Cloning And Functional Analysis Of A New Member Of RbcS Gene Family From Dunaliella Salina

RbcS is a gene, coding the small subunits of ribulose 1,5-bisphosphate carboxylase/ oxygenase. This enzyme is the highest protein contained in the chloroplast, locating in the chloroplast stroma, and can catalyze the photosynthesis to fix CO2. The small subunits are very important to the structure and function of the holoenzyme Rubisco, and they usually locate on one or several chromosomes in the form of gene family. Ordinarily, many plants contain about 4～10 RbcS genes, the sequences often are different with each other, but the protein function are usually the same. The RbcS gene family of the high plants often show differently expressions in different organs and different times of development.The control mechanism is also different, and it can occur in the transcriptional level or the post-transcriptional level. The combination of DNA-protein or the stability of its mRNA is also different, that is to say, exist in the gene family, is not just to enhance the expression of the gene.Salt stress is one of the important factors that affect the development of plants, many plants and the blue-green algae, salt can inhibit photosynthesis, but the Dunaliella Salina (D.salina) can enhance the photosynthesis. The research, reported by Liska and his partners , showed that when moving the D.salina from lower salt condition to the higher, it can strongly enhance the photosynthesis, fixing the CO2 to produce lots of glycerol to adjust the higher salt condition. RbcS is very important in the CO2 assimilation and the starch formation , but the regulate mechanism of the expression has not reported in the D.salina under the high salt conditin.Here, a pair of degenerate primers was designed according to the high conserved homologous amino acid sequences of the known RbcS of other organisms, and the RACE method was used to obtain the 5'and 3'ends sequences cDNA, and then a new full-length cDNA fragment named RbcS2 was obtained. According to the cDNA sequence of RbcS2, one pair of special primers was designed, and then the real time quantitative PCR was adopted to analyze the RbcS2 expression of light and salt under differnt times. This may be provide the experimental evidence for the reseach of the members of the gene family's structure and function, even for finding the molecular regulate and control mechanism of D. salina under salt stress. Methods1 A new partial cDNA of RbcS gene family amplified from the degenerate primers from D.salinaA pair of degenerate primers was designed according to the high conserved amino acid sequences of RbcS of D.salina, Chlamydomonas reinhardtii, Haematococcus pluvialis, Volvox carteri on the GenBank. A total RNA of D.salina was extracted with Trizol and then was reversly transcribed into the first strand cDNA using M-MuLV as the PCR template. After the partial cDNA of RbcS beening amplified, the PCR product was subcloned into pMD18-T vector and then was screened to identify its sequence after beening double digested. Homologous analysis of the deduced amino acid sequences was identified by BLAST and subsequently compared with the RbcS of other plants in the GenBank data.2 Cloning upstream and downstream sequence of RbcS2 from D. salina using RACEOne pair of special primers was designed according to the full-length cDNA sequence of RbcS2 to amplify 5'upstream and 3'downstream cDNA fragment from total RNA of D.salina by RT-PCR. The resulting PCR product was subcloned into pMD18-T vector and screened to identify its sequence after beening double digested.3 Jointing and analysis the full cDNA of RbcS2 from D. salinaA full-length cDNA sequence was jointed according to the results of degenerate cDNA,the 5'and 3'RACE cDNA. Homologous analysis of the cDNA fragment and the deduced amino acid sequences were analysed by BLAST and subsequently compared with the RbcS of other plants in the GenBank data. The codon preference was analysed by DENMEN. 4 Functional analysis of the RbcS2 from D. salinaAccording to the full-length cDNA sequence of RbcS2, one pair of special primers was designed, and another pair of special primers was also designed from the known house keeping gene GAPDH of D. salina as the internal control. Total RNAs of D.salina were extracted with Trizol reagent of light and salt under differntal times and then was reversly transcribed into cDNA using M-MuLV, and used the real time quantitative PCR to analyze its expression.Results1 A new partial cDNA of RbcS gene family amplified from the degenerate primers from D.salinaThe results showed that the obtained cDNA sequence was 209 bp with its 69deduced amino acids. The amino acid sequence shared high homologous with the RbcS of other plants, one result was 84% of the amino acids compared with the known RbcS1 of D.salina. All these had showed that one new partial cDNA of RbcS gene family had been amplified from the degenerate primers from D.salina.2 Cloning upstream and downstream sequence of RbcS2 from D. salina using RACEResults shown that the 5'RACE cDNA contained 302 bp, after removing the partial sequences that had been known and the unconding region, a sequence was obtained whose coding sequence and deduced amino acids was separately 168 bp and 56 amino acids, and the 5'UTR was about 90 bp. The 3'RACE cDNA contained 489 bp, after removing the partial sequences that had been known and the unconding region, a sequence was obtained whose coding sequence and deduced amino acid was separately 271 bp and 90 amino acids, and the 3'UTR was about 218 bp.3 Jointing and characterization the full cDNA of RbcS2 from D. salinaThe full-length cDNA was long 869 bp, a complete open reading frame was 561 bp and encoded 187 amino acids residue. Homologous analysis of the deduced amino acid sequences was analysed by BLAST and subsequently with other plants, The results showed that the putative amino acid sequence of RbcS2 shared high homology with other plants, one result was 77.89% of the nucleotide acid and 77.19% of the amino acid, that compared with the known full-sequence RbcS1 of D. salina, that is to say, a new full-length cDNA of RbcS gene family, named RbcS2, has been amplified from D. salina. The homology was higher with other microalgae than with the high plants.Analysis of synonymous codon bias in the RbcS2 gene shown that codons with high G+C content were used much more frequently than those with high A+U content, and it had higher G/C preference at the third codon position as other eukaryotic biology.4 Functiona analysis of the RbcS2 gene from D. salinaIt was responded to light, its expression can be photoinduced strongly. Under the dark condition, the expression of RbcS2 was relatively lower, after moving to the light, the transcription level of RbcS2 induced by light increased fast before the plateau phase was reached, and then went down gradually. It was responded to salt, and increased under high salt condition. The expression of RbcS2 mRNA under the high salinity (3.0 M NaCl) became higher than normal culture condition (1.5 M NaCl), at the same time, because of the influence of light, the gene expression under both the high salinity and the normal culture condition changed along with light and dark. Conclusion and discussionSalt stress can inhibit many plants' photosynthesis, but D.salina can enhance the photosynthesis. In order to research the especial function to enhance photosynthesis under the high salinity condition of D. salina, we had cloned a new member, named RbcS2, of the RbcS gene family, which was the keygene in the CO2 assimilation, and researched its function of light and salt tolerance by the real time quantitative PCR. The results showed that its expression could be induced by light and high salinity, compared with the dark condition, the transcription expression of RbcS2 could be induced by light in a period of time, and the expression of RbcS2 mRNA under the high salinity was higher than normal culture condition.It is concluded that RbcS2 may be a related gene with salt stress, whose expression increase under high-salt condition. The results may be provid the experimental evidence for D.salina, that the photosynthesis isn't decreased but enhanced for the molecular level' research, even for finding the molecular regulate and control mechanism of D. salina under salt stress.