Analysis Of Expressed Sequence Tags (ESTs) And Hsp90-3Gene In Senecio Scandens Buch.-Ham. Ex D. Don Based On Full-length CDNA Library

Objective:In order to understand the functional information of genetics, we constructed a full-length cDNA library in Senecio scandens Buch.-Ham. ex D. Don. Our results could play crucial roles in analyses of expressed sequence tags （ESTs）, and provide parameters for further exploring EST-SSRs （simple sequence repeats） in applications of molecular genetic map in Chinese traditional medicinal plants.Methods:Aliquots of total RNA extracted from leaves of seedlings using TRIzol-RNA Total RNA Isolation Kit in Senecio scandens Buch.-Ham. ex D. Don. We generated a full-length cDNA library from the antibacterial accession of Senecio scandens Buch.-Ham. ex D. Don material, using Creator SMART cDNA Library Construction Kit （CLONTECH） according to the manufacture’s suggestions. Titer of the library, full-length ratio and redundancy rate were evaluated by PCR amplification. Thus,600monoclonal sequences were randomly applied to perform further bioinformatics analysis based on satisfactory evaluations of the full-length-enriched cDNA library, using the software such as DNAMAN, Expasy Protparam, PROSITE, ProScale, CPHmodels-3.0and Swiss-model.Results:（1） As shown in the results, parameters of the quality of cDNA library were as follows:the capacity of the library （4.3X106cfu）, the titer of the library （1.3X106cfu/mL）, the average size of the inserted fragment （1.5kb）, the recombination rate （96.35%）, the full-length rate （64%） and the redundancy rate （3.28%）.（2）244unigenes of244effective ESTs sequence were found in the present study, with133non-annotated ESTs and80homologous sequences.Moreover,524full-length cDNA sequences were harvested in this study, involving467unigenes and5sequences associated with metabolism of pharmacological phenotypes.（3） Heat shock proteins （Hsp）, representing an important molecular chaperone in eukaryotic cells, was known to be a common response to development, stress resistance, signal transduction and evolutionary process in plants. The relationship between the structure and functional roles was elucidated in Hsp90based on generation of full-length cDNAs from Senecio scandens Buch.-Ham. ex D. Don. Sequence analysis of Hsp90-3 gene indicated that it shared93.71%identity with Arabidopsis thaliana （GenBank accession:NP₂00412.1）, encoding a protein composed of699amino acid residues with the predicted molecular weight of79.78kDa and the theoretical isoelectric point of5.08. Moreover, the distribution of Hsp90-3was involved in endomembrane system such as nucleus, peroxisomes, chloroplast thylakoid membrane, and choloplast matrix in the present study.Conclusion:（1） SMART technology was an effective way to construct a high quality of full-length cDNA library, and our results could be used for identification of functional genome, looking for new gene and researching on regulation of secondary metabolite biosynthesis expression in plants.（2） Numerous functional genes were related to a series of characteristics involving physiology, metabolic enzymes and resistance in Senecio scandens Buch.-Ham. ex D. Don.（3） Three-dimensional measurement of Hsp90-3protein revealed that it could play critical roles in molecular chaperone, signal transduction, transcriptional regulation and stress-response in higher plants.