Clone Of Ribosome-inactivating Protein Gene From Barley And Its Transformation Into Soybean

Soybean, one of annual legume plant, and originated in China. Nowadays, breeding and cultivating of soybean already widespread all over the world. As a frequent diet in East Asia, the sovbean owes rich nutrition and easy digestion. Mainly, the price is very low. Phytophthora root rot of soybean was regard as a common disease infect widely and hard therapy. It is able to cause much harm to soybean. Any stages during soybean life can be broken out and almost tissiues belong to soybean plant can be infected by soybean phytophthora root rot. The disease can greatly reduce production of soybean. The normal way against the disease achieved at some degree, but also with too much time work and serious pollution. The resistance to farm chemical and pesticide residues occured by tranditional way lead some hurt to people. Oppositely, the development on transgenic soybean material supply alternative way to solve the problem.Ribosome-inactivating Protein is a series of proteins that aginst virus, fungi and insect. It can harm ribosome during the cell, which lead to unable synethesis for proteins, final achieve the aim to kill the cell. Among nearly one hundred and fifty years of exploration and research, more than one hundred types of protein discovered, and the functional mechanism about RIPs had been found out primarily. Some of them already applied in some fields. Promoter plays a very important role during the gene transformation process, it is a high efficient way to express foreign gene by choosing favourable promoter. One of tissue specifie promoter, the root specific promoter applied to strart express foreign gene in the root tissue cell simply at special time,part and quantity, which not only aviold of some possible unexpected reaction in other tissue of plant, but also stop any waste metabolism appearing.In this study, oen of RIPs genes was cloned by PCR technology, and was connected into the plant expressed vector pCAMBIA1301-BADH that involved with aldehyde dehydrogenase BADH gene. Constructed safety ricombinant plant expressed vector pCAMBIA1301-BADH-RIP contained salt-tolerance selectable BADH as selected site. And the cotyledons of Soybean 'jinong 28'were chosed as explants for genetic transformation and were infected with the Agrobacterium involving marker-free expression vector containing RIPs and BADH binary genes. Selected and cultivated the recombinant tissue with selecting medium at 200mmol/L concentration of NaCl. The genomic DNA of transgenic plants were extracted and analyzed by PCR. Result showed that RIPs genes were transformed into the soybean successfully.The main results are following: (1) Safety recombinant plant expression vectors pCAMBIA1301-BADH-35S-RIP and pCAMBIA1301-RIP were constructed, which contain salt-tolerance selectable BADH.(2) By Agrobacterium-media transformation methord and pollen tube pathway, gene RIP which code ribosome-inactivating protein was transformed into the soybean cell. Obtained TO generation transgenic plants and seeds, and T1 generation transferred plant.(3) 18 transgenic plants of T1 generation were confirmed positively after PCR test.