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| Cloning of SbSKIP Gene from Sorghum (Sorghum bicolor) and Analysis of Drought-resistant Function in Tobacco (Nicotiana tabacum) |
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Abstract SKIP (ski-interacting protein) is a kind of metabolism related protein, playing an important role in regulating resistance to abiotic stress. In this study, the SbSKIP gene of Sorghum bicolor was cloned, whose cDNA encoding region included 1 485 bp and encoded 494 amino acids. Phylogenetic tree analysis showed that the SbSKIP gene of sorghum had the highest homology with Aegilops tauschii, and the second was Arabidopsis thaliana. According to phylogenetic tree analysis data, the amino acid sequence of SbSKIP had 52% similarity with Aegilops tauschii and 42% similarity with Populus trichocarpa (XP_002324443.2), Gossypium arboreum (KHG23995.1), Glycine max(ACY79501.1), Arabidopsis thaliana (NP_565151.1), respectively. Quantitative PCR analysis result showed that under the condition of simulated drought (16.5% PEG 6000), SbSKIP gene expression in leaves and roots did not changed significantly in 0 h, but it increased along with processing time. After 16 h of drought treatment, the expression significantly increased more than twice. And gene expression of sorghum leaf area was obviously higher than that of roots. We had constructed plant expression vector pSH-SbSKIP and transferred the vector to tabacco (Nicotiana tabacum) through leaf disc method transformation. And after resistance selection and PCR identification, we got a total of 25 strains of transgenic plants and harvested T0 generation seeds. TP4-4, TP4-9, TP4-13 had a high expression level of SbSKIP among transgenic plant. And the 3 different T0 plant strains TP4-4, TP4-9 and TP4-13 were used to analyze the seed germination and seedling drought tolerance at 0, 150 and 300 mmol/L mannitol simulated drought conditions, respectively. Results showed that the seed germination rate of transgenic plants were over 60% higher than that of wild type. As well as the root growth of wild type was obviously suppressed at the seedling stage under the 300 mmol/L mannitol treatment for 10 d. Under the natural condition with 20% PEG 6000, the seedlings which Long out of the 5~6 leaves were treated for 14 d. Results showed that the bottom of transgenic tobacco leaves margin slightly yellowed, and the bottom of wild type tobacco leaves mostly yellowed and even showed signs of wilting. And the average activity of super-oxide dismutase (SOD) of transgenic plants was 91.2% of that in the wild type, and H2O2, malondialdehyde (MDA) respectively than that of the wild type reduces the 42% and 32.7%, the content of soluble sugar was 1.1 times than that of wild type. Preliminary identification of SbSKIP gene expression to improve the drought tolerance of tobacco plants, and provides the basis for the further study SbSKIP gene function and the gene cloning and functional analysis and provide basic data for the creation of transgenic drought resistant materials.
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Received: 15 May 2016
Published: 06 August 2016
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