Construction Of Fruit Specific Expression Ipt Gene And Establishment Of Transformation System In Actinidia Deliciosa

Kiwifruit belongs to the family Actinidiaceae, genus Actinidia, which is one of the new fruits in the world. It is very popular owing to its special flavor and high content of vitamin C. However, fruit of some Actinidia species still need to be improved further. The aim of the research is to construct isopentenyltransferase (ipt) gene expressed mainly in the fruit using gene recombined technology to improve the kiwifruit qualities.Ipt is the key enzyme for the biosynthesis of cytokinins. The physiological function and role of cytokinins is multiplicate for plants, such as inducing and accelerating cell division, inducing histiocytic differentiation, promoting fruit growth etc. In practice, the ratio of fruit setting, the weight of fruit and the ratio of sugar to acid are increased with cppu, which is one of cytokinins. Fang reported that the weight of single fruit was increased to 20%~190% using cppu in kiwifruit. Some consequences of the manipulation of growth in wheat and triticale proved that the weight of seeds was effectively increased. As far as genetic transformation is concerned, the ipt gene has been used in many plants in present studies, such as rice, tobacco, cotton, tomato, etc. However, there are no such reports in kiwifruit. The Actinidin gene promoter has been isolated to ensure the ipt gene only expresses in fruits. Actinidin is a kind of cysteine protein enzyme only found in the fruit of kiwifruit. It is feasible to have the ipt gene driven by the Actinidin gene promoter. In the study, we use the pRA-9 plasmid as a starting plasmid to construct the ipt gene driven by kiwifruit fruit specific promoter-the Actinidin gene promoter.In addition, tissue culture is the groundwork of genetic engineering. Many studies show it is better for plant to regenerate with callus from seedling. So, it is necessary to establish the systems of seedling tissue culture. The growing characters of seedlings on 6 media without any hormone were investigated and the tissue culture systems of seedling was established, which is important for further research on genetic transformation of kiwifruit.The main results are as the follow:1. After enzyme digestion of pRA-9 and collection of Actinidin gene promoter (1.3kb) from agarose, we linked the promoter with pUC119 plasmid and transformed it into Exoli DH5a. Then we identified the obtained recombinant by PCR and double digestion. The results showed the cloning vector was successfully constructed.2. Using suitable enzymes to cut the plasmid pRA-9 and pGEM-T Easy, we recovered the fragments of Actinidin gene promoter (1.3kb) and ipt gene (0.72kb). Then linked these two fragments with pBI121 plasmid and transformed it into E.coli DH5a. The obtained positive recombinant is proved to be Actinidin-ipt chimeric gene by PCR and by double digestion.3. We have established the primary tissue culture systems of seedling for Actinidia deliciosa, and studied the growing characters of seedlings on 6 media without any hormone. The media are (1) MS+Sucorse 10g L-1; (2) MS+Sucorse 20g L-1;(3) MS+Sucorse 30g L-1; (4) 1/2MS+Sucorse 10g L-1; (5) 1/2MS+Sucorse 20g L-1; (6) 1/2MS+Sucorse 30g L-1 All the above media were added 100 mg L-1 inositol and 0.75 % agar. In this study, the best medium MS + Sucrose 20 g L-1 for the growth of seedlings was selected.4. As for the genetic transformation of green fluorescent protein gene (GFP) , the protoplast from leaves of A. deliciosa was used. Transient expression of GFP showedthat the gene was successfully transferred into A. deliciosa protoplasts by PEG method.In this thesis, the construction of isopentenyltransferase (ipt) gene expressed mainly in the fruit of Actinidia deliciosa was successfully completed, which can offer a useful chimeric gene for further researches on genetic transformation of kiwifruit. In theory, the chimeric gene only expresses in the young fruit after pollination, but it is a long time to go to confirm that.