Cloning And Expression Analysis Of XTH Gene Of Mirabilis Jalapa L.under Petroleum Stress

With the economic development and the largely demand for oil extraction,in the process of transportation and processing caused by the surrounding soil,water and other serious environmental pollution,become a environmental science to domain problem,as an environmentally friendly phytoremediation strategy has become a hotspot in polluting ecology.In this study,Mirabilis jalapa L.is used as the tested plant.According to preliminary results of transcriptome sequencing,the expression of XTH gene of Mirabilis jalapa L.growing up in oil pollution soil was significant different from that growing up in clean soil.Thus,we studied further on this gene.The gene function was analyzed by cloning the gene,bioinformatics analysis,localization and identification and related physiological indexes.The results of this study are as follows:1.The Mirabilis jalapa L.were cultivated in the soil with oil concentration of 0 g/kg,5 g/kg,10 g/kg,20 g/kg,30 g/kg and 40 g/kg for 12 h,24 h,36 h,48 h,72 h the Mirabilis jalapa L.roots to do the relevant physiological indicators of the determination.The activities of SOD,POD and CAT were the highest at 24h,and the contents of soluble protein and MDA were the highest in 24h.When the oil concentration was 20 g/kg,the activity of SOD and POD was higher than that of SOD and POD activities at the corresponding time points;Soluble protein and MDA content were also higher than the other time the corresponding concentration of soluble protein and MDA content.The activity of CAT was higher than that of other petroleum concentrations at 5 g/kg.SOD,POD,CAT activity and soluble protein content were treated with xyloglucan,but the content of MDA was opposite.2.The Mirabilis jalapa L.were cultivated in the soil at 0 g/kg,5 g/kg,10 g/kg,20 g/kg,30 g/kg,40 g/kg soil for 0 h,12 h,24 h,36 h,48 h,72 h,and the XTH gene was analyzed by real-time fluorescence quantitative PCR.The results showed that the relati’ve expression of XTH gene increased with the increase of time at the concentration of 5 g/kg,10 g/kg,20 g/kg,30 g/kg and 40 g/kg.When the oil concentration was 5 g/kg and 10 g/kg,the relative expression of XTH gene increased at 12 h-36 h,and decreased at 36 h-72 h.When the oil concentration was 20 g/kg,30 g/kg and 40 g/kg,the relative expression of XTH gene increased at 12 h-24h,and the relative expression was decreased at 24h-72 h.3.By bioinformatics software analysis,the coding length of the gene is 887 bp,encoding 285 amino acids,is a secretory protein.Through the MEGA 5.0 software to construct the phylogenetic tree,we can see that it has the closest relationship with PtXTH1,the PtXTH1 gene has xyloglucanoglycoside(XET)activity.It was also found that MirXTH protein had xyloglucanase activity(XET)activity by homologous sequence alignment,and the catalytic activity domain DEIDFEDFLG.4.The GFP-tagged MirXTH gene was transformed into onion epidermal cells by means of a gene gun,it was found that the gene was located on the protoplast by confocal microscopy.It was a gene related to cell wall metabolism and involved in regulating the ductility of cell wall.5.The XTH gene was ligated into pCAMBIA 1302 eukaryotic expression vector by in vitro amplification and then successfully transferred to Agrobacterium tumefaciens(LBA 4404),then preconceived into wild-type Arabidopsis,do transgenic validation of its function.