A Set Of Ligation-Independent Cloning Vectors For Monocotyledon And Application Of Antimicrobial Peptides In Rice Breeding For Disease Resistance

Scientists have used Agrobacterium-mediated genetic transformation to generate transgenic plants, and this method depends on the Ti plasmid. Traditional vectors and Gateway vectors are available currently. Traditional vectors are either laborious and slow, when Gateway system is quick but expensive. When use traditional vectors, we often encounter a variety of problems, such as inappropriate restriction endonuclease sites, difficulty of number of fragments connection. While Ligation-Independent Cloning vectors can make up for these deficiencies, and they are cheap. But LIC vectors used for transformation of monocots has not been developed.In this study, we constructed a set of Ligation-Independent Cloning vectors for Monocotyledon based on pCAMBIA and pGA vectors, and they can be used for gene expression analysis、protein localization studies and promoter function analysis. This set of LIC vectors is fast, and also inexpressive. Any fragment contained the15bp special sequence can be inserted into the vectors. Each set of vectors have Bar and Hyp marker gene for different plant transformation. Besides, the resistance marker gene is the Kan resistance gene, and Kan is relatively stablity, high screening efficiency. This set of vectors laid the foundation for next smooth experiment.Plant genetic engineering is regarded as an approach for providing resistance in rice. This approach is advantageous for introducing disease resistance into rice, since transgenic plants can acquire desired trait without any alteration of the orginal genetic background. In order to improve resistance to disease, different genetic strategies have been used which include utilization of antimicrobial genes,as well as plant disease-response pathway components. Antimicrobial peptides have a unique antibacterial mechanism, broad spectrum, and it is not easy to produce drug resistance. It is reported that many plants enhanced their resistance to disease when be introduced Antimicrobial peptide genes.In the study, the factors involved in genetic transformation of Agrobacterium transformation were optimized, and the efficiency has been greatly improved. Three antimicrobial peptide genes derived from the Chinese shrimp(ALF、WAP、Penaeidin) were transformed into rice, and5、7、2TO transgenic plants were obtained.Stable integration and inheritance were confirmed by hischemical staining assay, PCR screening and Southern blotting analysis. Based on the broad spectrum antimicrobial resistance of Antimicrobial peptide, we can analysis the disease-resistance of the transgenic plants,and it is desirable to be able to improve the disease resistance of rice.