| At present,it is generally believed that cryptophytic phycobiliproteins mainly exist as heterodimers composed of two different?subunits and two identical?subunits.While,a large number of previous experimental results of our laboratory have shown that?1 and?2 of PC645 from Chroomonas placoidea T13 are different subunits varied both in molecular mass and isoelectric point.But the reasons for these differences are still uncertain.Firstly,in this paper,the?subunit gene sequence and the number of PC645?subunits were determined at the gene level by sequencing the transcriptome of Chroomonas placoidea T13 and cloning of the?subunit gene of PC645.And the differences in PC645?2subunit expression under different light conditions were also explored.Secondly,at the level of protein complex of heterodimer as well as the subunit level,the absorption spectra,fluorescence spectra,circular dichroism and isoelectric point characteristics of PC645 with different proportions of?2 subunit and isolated?1 and?2 subunits were measured and compared.These allowed us to preliminarily confirm the similarities and differences of the two subunits in terms of spatial structure and chromophore types.Furthermore,the influence on the spatial structure and characteristics of the PC645 with different content of?2 subunits was also analyzed.The results of transcriptome sequencing of C.placoidea performed on the Illumina Hiseq high-throughput sequencing platform showed that the Clean Data reached 6.17Gb,and the percentage of Q30 bases was 93.90%.72,468 Unigenes were obtained after assembly of Clean Data using Trinity software.The NR,Swiss-Prot,KEGG,COG,KOG,GO and Pfam databases were aligned with Unigene using BLAST software,and a total of28,306 Unigene annotation results were obtained.According to the results of the Swissprot database,the?1 and?2 subunits of PC645 each has four genes,the?subunit gene just one.PCR primers were designed for the?subunit gene sequence given by transcriptome sequencing,so that the amplified fragment covered the entire amino acid sequence(177 amino acids)of the?subunit.The g DNA extracted by CTAB method and the c DNA obtained by reverse transcription were subjected to PCR amplification.The two?subunits gene sequences were amplified and purified,then were subjected to polyacrylamide gel electrophoresis.The results showed that the double-stranded DNA samples had only one band,the samples denatured by 95?had two single-stranded bands and one double-stranded band,the?subunit sequence reported in 1990 and 2014were not found in the two DNA samples.It suggests that the two?subunits may be encoded by the same gene,and there is no difference at the RNA level.The sequence of the?subunit gene amplified by g DNA and c DNA was completely consistent,indicating that the gene has no intron.Therefore,the difference of the two?subunits should not arise from the splicing process after transcription.The low light of level 0 12h is conducive to the accumulation of PC645 in C.placoidea,at this condition,Chl a/c-light-harvesting complex scale is relatively minimal,a larger proportion of light energy capture depends on phycocyanin;The effect of increased light intensity on Chl a/c-peripheral light-harvesting complex in C.placoidea during the log-growth phase was not significant,mainly affects the water soluble phycobiliprotein,it is speculated that the phycobiliprotein may play a greater regulatory role in the high-light adaptation mechanism of C.placoidea.The C.placoidea cells cultured under 6 different light conditions(level 0 12h,level 0 24h,level 1 12h,level 124h,level 2 12h,and level 2 24h)were broken and subjected to Sephadex G-100chromatography twice.PC645 samples with a ratio of A645/A280>7 and a proper purity after checking by Native-PAGE electrophoresis were collected and subjected to SDS-PAGE electrophoresis to analyze the contents of two?subunits.The results showed that the light culture conditions effected on the relative content of?2 subunit in PC645 of C.placoidea.PC645 exhibit a highest ratio of?2subunit cultured at level 1 12h and 24h.Too low or high intensity of light were not conducive to the accumulation of?2 subunits in PC645 samples,but showed no noticeable effects on the relative total content of?subunit.The contents of ?2 subunit among the 70%?100%PC645 samples obtained by ammonium sulfate fractionation precipitation were different,which caused the difference in water solubility of these PC645 samples.Therefore,it is proposed that PC645 has different forms of heterodimers.The 100%PC645 with the strongest water solubility only contains?1?2?1?1,while the 70%?90%samples with relatively weak water solubility may consist of different proportions of?1?2?1?2 and?1?2?1?1.The?1?2?1?2 is more hydrophobic than?1?2?1?1 because the?2 subunit with a more acidic isoelectric point that could neutralize the charges of the alkaline?subunits.Thus?1?2?1?2may play a more important role in the process of interaction and energy transfer with the lipid soluble chlorophyll-protein complex on the membranes.The 70%?100%PC645 samples contain different proportions of?2 subunits exhibited the same CD spectra at far ultraviolet and visible range,but different at near ultraviolet region,indicating the same chromophore types and protein microenvironment but different tertiary structure of the apoprotein.The two isolated?subunits had similar absorption spectra,fluorescence spectra as well as visible region CD spectra.But the differences of far ultraviolet CD spectra suggested that their spatial structures are different.Since?1and?2 subunits are expressed by the same gene,and consist of the same number of amino acid residues and similar sequence,therefore,in a sense,they are considered as conformational isomers. |
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