Molecular Cloning And Expression Regulation Of Cryptochrome Genes In Chlamydomonas Sp. ICE-L

Light is not only the energy source of photosynthesis,but also the signal source of growth and development.The growth and development of algae is closely related to light.Cryptochromes are photoreceptors that receive blue light(440?460 nm)and near-ultraviolet UVA light(320?400 nm).Cryptochromes belong to the cryptochromes and the photolyase family(CPF),which participates in photoreactions and regulation of biological clocks in animals,plants and microorganisms.At present,the research on cryptochrome mainly focuses on terrestrial plants,and great progress has been made in the research on the structure,biological function,photoexcitation and signal transduction regulation mechanism of plant cryptochrome.However,there are few studies on cryptochrome from marine microalgae,especially from polar microalgae.Chlamydomonas sp.ICE-L has grown and reproduced in low temperature,high salinity,seasonal illumination and strong ultraviolet radiation.And it has a strong adaptability to low-temperature and high-salinity stress.The study of it can provide a basis for the research of Antarctic habitat and other species.This paper is the first to study the cryptochrome of Chlamydomonas sp.ICE-L,which will not only help to understand the function and mechanism of the cryptochrome of Chlamydomonas sp.ICE-L,explain the light regulation process of marine algae,but also lay the foundation for studying the adaptability of polar algae to extreme environments.Objectives:This study mainly focused on cloning and expression regulation of cryptochrome genes in Chlamydomonas sp.ICE-L.In order to clarify the response of Chlamydomonas sp.ICE-L cryptochrome blue light signal and its role in the regulation of rhythmic light,and to explore the light-induced regulation function of Antarctic ice algae cryptochrome,and to explore the regulation mechanism of Chlamydomonas sp.ICE-L biological clock,To lay the theoretical foundation for further exploring the molecular mechanism of Antarctic algae environmental adaptation.Methods:1.According to the transcriptome sequencing results of Chlamydomonas sp.ICE-L,CiCRY-DASH1 and CiPlant-CRY1 were cloned by molecular biology technology and analyzed by bioinformatics.2.Temperature,salinity,light quality,photoperiod and ultraviolet conditions were selected to analyze the genes of CiCRY-DASH1 and CiPlant-CRY1 by real-time quantitative PCR,and detected their transcriptional regulation levels.3.To construct prokaryotic expression system of CiCRY-DASH1 and CiPlant-CRY1.4.Water-PAM was used to determine the maximum photochemical efficiency(Fv/Fm)and non-photochemical quenching(NPQ)of physiological parameters of Antarctic ice algae cells under different conditions(ultraviolet UVA and UVB,different light quality and photoperiod).Results:1.The full length of cryptochrome genes CiCRY-DASH1 and CiPlant-CRY1 were cloned by molecular biology technology.CiCRY-DASH1 has 1836 bases,encoding 608amino acids,and its protein molecular weight is 67.35 kDa.CiPlant-CRY1 has 1452 bases,encoding 483 amino acids,and its protein molecular weight is 52.32 kDa.Phylogenetic tree analysis showed that CiCRY-DASH1 and CiPlant-CRY1 proteins had the closest evolutionary relationship with Chlamydomonas reinhardtii and C.subellipsoidea C-169.2.Real-time quantitative PCR analysis of the CiCRY-DASH1 and CiPlant-CRY1genes of Chlamydomonas sp.ICE-L was completed.The results of real-time quantitative PCR showed that CiCRY-DASH1 and CiPlant-CRY1 had a certain trend during low-temperature treatment and showed transcriptional changes at the optimum temperature.The biological clock was temperature-compensated,which further confirmed that they were key components of biological clock.The CiCRY-DASH1 and CiPlant-CRY1 genes of Antarctic microalgae had the highest expression level at the optimum salinity of 32‰and demonstrated certain regularity.The expression levels of the CiCRY-DASH1 and CiPlant-CRY1 genes under different light forms(white,blue,green,yellow and red light)were found to effectively respond to various light quality treatments,especially blue,yellow and red light.This implied that the protein may be involved in light signal transduction pathways under blue light,yellow or red light.Moreover,the CiCRY-DASH1 and CiPlant-CRY1 can positively respond to extreme polar day and night treatment and exhibit a certain circadian rhythm.Under ultraviolet stress,the CiCRY-DASH1 and CiPlant-CRY1 were sensitive to UVB and violet light.3.Prokaryotic expression systems of the CiCRY-DASH1 and Ci Plant-CRY1 of Chlamydomonas sp.ICE-L were constructed.The prokaryotic expression vectors pEASY~?-Blunt E2-CiCRY-DASH1 and pEASY~?-Blunt E2-CiPlant-CRY1 were successfully constructed and transformed into Transetta DE3 expressing competent cells,which were induced at 16?for 12 h under the IPTG.The target proteins CiCRY-DASH1 and CiPlant-CRY1 with the same molecular weight as the predicted protein were obtained by SDS-PAGE electrophoresis analysis.The expressed target protein mainly existed in the form of inclusion bodies in the precipitate.4.The chlorophyll fluorescence analysis of Chlamydomonas sp.ICE-L was completed.Studies showed that UVB,blue light and polar day were the stress conditions that might affect the physiology and metabolism of algae in the polar environments.Under the conditions of UVB,blue light and polar day,the Fv/Fm of Chlamydomonas sp.ICE-L decreased gradually with the prolongation of treatment time,and its NPQ increased correspondingly,indicating that the PSII reaction center of Chlamydomonas sp.ICE-L had photoinhibition under UVB,blue light and polar day conditions.Conclusion:In this study,cryptochrome genes Ci CRY-DASH1 and CiPlant-CRY1 of Chlamydomonas sp.ICE-L were cloned and analyzed by molecular biology technology.By studying the expression pattern of Chlamydomonas sp.ICE-L cryptochromes under different stress conditions,it was found that Ci CRY-DASH1 and Ci Plant-CRY1 were key components of the biological clock.And the CiCRY-DASH1 and CiPlant-CRY1 might be involved in light signal transduction pathways under blue light,yellow or red light.Moreover,the CiCRY-DASH1 and CiPlant-CRY1 can positively respond to extreme polar day and night treatment and exhibit a certain circadian rhythm.The prokaryotic expression provided a certain material basis for the subsequent functional characterization of cryptochrome genes.Chlorophyll fluorescence analysis showed that UVB,blue light and polar day were the stress conditions that might affect the physiology and metabolism of algae in the polar environments.