Transformation Of Maize With PR Genes TLP And Ace-AMP1Confer Resistance To Sheath Blight

Banded leaf and sheath blight (BLSB) is one of the important diseases of maize, which seriously affects the yield and quality of maize. Although chemical and agricultural control have an effect on BLSB, it is easy to generate resistant pathogens and cause pollution to the environment. Thus using the way of genetic engineering to breed disease-resistant maizes has become the hot spot of the researches. Most genes of the pathogenesis-related protein (PRs) family have broad-spectrum antifungal properties, which have been widely used to generate transgenetic resistant plants. In order to improve the maize resistance to BLSB, we transferred the rice thaumatin-like protein gene TLP belonging to PR-5protein family and onion lipid transfer protein gene Ace-AMP1belonging to PR-14protein family into maize by Agrobacterium-mediated transformation at the same time. Our major results are listed below.1. Construction a binary gene expression vector harboring two target genes. Gene TLP was acquired by RT-PCR from total RNA of rice leaves. We designed two long primers according to the signal peptide sequences of the gene Ace-AMP1, and on this basis, we utilized over-lapped extension PCR technique to obtain the signal peptide fragments. The mature peptide fragment of gene Ace-AMP1was cloned from a plant expression vector of PET-32a (+)-Ace-AMP1which was constructed before. When we got the signal peptide fragment and the mature peptide fragment of the gene Ace-AMP1, we amplified the full length gene Ace-AMP1by the technique of over-lapped extension PCR. Through putting the two target genes into two different expression cassettes of plant expression vector pCAMBIA3301, while making the gene TLP into the downstream of corn ubiquitin promoter and the gene Ace-AMP1into the downstream of Cauliflower Mosaic virus CaMV35S gene promoter, we successfully created the plant binary expression vector pCAMBIA3301-TLP-Ace.2. About80transgenic maize plants (TO) were obtained by transferring the bivalent gene expression vector into immature embryo and embryonic callus induced by immature embryo of maize lines A188and Hi II using Agrobacuterium-mediated transformation. Nested PCR analyses showed that28transgenic maize plants appeared PCR positive which had the two target genes.3. A0.01%basta solution was painted to the transgenic maize leaves to test the expression of the bar gene. The bar gene positive seedlings were used as materials for further PCR detection to confirm the presence or absence of two target genes. We got herbicide-resistant transgenic plants.4. R. solani kuhn were inoculated to the leaves of transgenic plants that were positive for the PCR detection and resistant to herbicide. Photographic observation after24h,48h and72h was performed. We compared the transgenic and non-transgenic plants, and found that part of the transgenic maizes performed resistance to R. solani kuhn and had a less severe disease.5. Seeds of transgenic plants (TO) were harvested by self or sister cross pollination.