino-terminal photolyase homology region (PHR) that binds to the flavin chromophore, and a carboxy-terminal Plants 2022, 11, 2213 2 of 12 C-terminal extension responsible for signaling [3]. According to sequence similarity, plant CRY family genes can be divided into three subfamilies: plant CRY, animal CRY, and CRY-Drosophila, Arabidopsis, Synechocystis, Homo (CRY-DASH) [4,5]. The light signal transmission mediated by plant CRY photoreceptors mainly includes three molecular processes: blue light-induced photoreduction, conformational change to form active dimers, and binding to downstream proteins to transmit light signals [6]. Despite advances in characterization of CRYs, their photochemical, regulatory, and light-induced structural changes remain to be addressed.
At present, the research on marine algal CRY has just started, and there are few reports on the low-temperature algal CRY, and in-depth research is urgently needed. Light is the most influential driver of Antarctic ice algal communities. Blue light can penetrate to substantial depths in seawater. Blue light receptors play an important role in the adaptation of algae to the changing marine environment [7]. At present, the research on algal CRY mainly focuses on CryP and CPF1. CryP is involved in regulating the input pathway of the circadian clock, has an inhibitory effect on the reset of the circadian clock by blue light, and is also associated with circadian rhythm changes, affecting the accumulation of light-harvesting proteins [8]. Volvox carteri and Chlamydomonas reinhardtii each have two CRY-DASHs, but they have not been characterized to date [9]. Antarctic ice algae can thrive in the Antarctic polar day and night and sea ice environment with strong ultraviolet radiation, while room temperature marine algae cannot survive. Antarctic ice algae must have special physiological mechanisms to maintain their survival and reproduction [10]. Obviously, compared with normal temperature plant CRY, Antarctic ice alga CRY can regulate physiological functions in the sea ice environment with extreme light-dark cycles and strong ultraviolet radiation, and must have a special mechanism of action.
We have cloned the complete open reading frame (ORF) of CiCRY-DASH1 (GenBank ID MK392361) from the Antarctic ice alga Chlamydomonas sp. ICE-L, and performed related characterizations [11]. The CRYs of Chlamydomonas sp. ICE-L may be related to how ice algae adapt to extreme Antarctic environments. Here, we study a novel plant-type CRY gene CiPlant-CRY1 in Chlamydomonas sp. ICE-L, which may provide a new perspective for the adaptation of Antarctic ice alga CRY to the polar marine environment and its physiological characteristics.

Bioinformatics Analysis of CiPlant-CRY1
As shown in Figure 1, the amino acid sequence of CiPlant-CRY1 contains the PHR domain, a protein conserved functional domain shared by CRY, while the C-terminal extension is not found.
Bioinformatics analysis showed that CiPlant-CRY1 had 1452 base pairs, encoding 483 amino acids, and its molecular formula was C 2293 H 3498 N 648 O 729 S 16 and relative molecular weight was 52.32 kDa, and as an acidic protein, its PI was 4.90. Its instability index (II) was calculated as 37.82, which indicated that the protein was stable. Its average coefficient of hydrophilicity GRAVY was −0.641, and its aliphatic index was 64.47. The secondary structure of CiPlant-CRY1 showed that helix accounted for 20.29%, strand for 2.69%, and loop for 77.02%. Analysis of its transmembrane profile by TMPred showed that there may be no transmembrane proteins. In addition, the subcellular localization of the CiPlant-CRY1 gene was predicted by the WoLF PSORT online tool, showing that CiPlant-CRY1 is likely to be located in the nucleus. amino acid sequence alignment analysis showed that CiPlant-CRY1 of Chlamydomonas ICE-L had the highest homology (34.48%) with C. reinhardtii, indicating that the spec had the closest affinity. It also had homology with Gonium pectoral (34.10%), Monoraph ium neglectum (32.57%), and Raphidocelis subcapitata (29.12%), and it had the lowest hom ogy (27.97%) with C. subellipsoidea C-169. The phylogenetic tree ( Figure 2) showed t CiPlant-CRY1 belonged to plant type CRY, and was most closely related to the plant ty CRY of Chlamydomonas reinhardtii.  As shown in Figure 1, the conserved amino acid domains are drawn in three frames, and these conserved amino acid domains are located in the FAD binding site region. The amino acid sequence alignment analysis showed that CiPlant-CRY1 of Chlamydomonas sp. ICE-L had the highest homology (34.48%) with C. reinhardtii, indicating that the species had the closest affinity. It also had homology with Gonium pectoral (34.10%), Monoraphidium neglectum (32.57%), and Raphidocelis subcapitata (29.12%), and it had the lowest homology (27.97%) with C. subellipsoidea C-169. The phylogenetic tree ( Figure 2) showed that CiPlant-CRY1 belonged to plant type CRY, and was most closely related to the plant type CRY of Chlamydomonas reinhardtii.

