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Construction And Analysis Of Suppression Subtractive CDNA Library Of Up-regulated Genes From Ulva Pertusa Under Emersed Stress

Posted on:2013-08-05Degree:MasterType:Thesis
Country:ChinaCandidate:F YangFull Text:PDF
GTID:2253330395979935Subject:Marine biology
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To investigate functional genes differentially expressed in Ulva pertusa under emersed stress, a cDNA library of up-regulated genes was constructed with the cDNAs from U. pertusa treated with emersed stress simulation in the laboratory as tester and the cDNAs from U. pertusa in the normal condition as driver, by using the suppression subtractive hybridization(SSH) method.160clones were selected randomly to be sequenced from the cDNA library,150ESTs were obtained and accounted for93.8%of the total sequenced samples. Among thsse ESTs,21fragments were redundant sequences and other137fragments were non-redundant sequences which were all independent ESTs(Singleton). Results of analysis by some softwares and alignments indicated that the length of the ESTs were ranged from100to900bp and the average length was364bp. The peptises deduced from the ESTs had20%to60%similarities with amino acid sequences of the known functional proteins. It was found that22ESTs selected from the of alignments had more higher similarities with the sequences of higher plants and algae that genomes sequencing had been completed. The ESTs according to the functional annotation showed that, cell growth and development accounted for22.63%; protein synthesis and degradation accounted for5.84%; metabolism accounted for7.30%; stress response accounted for5.11%; photosynthesis and photoinduction accounted for5.11%; transcription regulation accounted for3.65%and signal transduction accounted for6.57%; there were60sequences with unknown function and no searching result and they accounted for24.09%and19.71%of total non-redundant sequences respectively, the ratio was quite high. The result of codon usage bias for22ESTs forecast and analysis showed that the nucleotide in the third position of codons was no significant differences(p>0.05) in U. pertusa and the GC frequency of ESTs sequences was50.66%, the usage of stop codon in U. pertusa preferred to TGA obviously.The expression levels of some selected genes, including Phytoene desaturase, Calmodulin,14-3-3-like protein and Osmotic stress-activated protein kinase from the cDNA library before and after emersed treatment, were measured by real-time fluorescence quantitative PCR, the results indicated that the expression of the genes were all up-regulated with the treated time polonged. The expression and regulation of those different genes might play an important role in the molecular response mechanism of the process of stress tolerance for U. pertusa emersed in the intertidal zone, they also provided some reference points for interpreting the regulatory network and molecular mechanism for stress tolerance resisting of intertidal algae.
Keywords/Search Tags:Ulva Pertusa, Emersed Stress, Suppression Subtractive Hybridization(SSH), cDNA Library, Real-time Fluorescence Quantitative PCR
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