Studies On Photoprotective Function And Mechanism Of Dscbr Gene In The Green Alga Dunaliella Salina

Photooxidative damage can result in the release of superoxide radicals or singletoxygen. In order to maintain their normal function under light stress conditions,plants and algae have developed multiple repair and protection systems. ELIPs (forearly light-induced proteins) in higher plants, as one of specific light stress proteins,can be considered to be part of these repair and protection responses, CBR (forcarotene biosyntheses related protein), an algal homologue of plant ELIPs, have asimilar function. The cDNA sequence of cbr gene has been cloned by our lab,designated Dscbr gene. On the basis of these studies, in this paper, we had anoriginal research on regulation of transcription, photo-protective andpigment-binding functions ofDscbr gene. The main results are as followed.Ⅰ. Dscbr mRNA was induced only transiently by light stress and NaCl shocks.The relative abundance of Dscbr mRNA was analysed using Real-TimeQuantitative PCR when Dunaliella Salina cell was exposed to light or NaCl shocks.The relative abundance of 18s rRNA was also determined and used as the internalstandard. (1) The results showed Dscbr mRNA levels correlated with the lightintensity. The transcript amounts of Dscbr which were induced by a 2h HL-treatmentat fluxes of 800μmol m^-2s^-1 were highest but decreased to about 60％at a 1500μmolm^-2s^-1 light intensity. (2) The result showed that Dscbr mRNA was induced onlytransiently by light stress. The mRNA amounts were estimated as a function of timeafter transferring the cultures from HL to LL (recovery) conditions. The results ofsuch an experiment demonstrated that Dscbr mRNA was short-lived. Under the treatment of high light, the mRNA accumulated rapidly in 2h. When light wasdiminished to the normal condition, the mRNA degraded quickly in 1h. After 2h,Dscbr mRNA expression was recovered to the normal level. (3)Dscbr mRNA levelsobviously depended on NaCl concentration in the culture. The same results wereobtained in both treatments. In the first 2-4 hours of high and low salt shocks, theexpression of the mRNA was both very high. After 6 hours, the mRNA levelsdecreased to the normal in parallel. Because D. salina had an ability to adapt tohypersaline environment, it was damaged more severely by low NaCl(0.5M) stressthan by high NaCl(3.0M), low NaCl could induce higher Dscbr transcript level.(4)The transient expression suggested that Dscbr maybe act an importantprotection/repair role in the early phage of light stress. The accumulation of Dscbrtranscripts was regulated by photooxidative damage induced from all kinds ofstresses and not by certain specific factors.Ⅱ. By the floral-dip method, Dscbr gene was transformed into Arabidopsis elip/cbrmutation.In order to assure the Dscbr gene function, we used an Arabidopsis elip/cbrgene mutant (RIKEN BioResource Center No. PST20631) as transgenic plantmaterial. Firstly, the vector pBI121-Dscbr was constructed. Then the pBI121-Dscbrvector was introduced into Agrobacterium tumefaciens EHA105 using thefreeze-thaw method. Thirdly, the Arabidopsis elip mutation was transformed on alarge scale by the floral-dip method. Finally, we got a convenient and dffectivemethod about seed sterilization and indoor culture of Arabidopsis for the laboratories.This method was applied to select the transformed plants with kanamycin marker.Ⅲ. Constitutive expression of Dscbr genes in Arabidopsis mutant restored thephotooxidative tolerance of the plants to the wild-type level and the findingsindicated that Dscbr fulfill a photoprotective function.We got 76 kanamycin resistant plants. The transformed plants were transplantedinto vermiculite: sand (1:1) medium. Transgenic plants with Dscbr gene wereobtained and their target gene was detected through PCR analysis in this study. Forfunction analysis, the Arabidopsis mutant was exposed to high light and low temperature stresses, resulting in leaf bleaching and extensive photooxidativedamage. Constitutive expression of Dscbr genes in mutant after light stress resultedin Dscbr accumulation and restored the phototolerance of the plants to the wild-typelevel. Our findings indicated that Dscbr fulfill a photoprotective function and involveprotection and/or repair of photosynthetic apparatus during high-light stress. Thisresult is in agreement with elip gene of higher plants (Hutin et al.2003).Ⅳ. Using E. coli expression and protein purification system, Dscbr gene wasexpressed and purified.In order to study the mechanism of Dscbr fulfilling the photoprotective role,E.coli expression and protein purification system was used in the paper. Dscbr genewas subcloned into expression vector PET-32a. E.coli BL21 was transformed withthis PET-Dscbr vector and the fusion protein DsCBR was purified throughelectroelution in dialysis bags. The basic works would help us to do farther researchin DsCBR protein.Ⅴ. We established a high-performance reconstituting system to reconstitute purifiedDsCBR proteins with pigments in vitro. The results showed that DsCBR could bindchlorophyll a, chlorophyll b and carotene and then formed pigment-proteincomplexes.It is suggested that CBR/ELIP is involved in the PSⅡrepair process throughphotoprotection (Jin et al. 2001). The mechanism of CBR/ELIP function may berelated to transient binding of pigments as "pigment-carrier" proteins. In thisresearch, a modified method (freezing under liquid nitrogen and thawing under roomtemperature) was used to reconstitute purified DsCBR proteins with pigments invitro and a high-performance reconstituting system was developed to study structureand function of DsCBR. LDS-PAGE analysis suggested that DsCBR proteins couldreconstitute with cell pigments from Dunaliella salina. It has been showed thatDsCBR could bind chlorophyll a, chlorophyll b and carotene and then formedpigment-protein complexes. The possibility of "pigment-carrier" working model forELIPs/CBR was raised by this result. It was postulated that the mechanism of Dscbrfulfilling photoprotection function may be as followed: Under light stress conditions, the damaged pigment-protein complexes released free pigments. These pigments arebound to the DsCBR and then transferred to chlorophyllases are degraded by specificproteases or they can be reused for ligation with the newly synthesized polypeptides.The paper is important in theory to study the function of cbr gene in algae andthe mechanism of photoprotective function in higher plant and algae.