Co-expression Of Mutiple Genes Mediated With Lp4-2A In Plants

Phytopathogens caused enormous losses in cultivated and stored crops in the world. Pesticides and antimicrobials are not only significantly regarded as serious environmental contaminants but also increase production costs, and they contribute to the increase in antimicrobial-resistant species. Fusarium head blight caused by species of Fusarium is an prevalent disease of cereal crops, which not only can cause yield loss of crop, but also can potentially threat on human and ainimal health by producing a variety of mycotoxins. Antimicrobial peptide is a class of small molecule peptide coded by some genes in the biological cells, and can effectively enhance the host’s ability to resist invasion of pathogens. Therefore it will have abroad application prospects in plant disease resistance genetic engineering. Substantial increase in resistance-related gene expression in plants is in the primary objective of the project. It is a more important strategic by using multi-gene co-expression approach to improve protein expressionLP4 is a naturally occurring linker peptide (LP4) originating from a polyprotein occurring in seeds of Impatiens balsamina and encoding 29 amino acids, Expression in eukaryotes, there will be a processing between in the first and second amino acids when expression in eukaryotes. The FMDV 2A region is a very small protein of only 20 amino acids, it can be an ideal sytem for co-expressing multiple genes in a single open reading frame in both mammalian and plant cells, tides from other viruses. This peptide can mediate polyprotein ’cleavage’ by a unique non-proteolytic mechanism thought to involve an intra-ribosomal ’skip’ during translation, such that a peptide bond is not formed between aminoacids 19 and 20 of 2A, yet translation continues. The hybrid linker peptide is a combination between the first The first 2-9 amino acids of LP4 and the 20 amino acids FMDV 2A sequence. To pBlu-scriptKS (+)-based, respectively, connected in turn to hybrid peptide Lp4-2A to NPRI gene, cecropin Pep3, MsrA1, Ace, RSD2 to connect to multiple cloning sites, in which different antimicrobial peptides have different signal peptide, and then assembled MG fusion gene expression vector in pTKAc under the CaMV35S promoter, which build into Lp4-2A connected by the five disease-related genes in the same reading frame (ORF) of the plant expression vector. Through the integration of Agrobacterium-mediated gene transfer into Arabidopsis thaliana, the level of nucleic acid and protein identification, and transgenic plants to Fusarium graminearum to identify the spray inoculation to assess their resistance.The results showed that transferring the dicotyledon expression vector into Arabidopsis thaliana by the Agrobacterium-mediated Floral dip method, and there have 22 positive through PCR identification of transformed plants. Six were to be higher expression of transgenic plants by ELISA screening after extraction of the T2 generation of PCR-positive identification of the total protein in leaves. RT-PCR identify and Western blot analysis confirmed that the normal MG transcription and protein expression as well as the correct prosessing of Lp4-2A in the plants.T2 generation of transgenic plants was carried out to deal with flowering spray inoculation The results showed that the resistance of MG transformed plants is much larger than wild-type and GFP transformed plants, and stronger than single-gene transformed plants with RSD2. The results of these studies provide evidence that co-expression of multiple genes mediated with Lp4-2A will be carried out in crop.