Fusion Of P. Trifoliata CBF And GFP And Transformation Into Tobacco And Trifoliate Orange

Low temperature is one of the most important factors which restricts plant growth and distribution of the plants.As a species of subtropical fruit tree,citrus plant is restrained by low temperature at cultural area.Natural recurrent low temperature often decreases productivity and quality of fruit,and even causes death in severe case,which results in a great loss of yield and commercial value. So far,the problem of freezing harm occurred in oranges has not dissolved efficiently in the world yet.Thus,breeding the varieties for tolerance to low temperature by using the technology of genetic engineering of plant molecule biology,has become the effective way to dissolve it ultimately.Plant cold resistance,as a complex quantitative trait,is a result of cooperation with multiple genes,suggesting that single gene transformation may only partly enhance the cold tolerance of the plants,to some extent.Therefore, transformation of a series of genes related to cold acclimation,is a key factor in regulating the gene expression.CBF,induced by low temperature rapidly,can improve transcriptional levels of these genes by binding to CRT/DRE element specially,resulting in the synthesis of saccharide,proline,proteins contributed to the stabilization of biomembrane,and some other substances,which subsequently enhanced cold tolerance of the plant.Further studies on gene transformation testified that cold tolerance of the plants containing high expressed foreign CBF gene is improved greatly.This suggests a new way of improving cold tolerance of plant by genetic engineering technique.Trifoliate orange(Poncirus trifoliata(L.) Raf.),is a rootstock commonly used for citrus production as it high tolerance to cold even the extreme low temperature -20℃.Previously studies in our laboratory have separated the CBF gene in P.trifoliata,but the knowledge of its function and expression mode had not been fully understood yet.It is necessary to choose the appropriate promoter in research of gene expression.Promoter plays the most important role in gene expression.Therefore,this study was to explore the function and expression mode of CBF gene,in order to utilize the CBF gene in enhancing the cold tolerance of citrus plants. This study cloned CBF from trifoliate orange,sequenced the CBF gene including its promoter,performed the fusion expression of the promoter with the GFP gene,and then transferred them into tobacco and P.trifoliata in order to observe the space-time expression mode.The details of these results are as follows:①Construction fusion gene CG..Using P.trifoliata DNA as the template, CBF and its own promoter as a complete sequence were cloned from P. trifoliata.Import amplified product of 5'introduction into EcoRI restriction sites and import 3' digestion into SalI points.Results of sequencing showed that amplified sequence and the sequences of the target gene had the same expression.After digestion the CBF and GFP sequence have the same sticky end,with T4-ligase two fragments connected as a template.Using CBF upstream primer,GFP downstream primers PCR amplified.It was noticed that integration gene shift,amplified product can be verified via sequencing as the purpose fusion gene,named CG.For a total length of 2.4 kb,it is a self-Poncirus CBF gene promoter,fusion gene of P.trifoliata CBF and the GFP.②Construction fusion gene expression vector CG2301.pCAMBIA2301 and fusion gene CG were digested with double restriction enzyme EcoRI and Bstp based on expression carrier pCAMBIA2301,then were linked with T4 ligase and fusion gene expression carrier CG2301 were constructed.This carrier contains fusion gene of CBF and GFP and was promoted by CBF self promoter, which results in Kan tolerance.③Fusion gene expression vector CG2301 was transformed into tobaccos by leaf-law via agrobacterium- mediated,which had received 43 resistance seedlings having roots.13 of them were amplified by PCR identification to show that the target gene has transformed into tobacco successfully.④Fusion gene expression vector CG2301 was transformed into P.trifoliata by hypocotyl via agrobacterium-mediated,which had received resistance seedlings.2 of 14 resistance seedlings were amplified p by PCR identification to show that the target gene has transformed into tobacco successfully.⑤Expression patterns of fusion gene CG in transgenic tobacco plants were studied.Tthe plants were treated by different low temperature between 4℃and -5℃temperature at interval time,and gene expression instance in different organs were observed using fluorescence microscopy.The results showed that the fluorescence firstly expressed in the root(under 4℃after 2 h and under -5℃after 15 min),followed by the leaves(under 4℃after12 h and under -5℃after 15 min with a small a mount of fluorescent can be detected),stems expressed at last(under 4℃after 48 h and under -5℃after 30 min with a small amount of fluorescent can be detected).Fluorescence in the leaves firstly appeared in the organ of vein,and increased with time,indicating that the expression increased at that condition.When a wound occurred in leaves,much fluorescence can be detected around the wound.In the roots and stems, fluorescence can be constantly strengthened with time when they received the same low-temperature stress.