| Abiotic stress such as high salt, drought and chilling seriously affects the growth of crops, and causes the significant loss of agricultural economy. It is very important to study plant stress tolerance. Olimarabidopsis pumila belongs to cruciferae, which was widely distributed in semi-arid and semi-salinized regions of the Xinjiang, and compare with Arabidopsis thaliana, O. pumila were more tolerance to salt. Here, we aim to mine salt tolerance gene by analysis c DNA library of O. pumila.Object: The normalized c DNA library of O. pumila was used to sequence c DNA clong at random, and analyse expressed sequence tags(EST) by bioinformatics. The destination library of O. pumila was built by Gateway technology, and transformed into A. thaliana to construct a transformant library to mine the salt-tolerance genes from O. pumila library, and proceed to analyse preliminary gene function.Methods: About 20 000 clones randomly selected from c DNA library of O. pumila, sequencing and analysis of ESTs were perform, including removal of low quality ESTs and pollution of the stitching, sequence assemble, BLAST analysis, functional annotation, GO classification, COG classification, and KEGG pathway analysis. Gene expression level under salt stress of stress resistance genes and the NAC transcription factors in the library were analyzed by quantitative Real-time PCR(q RT-PCR).The O. pumila destination library was constructed by Gateway technology, and was mobilized into Agrobacterium tumefaciens GV3101 and transformed in A. thaliana. The A. thaliana seeds were harvest for high throughput screening.Two NAC transcription factor genes Op NAC026 and Op NAC083 were cloned by RT-PCR, and analyzed by bioinformatics. Their expression pattern under highly salt stress was performed by q RT-PCR method. The over expression vectors of plant were constructed, and transformed in tobacco and detect the physiological indexes.Results:(1) By sequence analysis, 16 146 high-quality ESTs were generated and assembled into 8 893 unique sequences including of 2 486 contigs and 6 407 singletons. 4 777 unigenes were categorized according to the gene ontology(GO) hierarchy, 3 651 unigenes were categorized according to the COG, 8 301 unigenes were related to KEGG path way.(2) The capacity of primary expression library and normalized expression library was 4.61×104 clone formed unit(cfu) and 5.96×104(cfu), respectively. Two transgenic plants were generated.(3) The full-length cDNA of Op NAC026 was 1 327 bp, the open reading frame(ORF) of which was 906 bp in length and encoded a putative 301 amino acids. Phylogenetic analysis revealed that Op NAC026 had high sequence similarities with At NAC026 and At NAM of A. thaliana. Op NAC026 displayed a much broader expression range at different O. pumila tissues, with a highest expression in leaves. Op NAC026 transcription level was up-regulated as 24 h of Na Cl treatment, 12 h of 20 % PEG-6000 treatment, 6 h of ABA treatment and 8 h of 4 ℃ treatment, respectively. Our research indicated that transgenic tobacco of Op NAC026 had more potential for salt tolerance.(4) The Op NAC083 ORF was 723 bp, encoded a putative 240 amino acids. Phylogenetic analysis revealed that Op NAC083 had high sequence similarities with At NAC083 and ANAC083 of A. thaliana and they belong to the same clade. Op NAC083 displayed a much broader expression range in O. pumila different tissues, with a highest expression in leaves. Op NAC083 transcription level was up-regulated as 2 h of Na Cl treatment and 6 h of ABA treatment. However, its transcription was inhibited by PEG-6000 and 4 ℃ treatment. Our research indicated that transgenic tobacco of Op NAC083 had more potential for salt tolerance. |
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