Proline Cis-Trans Isomerase-cyp And Fkbp Of Gracilaria Lemaneiformis, Gene Cloning And Expression Analysis Under Heat Stress

Gracilaria lemaneiformis is an important agarophyte. Breeding of hightemperature resistant strains for prolong the cultivation period and increase productionis of great significance. In order to breed high temperature resistant strains moreeffectively, the molecular mechanism of G. lemaneiformis response to heat stressbecomes to an important problem to be researched. In our previous work, the ESTs ofcyp and fkbp of peptidyl-prolyl cis-trans isomerase(PPIase) were screened from thedifferentially expressed cDNA library of G. lemaneiformis under high temperaturestress, which indicated that PPIase played an important role in the response of G.lemaneiformis to heat stress. PPIase can catalyze proteins with proline residues foldingto natural spatial structure，so it has the ability to resist the environmental stresses, andregulate the immune response. The research of PPIase will lay foundation for study themolecular mechanism of heat response in G. lemaneiformis.In this experiment, cyp and fkbp genes of G. lemaneiformis were first cloned, andtheir copy number in genome was detected. At the same time,,expression of the twogenes in three strains-wide type, cultivar981and07-2of G. lemaneiformis underhigh temperature stress were measured and compared. The results showed:①The cypgene sequence is613bp long, and the GC content is55.2%.cDNA sequence is498bplong, encoding165amino acids. Compared to the cDNA sequence, the DNA sequencewas found to contain2exons and1intron. The cDNA sequences of cyp of the threestrains-wild type,981, and07-2are the same. The homology analysis shows that thehomology of cyp between G. lemaneiformis and Chlamydomonas reinhardtii (GenBankNo. XM001702591) is the highest（71%）. The cloned fkbp gene sequence is592bplong, and the GC content is51.4%，cDNA is of495bp long, encoding164amino acids.Compared to the cDNA sequence, the DNA of fkbp was found to contain2exons and one intron. The sequence homology analysis indicated that G. lemaneiformis was notclustered with no other algae.②With fkbp and cyp genes as probes of southernblotting，the copy number of the genes in the wild type,981, and07-2were detected tobe only one in the genome.③Fluorescence quantitative PCR was used to detect theexpression patterns of fkbp and cyp in G.. lemaneiformis under high temperature stress.The results showed that the cyp gene expression of the three strains under the conditionof32℃heat shock presented the change rule of―increase-decrease-increase‖, and thelowest level was appeared at36h. Expression quantity of07-2was higher than981,and even higher than the wild type. The expression of fkbp gene in the three strainssunder32℃heat condition showed different trends. The expression pattern of fkbp inwild type is―increase-decrease-increase‖and the quantity of expression is alwayshigher than that of the control group without heat stress; while in981and07-2, theexpression showed a trend of "decrease-increase-decrease", and the express quantitieswere inferior to that of the wild type. These researches showed that under the hightemperature stress, the wild type mainly increased the expression of fkbp, and thecultivar07-2mainly increased the expression of cyp gene to catalyze proteinscontaining proline residue to fold to natural structure in order to resist to heat stress.This experiment lays the foundation of studying mechanism of G. lemaneiformisresponse to high temperature, and also provides molecular basis to increase the heatresistant ability of G. lemaneiformis.