Effects of Different Stress Conditions on the Expression of CiPlant-CRY1
The effects of temperature stress on CiPlant-CRY1 at the transcriptional level are shown in

Effects of Different Stress Conditions on the Expression of CiPlant-CRY1
The effects of temperature stress on CiPlant-CRY1 at the transcriptional leve shown in Figure 3

Effects of Different Stress Conditions on the Expression of CiPlant-CRY1
The effects of temperature stress on CiPlant-CRY1 at the transcriptio shown in Figure    CiPlant-CRY1 at salinity of 16‰ reached the highest value of 14.111 at 6 h, but was still slightly lower than 32‰. At the normal salinity of 32‰, the expression level of CiPlant-CRY1 reached the highest value of 15.549 at 6 h. At salinity of 64‰, the expression level of CiPlant-CRY1 reached the highest value of 12.155 at 12 h (p < 0.01). The expression level of CiPlant-CRY1 at salinity of 96‰ was higher than other experimental groups at 8 h and 24 h (p < 0.01).     The relative expression of the CiPlant-CRY1 in different photoperiods is shown in Figure 6, and the expression level of CiPlant-CRY1 fluctuated greatly. In the normal light, the expression of CiPlant-CRY1 reached a maximum value of 2.667 on the 4th day and then decreased. In the polar day conditions, the expression of CiPlant-CRY1 reached a maximum value of 3.499 on the first day (p < 0.01). In the polar night conditions, the expression of CiPlant-CRY1 was higher than other experimental groups at 0.5, 2, 8, 12, and 30 days, and reached the maximum value of 5.606 on the second day. The relative expression of the CiPlant-CRY1 in different photoperiods is shown in Figure 6, and the expression level of CiPlant-CRY1 fluctuated greatly. In the normal light, the expression of CiPlant-CRY1 reached a maximum value of 2.667 on the 4th day and then decreased. In the polar day conditions, the expression of CiPlant-CRY1 reached a maximum value of 3.499 on the first day (p < 0.01). In the polar night conditions, the expression of CiPlant-CRY1 was higher than other experimental groups at 0.5, 2, 8, 12, and 30 days, and reached the maximum value of 5.606 on the second day. The relative expression of the CiPlant-CRY1 in different ultraviolet lights is shown in Figure 7, and the expression levels of CiPlant-CRY1 first increased, then decreased. The expression level of CiPlant-CRY1 changed slightly in the normal light conditions. The expression levels of CiPlant-CRY1 did not change much in UVA irradiation, but most of them were higher than that in normal light. UVB has the greatest effect on the expression of CiPlant-CRY1, which reached the highest value of 48.128 at 2 h (p < 0.01). In the violet light, the expression level of CiPlant-CRY1 increased gradually within 4 h and reached a maximum value of 29.947. The relative expression of the CiPlant-CRY1 in different ultraviolet lights is shown in Figure 7, and the expression levels of CiPlant-CRY1 first increased, then decreased. The expression level of CiPlant-CRY1 changed slightly in the normal light conditions. The expression levels of CiPlant-CRY1 did not change much in UVA irradiation, but most of them were higher than that in normal light. UVB has the greatest effect on the expression of CiPlant-CRY1, which reached the highest value of 48.128 at 2 h (p < 0.01). In the violet light, the expression level of CiPlant-CRY1 increased gradually within 4 h and reached a maximum value of 29.947.

Protein Expression
The recombinant strain Transetta (DE3)-pEASY ® -Blunt E2-CiPlant-CRY1 was induced to express by IPTG and subjected to SDS-PAGE (Figure 8). The DE3-containing bacteria were induced overnight at 16 • C with 0.5 mmol/L IPTG as a negative control. The result showed that there was an obvious protein band (in the box) in lane 6, which was located in the precipitate. Its relative molecular weight is consistent with the predicted protein relative molecular weight of 52.32 kDa, which can be judged as the CiPlant-CRY1 protein band. However, it also expressed in uninduced bacteria, and the reasons may be as follows: (a) this protein is well expressed and can be expressed without induction, (b) this protein is similar in size to a protein in the original strain, and the two protein bands run into a single band.

Protein Expression
The recombinant strain Transetta (DE3)-pEASY ® -Blunt E2-CiPlant-CRY1 was induced to express by IPTG and subjected to SDS-PAGE ( Figure 8). The DE3-containing bacteria were induced overnight at 16 °C with 0.5 mmol/L IPTG as a negative control. The result showed that there was an obvious protein band (in the box) in lane 6, which was located in the precipitate. Its relative molecular weight is consistent with the predicted protein relative molecular weight of 52.32 kDa, which can be judged as the CiPlant-CRY1 protein band. However, it also expressed in uninduced bacteria, and the reasons may be as follows: (a) this protein is well expressed and can be expressed without induction, (b) this protein is similar in size to a protein in the original strain, and the two protein bands run into a single band.

