Construction Of A CDNA Library From The Ephemeral Plant Olimarabidopsis Pumila And Preliminary Analysis Of Expressed Sequence Tags

Olimarabidopsis pumila blongs to Cruciferae Olimarabidopsis, and its flower is yellow, andfruit coat. O. pumila is close relative of the model plant Arabidopsis thaliana, but it is moretolerant to salt stress than Arabidopsis thaliana O. pumila is widely distributed in semi-arid andsemi-salinized regions of the Xinjiang.Object: To isolate salt tolerance gene from this species, a normalized and full-length cDNAlibrary was constructed from the leaf tissues of O. pumila treated with500mmol/L NaCl by usingcap antibody to enrich full-length cDNA, and the use of gateway technology allowed libraryconstruction to be performed without traditional restriction enzyme cloning methods. Clonesrandomly selected were sequenced in the library. This study will provide an important resource forsalt-tolerant gene mining, comparative genomics, and genome evolution among Cruciferaespecies.Methods: A normalized and full-length cDNA library was constructed from the leaf tissues of O.pumila that was treated with500mM NaCl by gateway technology and genome saturated hybridtechnology.1151clones randomly selected from normalization library, sequencing and analysisof sequencing of EST, including removal of vector sequence and the low quality of sequence,sequence splicing and assembly, and analyzed by BLAST (basic local alignment search tool),functional annotation and classifications, and analyzed signaling pathway. The expression profileof contigs respectively under highly salt stress levels were analyzed by RT-PCR and qRT-PCRtechnical. Thefull-length gene of OpNHX1was identified by RT-PCR and5’-RACE technology.The plant expression vector and yeast expression vector were constructed, and transformed intoA.thaliana and yest mutant respectively by floral diping and acetic acid lithium respectively. Thephenotype of salt resistance of transgenic Arabidopsis was analyzed to verifiy gene function. Acalmodulin protein like gene OpCML13was cloned from normalized library by RT-PCRtechnique.Results:(1) The titers of the primary and the normalized cDNA library were1.6×106cfu/mL and6.7×106cfu/mL, respectively. The recombination rate of normalized cDNA library about95%,insert fragment average size is more than1kb.(2) By sequence analysis,894high-quality ESTs were generated and assembled into736unique sequences consisting of72contigs and664singletons. The736unigenes were similar to A.thaliana, A. lyrata, or Thellungiella salsuginea. The resulted unigenes were categorized accordingto the gene ontology (GO) hierarchy involving a wide range of biological processes. Analysis ofESTs related to various stresses showed that more stresses resistance related genes exist in the O.pumila. All ESTs data are commited to GenBank, and accession number: JZ151519~JZ152412.(3) Expression analysis to high abundance of16contigs in normalization library wereperformed, with different concentration of NaCl treatment for24h. RT-PCR results showed thatthe expression of contig10,11,14increased with increasing concentration of NaCl.Under200mmol/L NaCl treatment different time, RT-PCR results showed that the expression of contig7,9,11,16increased with the increasing of time. Under100μmol/L ABA treatment different time,RT-PCR results showed that the expression of contig1,5,9,15,16increased with the increasingof time.(4) The full-length cDNA of OpNHX1was2153bp, the open reading frame (ORF) of whichwas1605bp in length and encoded a putative534amino acids. Phylogenetic analysis revealedthat OpNHX1had high sequence similarities with AtNHX1and EhNHX1and they belong to thesame clade. OpNHX1expression was detected in roots, stems, leaves, flowers and siliques of O.pumila, with the highest expression in stems. The expression of OpNHX1was induced by salt,PEG, cold, and abscisic acid. Importantly, overexpression of OpNHX1in A. thaliana improved the survival rate of transgenic plants under salt stress. Heterologous expression of OpNHX1inW303-1B Δnhx1yeast Na+/H+antiporter mutants could complement the mutant phenotype.(5) The ORF of OpCML13gene contained447bp encoding a peptide of148amino acidsresidues. The structural analysis of OpCML13showed that it included four EF-hands motifs, andit is a hydrophilic protein. Phylogenetic tree revealed that OpCML13showed closer genetickinship with AtCML13and AtCML14of Aarbidopsis thaliana indicating that they belonged to thesame evolutionary branch. The tertiary structural analysis of OpCML13through Swiss-modelshowed that it contained eight α-helixes and ten β-turns. Expression analysis by RT-PCR indicatedthat OpCML13displayed a much broader expression range in different tissues in O. pumila, with amaximum expression in root and the expression of OpCML13is induced by salt, PEG, cold, andabscisic acid.Conclusion: A good-quality primary and normalized cDNA library was constructed,894high-quality ESTs were generated and assembled into736unique sequences consisting of72contigs and664singletons. Expression analysis to high abundance of16contigs in normalizationlibrary showed that a large number of salt-tolerant genes in the library. The OpNHX1andOpCML13genes of O. pumila were cloned. Genetically modified studies showed that OpNHX1can improve salt tolerance ability. OpCML13was induced by salt, PEG, cold, and abscisic acid,indicating that played an important role in adversity stresses.