| Watermelon(Citrullus lanatus) is an economically important horticultural crop with extensive cultivation and consumption. Low temperature is a common environment stress factor limiting agricultural production. Being a thermophilic crop, watermelon frequently suffers from the low temperature stress in the early Spring and off-season cultivations resulting in great economic loss. Elucidating the functions of low temperature responsive genes and their regulating networks in watermelon not only can help us understand the tolerant mechanism of watermelon to low temperature, but also can provide valuable information for the genetic improvement of low temperature tolerant watermelon cultivars. Results on model plants show MYB transcription factors play important regulating roles in response to low temperature stress. In our previous RNAseq analysis, we identified a MYB transcription factor(Cla007586), which was signiciantly upregulated in watermelon suffering from low temperature stress. Whether this MYB transcription factor has the function of regulating low temperature tolerance and how it performs the function in watermelon are still unknown. To address these problems, we cloned this gene, overexpressed it in tobacco, and analyzed the responses of transgenic plants to low temperature stress. Meanwhile, we also screened out some proteins interacting with its coding protein in watermelon by yeast two-hybrid system. The results are as the following.1. Sequence analysis showed that Cla007586 is a typical R2R3-MYB transcription factor. Its open reading frame(ORF) is 861 bp in length, encoding 286 amino residues with an predicted molecular mass of 32.6 kDa and an isoelectric point of 5.79. Both trancriptome data and q RT-PCR analysis revealed that Cla007586 was up-regulated by low temperature stress.2. Analysis of protein subcellular localization showed that Cla007586 was located in the nucleus. We constructed plant expression vector pK7FWG2-Cla007586 and transformed it into Nicotiana benthamiana to express transiently. Then, under the confocal microscopy we found that the green fluorescence distributed in the nucleus and overlapped with the fluorescence of the nucleus dye DAPI in the leaf epidermal cells.3. The full-length ORF of Cla007586 was subcloned into the plant expression vector pHellsgate8 and transfromed into tobacco(Nicotiana tabacum L. cv. SR1) by Agrobacterium tumefaciens LBA4404-mediated leaf disc method. By PCR and RT-PCR validation, two transgenic tobacco plants, OE3 and OE5, were selected and their T1 seeds were harvested.4. Responses of transgenic tobacco plants to low temperature stress were analyzed. The transgenic plants and the wild type plants showed no differences on phenotypes under normal growth condition. However, compared to the wild type plants, the leaves of transgenic seedlings suffered more serious water loss, severe wilting, higher membrane damage and membrane lipid peroxide degree under 4℃ low temperature stress.5. The proteins that interact with Cla007586 were identified in watermelon by yeast two-hybrid system. A normalized cDNA library derived from watermelon leaves was constructed and transformed into Y187 strains. Meanwhile, the bait strains Y2 HGold including pGBKT7-Cla007586 vector were constructed. Transcription activation and toxicity tests showed that bait vector p GBKT7-Cla007586 was nontoxic and inactive under the concentration of 150 ng/μL AbA. Through yeast mating, 13 candidate interacting proteins were identified, and two of them, namely A02 and A10, were selected for further analysis. A02 encodes Omega-3 fatty acid desaturase, which can change the content of unsaturated fatty acid in the membrane by catalyzing 16:2 or 18:2 to 16:3 or 18:3 and then enhance plant tolerance to low temperature stress. A10 encodes heat shock transcription factor.These two genes were all up-regulated in response to low temperature in watermelon, indicating that they may interact with Cla007586 to regulate the response of watermelon to low temperature stress at transcription level. |
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