Protein Expression
The recombinant strain Transetta (DE3)-pEASY ® -Blunt E2-CiPlant-CRY1 was induced to express by IPTG and subjected to SDS-PAGE (Figure 8). The DE3-containing bacteria were induced overnight at 16 °C with 0.5 mmol/L IPTG as a negative control. The result showed that there was an obvious protein band (in the box) in lane 6, which was located in the precipitate. Its relative molecular weight is consistent with the predicted protein relative molecular weight of 52.32 kDa, which can be judged as the CiPlant-CRY1 protein band. However, it also expressed in uninduced bacteria, and the reasons may be as follows: (a) this protein is well expressed and can be expressed without induction, (b) this protein is similar in size to a protein in the original strain, and the two protein bands run into a single band.

Discussion
Antarctic ice algae thrive in polar sea ice, which is an extremely important biosphere habitat on Earth. The sea ice environment is characterized by low temperature, no light or low light, and seasonal dynamic changes. During the seasonal changes in sea ice formation and melting, eukaryotic phytoplankton living in sea ice habitats experience dramatic changes in environmental factors such as light, temperature, and salinity, and a variety of microalgae are able to adapt to these changes and form distinct sea ice biomes, known as ice algae [12]. The unique physiological characteristics of ice algae are closely related to their ability to adapt to extreme environments of sea ice [13]. CRY has also been extensively studied in green algae, which appear to have a more complex set of CRYs than higher plants [7,8,14]. In addition to canonical plant CRY and CRY-DASH, green algae also have plant-like, animal-like, and cryptochrome/photolyase family (CPF) proteins [15]. At present, most of the studies on algal CRY focus on normal temperature algae, while research on low-temperature algal CRY is less common. We have reported the cloning and expression characteristics of the CiCRY-DASH1 gene in Chlamydomonas sp. ICE-L [11]. In this paper, the CiPlant-CRY1 gene of Chlamydomonas sp. ICE-L was characterized. The amino acid sequence of the CiPlant-CRY1 gene only contained the conserved protein PHR domain shared by CRY, but no C-terminal extension functional domain. For plant CRYs, it has been shown that the C-terminal extension is partially destabilized upon illumination [16]. However, the motifs conserved in the C-terminal extension of land plants are not found in plant CRY from Chlamydomonas reinhardtii [7].
Plant CRY has recently been implicated in adaptations to temperature variation, including temperature compensation of the circadian clock [17]. Temperature is an essential factor affecting biological rhythm, and the biological clock has a temperature compensation effect, which means it can remain stable under certain temperature conditions [18,19]. The transcriptional regulation of the CiPlant-CRY1 gene showed a certain change trend at −5 • C, 0 • C, and 5 • C, and the transcription level at −5 • C, 0 • C, and 5 • C was significantly higher than that at 10 • C, indicating that temperature can affect the expression of the CiPlant-CRY1 gene. The suitable temperature for the growth of Chlamydomonas sp. ICE-L is 4-6 • C, and its biological rhythm will be destroyed when the temperature exceeds the suitable growth temperature.
Antarctic ice algae can survive and multiply in sea ice salt sacs. Dehydration, caused by the high brine salinities, is a major stressor for ice algae, which may experience salinities three times that of seawater. Conversely, when the ice melts, the released ice algae will be suddenly exposed to hyposaline conditions close to freshwater values [12]. The expression of the CiPlant-CRY1 gene in Chlamydomonas sp. ICE-L was the highest at the optimal salinity of 32‰ and showed a certain regularity. In addition, its expression level under the salinity of 16‰ and 96‰ was significantly lower than that of the optimal growth salinity, indicating that salinity can affect the expression level of the CiPlant-CRY1 gene.
In plants and algae, light serves both as the energy source for photosynthesis and a biological signal that triggers cellular responses via specific sensory photoreceptors [20]. To perceive fluctuations in light quality, quantity, and timing, plants and algae have evolved diverse photoreceptors including CRY [21]. Changes in the expression level of the CRY gene under different light qualities (white, blue, green, yellow, and red light) indicate that it can effectively respond to different light qualities, especially blue and yellow light. This reveals that CiPlant-CRY1 may not only be categorized with respect to a potential role as a blue light receptor, but yellow light activation of CiPlant-CRY1 needs to be considered as well in the design of future experiments [22]. Moreover, CiPlant-CRY1 can maintain a high level of expression through the treatment of blue light, which indicates that CiPlant-CRY1 is involved in the formation of light morphology, regulation of pigment biosynthesis, light capture complex synthesis, cell cycle, etc., as a blue light receptor [8].
Antarctica represents an extreme light regime with an annual light cycle including periods of polar day and polar night. The expression of the CiPlant-CRY1 gene is highly responsive to long-term light and dark treatments, and shows a certain circadian rhythm, indicating that its expression is regulated by circadian rhythm [19]. Light/dark cycles entrain the Chlamydomonas circadian clock, and CiPlant-CRY1 belongs to the biological clock component [23,24]. Therefore, analyzing the circadian expression of CiPlant-CRY1 in long-term light and dark environments can help to understand the mechanisms of plant circadian regulation.
CRYs are photolyase (PHO)-like flavoproteins and together constitute a CPF that has been found in all evolutionary lineages [4,25]. The CPF includes structurally similar proteins that function differently and use near-ultraviolet (UV)/blue light photons as an energy source to mediate either DNA repair or perception and signal transduction of external light signals [22,26,27]. The ozone hole over Antarctica may produce massive amounts of UV radiation. The expression level of CiPlant-CRY1 was most affected by UVB irradiation. CRY blue light signaling shows some similarities to UVR8 UV-B signaling [28]. We speculate that the CiPlant-CRY1 gene is sensitive to UVB and may be related to the adaptation of Antarctic ice algae to strong ultraviolet radiation environments.

