Transfomation Of Fuse Gene3α-HSD/CR-aiiA And Two Singnal Genes3α-HSD/CR And AiiA In Tobacco And Expression Analysis

With the rapid development of human economy, a lot of waste gas, waste water and wasteswere discharged into the environment, which leads to increasing environmental pollution.3α-HSD/CR gene, a key enzyme3α-hydroxysteroid dehydrogenase/carbonyl reductase(3α-HSD/CR) gene from Comamonas testosteroni, is very good degradation function for thesteroid compounds of environmental pollutants. aiiA gene is an acyl homoserine lactones(AHLs)gene from Bacillus thuringiensis(Bt.). AiiA protein can open the lactone bonds of AHLs producedby Erwinia carotovora, leading to AHLs lost signal molecules function which decreases thevirulence of bacterial pathogens. In recent years, with the rapid development of the plant genicengineering," superorganism " is major concerned and transgene develops from the single gene tomultiple genes. But, it is difficult to transform multigene for the lack of the capacity and thenumber of vector. The technology of fusion genetic transformation and constructing fusionexpression vector is developed, which has opened up a new way of multigene genetictransformation.In this study, the plant expression vectors containing fusion gene3α-HSD/CR and aiiA wereconstructed and transferred to tobacco plants, then studied the exprenssion of fusion gene intobacco plants. The main methods and results were as follows:1. The plant expression vectors of fusion gene3α-HSD/CR-aiiA were constructed and namedpTCK303-3α-HSD/CR-aiiA. In addition, two plant expression vectors respectively containing3α-HSD/CR and aiiA were constucted as control to study the expression of fusion gene. Theresults of restriction endonuclease digestion and sequencing showed that the vectors weresuccessfully constructed.2. Fusion gene3α-HSD/CR-aiiA and two singnal genes3α-HSD/CR and aiiA weretransferred to tobacco plants by agrobacterium mediated method. The resoults of PCR and GUShistochemical staining showed that transgenic tobacco plants containing these genes weresuccessfully obtained.3. The results of enzyme-linked immunosorbent assay (ELISA) showed that3α-HSD/CRprotein were sucessfully expressed in transgenic tobacco plants containing3α-HSD/CR-aiiA and3α-HSD/CR. 4. The cholesterol degradation test showed that the rate of cholesterol degradated bytransgenic tobacco plants containing3α-HSD/CR-aiiA and3α-HSD/CR is about70%, much higherthan the control.5. The leaves from the transgenic tobacco plants containing3α-HSD/CR-aiiA and aiiA wereinoculated Erwinia carotovora, and the results showed that transgenic tobacco leaves from thebody had the inhibition to soft rot caused by Erwinia carotovora.6. The cholesterol degradation test and the disease resistance test revealed that: transgenictobacco plants containing fusion gene3α-HSD/CR-aiiA expressed both3α-HSD/CR and AiiAproteins.So as to transgenic tobacco plants of restorated envirment and resistanted diseases wereobtained.