| Sugarcane is one of important sugar crop and economic crop. It is a pathway of sugarcane and canesugar development to increase sugar content, yield, and stress resistance of sugarcane by breeding new cultivars and improving culitivation techniques. Ethylene plays important regulational effects in plant growth and development. There are some researches on signal transduction and related genes of ethylene regulation, and some ethylene receptor genes had been gained, but its regulational mechanism is unclear. Ethephon increased significantly sugarcane yield, sugar content, and stress resistance, but it regulational molecular mechanism is unclear. The expression, transformation and promoter sequence of SoERSl were studied with ROC22as material. The main results are as follows.1. Expression analysis of SoERSl. The leavels of SoERSl expression was mature leaf> young leaf>old leaf in elongation stage and process maturity stage in ROC22. The expression of SoERS1was higher in old stem than in young stem and mature stem, that of mature stem was higher than that of young stem in elongation stage, but it was reverse in process maturity stage. SoERS1expression could be detected in different tissues at the beginning of flowering, it showed that SoERS1expression may be constitutive expression. Drought and dark treatments inhibited SoERS1expression of mature leaf. SoERS1expression increased significantly in mature leaf at1st d after100mg/L ethephon treatment. The expression of SoERSl in mature leaf of seedling stage decreased firstly, then increased after6℃low temperature stress, but the expression increased in the leaf of recovery growth at2d after low temperature stress.2. The construction of plant expression vector. According to sugarcane ethylene receptor gene (GQ369007) sequence from ROC20, primers were designed by coding region sequence, and the complete coding frame sequence of ERS was cloned, and it was named as SoERSl. Xba I and Sma I restriction enzyme sites were added to upstream and downstream primers, sense expression vector pBIERS of SoERSl was built. SoERS1gene siRNA interference segment was forecasted and analyzed by siRNA Target Finder software, upstrem403bp of coding region was selected as interference segment.403bp was inserted into the intermediate vector176with forward and reverse type, and then intermediate vector176was double digested by EcoR Ⅰ and Hind Ⅲ. The target gene was connected to pCAMBIA1300to build RNAi interference vector p1300ERS.3. SoERSl transformation and prokaryotic expression.Sense expression vector pBIERS was transformated into tobacco K346by Agrobacterium-mediated method. SoERS1gene had been integrated into tobacco genome DNA by PCR detection of target fragment and vector GFP gene sequences. Transformation efficiency was61%. Spraying200mg/L ethephon on transgenic tobacco at seeding stage increased ethylene release rate in leaf.No fusion expression vector pETERS1and fusion expression vector pETERS2did not expressed in prokaryotic strain BL21(DE3) and Rosetta (DE3). It may be relate to high rare codon content in gene sequence, or some chracters of enzyme protein unsuitable for the expression in prokaryotic cells.4. Cloning and analysis of promoter sequence of SoERS1. Gene promoter sequence of SoERSl was cloned by chromosome walking technology based on thermal asymmetric interlaced PCR principle, and it was1410bp sharing82%and80%homology with the sequence of ERS25and ERS14of corn, respectively. The accession number is KC862312in GeneBank. The gene promoter sequence of SoERSl is suggested that it has promoter acting element TATA-BOX and CAAT-BOX, light-responsive elements, drought-induced MYB binding sites, methyl jasmonate-responsive element, salicylic acid response element, endosperm expression cis-regulatory elements, heat stress responsive elements et al.,its expression may be affected by the regulation of physiological cycle, hormone, drought, light and other factors by PlantCARE and PLACE online analysis. |
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