Algal Culture
The Antarctic ice alga Chlamydomonas sp. ICE-L was isolated from the floating ice near the Zhongshan Research Station of Antarctica (69 • S, 77 • E) [29]. Cultures were routinely grown in autoclaved Provasoli seawater medium [30] at a light density of 40 µmol m −2 s −1 with a diurnal cycle of 12 h light and 12 h dark at a temperature of 5 ± 1 • C.

RNA Extraction and cDNA Synthesis
Total RNA was extracted using TRIzol reagent (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. RNA integrity was determined by electrophoresing the sample on a 1% agarose gel, while RNA quantification and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA synthesis was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China).

Cloning and Sequencing of CRY Complete ORF
Specific primers (Table 1)
CiPlant-CRY1-specific primers (Table 1) were designed using Primer Premier 5.0 software (PREMIER Biosoft International, San Francisco, CA, USA). The housekeeping gene, 18S ribosomal RNA, of Chlamydomonas sp. ICE-L was used as an internal control. qRT-PCR was performed with a LightCycle ® 96 Instrument (Roche Diagnostics, Indianapolis, IN, USA) and TransStart ® Green qPCR SuperMix (TransGen Biotech, Beijing, China). The qRT-PCR amplification programs included an initial denaturation step of 94 • C for 30 s; followed by 42 cycles of 94 • C for 5 s, 58 • C for 15 s, and 72 • C for 10 s. The data were further analyzed by the comparative Ct (2 −∆∆CT ) method [33]. The LSD test and average ± SD of each experiment were calculated and analyzed using IBM SPSS Statistics 22.0 data analysis software (IBM Corp., Armonk, NY, USA).

Protein Expression
The PCR product was ligated into a pEASY ® -Blunt E2 Expression Vector (TransGen Biotech, Beijing, China), which was then transformed into a Trans1-T1 Phage Resistant Chemically Competent Cell (TransGen Biotech, Beijing, China). Subsequently, the correctness of the CiPlant-CRY1 sequence and ligation direction was verified. The plasmid was extracted from the bacterial solution with correct expression direction and then transformed into a Transetta (DE3) Chemically Competent Cell (TransGen Biotech, Beijing, China). Finally, isopropanol-β-D-thiogalactoside (IPTG) was used to induce the expression of the CiPlant-CRY1 protein in the bacterial solution with the correct expression direction.

Conclusions
In this study, a novel plant type CRY gene CiPlant-CRY1 was first cloned and analyzed from Antarctic ice alga Chlamydomonas sp. ICE-L. qRT-PCR analysis revealed that CiPlant-CRY1 is an important photoreceptor gene in response to blue or yellow light. Its expression is regulated by circadian rhythm and may be related to the adaptation of ice algae to strong ultraviolet radiation environments. The heterologous expression of the recombinant CiPlant-CRY1 gene laid the foundation for further functional studies. This study provides a novel view of the mechanisms by which Antarctic ice algal CRY photoreceptors adapt to extreme sea ice environments.
Author Contributions: Conceptualization, Z.Z.; validation, Y.Z.; formal analysis, Y.Z. and X.Z.; investigation, Y.Z. and X.Z.; writing-original draft preparation, Y.Z. and Y.B.; funding acquisition, Z.Z. and J.M. All authors have read and agreed to the published version of the manuscript.