Molecular Cloning And Functional Analysis Of Anti-freeze Genes From Chlamydomonas Sp.ICE-L

Antarctic ice algae Chlamydomonas sp. ICE-L is an excellent material for thegreen photosynthetic organisms stress acclimation mechanism study because of thefreezing environment it inhabits.To conduct a transcriptom analysis of Chlamydomonas sp. ICE-L under freezingstress conditions and explore the molecular biology mechanism of ice alga acclimationto the stress, we conducted a transcriptom analysis through batch real-time PCRanalysis. Total304genes which matched to the known sequences in the GenBank werechoosn for real-time PCR analysis under freezing stress. The expression of62geneswere up-regulated and29were down-regulated at freezing conditions compared to7℃control.The results indicated that, under freezing stress, some glucose metabolism-relatedgene expression was significantly upregulated, such as starch synthase gene.Photosynthesis is very important for sea ice photosynthetic organisms to adapt to thefreezing environment, our study also confirmed that in order to increase light-harvestingefficiency, the expressions of several light-harvesting proteins and photosynthesis-related protein were significantly up-regulated. In order to maintain the stability of thestructure of a variety of protein molecules under the frozen stress conditions, a varietyof molecular chaperone genes expressions, such as HSP70and HSP90, weresignificantly up-regulated. In order to ensure the smooth process of transcription,translation, the cold activation of RNA-binding protein gene and RNA helicase geneexpression levels were significantly increased, and these genes play a very importantrole in the process of adaptation to the low temperature of the Antarctic ice algae.Among the up-regulation expressed genes, nitrate reductase (NR) and cold inducedglycine-rich RNA-binding protein (GRP) were selected for further characteristic andfunctional study.Nitrate reductase plays an important role in the abiotic stress adaptation in plantsby regulating nitrogen metabolism. The full-length of Chlamydomonas sp. ICE-L NRgene contained an open reading frame of2,589bp encoding a nitrate reductase of863amino acids. Phylogenetic analysis showed that the gene was homologous to knowngreen algae NRs with identity of63%,61%,60%and54%to Volvox carteri, Chlamydomonas reinhardtii, Dunaliella tertiolecta and Chlorella vulgaris respectively.Sequence alignment indicated that there were differences among the active sites ofgreen algae, which may correspond to their uniquely enzymatics characteristics. Inaddition, the tertiary structure of ICE-L NR protein sequence was predicted. Theexpressions of ICE-L NR gene under different temperatures（-20℃，-12℃，0℃，7℃，16℃） and times （0.5h，1.5h，3h，6h，12h）were analyzed throughreal-time PCR. The results indicated that with the decrease of temperature, theexpression of ICE-L NR gene was up-regulated. It was4folds higher under-20℃compared to16℃. The expression level was the highest after freezing treatment for3hwith a relative change of3.1. ICE-L nitrate reductase activity was measured by themethod of in vitro measured activity and found that at low temperature, nitratereductase activity remained at a high level.Cold activation of RNA binding proteins are important transcription factors play animportant role in many species of cold adaptation process. An GRP gene ofChlamydomonas sp. ICE-L, was identified from the cDNA library and sequenced. Thefull-length of ICE-L GRP gene contained an open reading frame of468bp encoding aprotein of155amino acids. Phylogenetic analysis showed that the gene washomologous to known Physcomitrella patens Subsp and Prunus avium. Sequencealignment indicated that there were two conserved RRM domains among the active sitesof green algae, which may correspond to their low-temperature sensitive characteristics.In addition, the GRP gene was ligated into plasmid pET-28a(+) and transformed into E.coli BL21(DE3). SDS-PAGE analysis showed that there was substantive expression ofGRP in E. coli.A large number of functional genes relevant to freezing adaptation has beendiscovered. This study will be helpful in revealing adaptive mechanism of ice alga tothe extreme environments of Antarctic. And will be helpful in finding the potentialapplications of the functional gene of the Antarctic ice algae in improving the anti-stressability of economic crops.