Transformation Of Maize With Anti-apoptotic Genes P35and Iap Confers Resistance To Sheath Blight

Banded leaf and sheath blight (BLSB) of corn is a wide range of worldwide fungal diseases, which is caused by Rhizoctonia solani kuhn. The pathogen is strong saprophytic and have a wide host range. By now, any variety of corn that is resistant to BLSB have not yet been found. Currently, the methods of managing BLSB have mostly depended on chemicals and agricultural control. In order to gain new corn germplasm with broad spectrum and durable resistance to diseases caused by necrotrophic pathogens, we transferred anti-apoptotic genes p35and iap which are originated from Autographa californica nuclear polyhedrosis virus and Spodoptera exigua separately into maize by Agrobacterium-mediated transformation.In our researches, we constucted two vectors for gene engineering, which are fusion gene expression vector and binary expression vector. Anti-apoptotic genes p35and iap were separately cloned from pCAMBIA1305.1-p35and pBI121-iap using PCR with three primers designed according to the encoding region sequences. The gene p35is900bp in length and the gene iap is1137bp in length. For the purpose of improving the expression of these two genes, we utilize overlapping PCR technology to create a DNA fusion segment harboring genes p35and iap, which was drived by maize ubiquitin promoter UBI-1. Linker polypeptide LP4/2A was applied to join genes p35and iap in building fusion gene expression vector. Besides, kozak sequence was introduced into gene p35which was placed in front of fusion gene to optimize the start codon of gene p35. In addition, we made use of Ω sequence which is non-translation leader sequence with abundant TTAAC originated from TMV as enhancer, putting between iap gene which was placed behind fusion gene and UBI-1promoter. Fusion gene p35-LP4/2A-iap was inserted into plant expression vector pCAMBIA3301so as to create fusion gene expression vector pCA3301-p35-LP4/2A-iap. When constructing the binary expression vector, genes p35and iap were separately placed in a set of expression cassette, and p35gene was linked into the multiple clone site of pCAMBIA3301and driven by promoter UBI-1.The iap gene was placed at the position of the gene gus of pCAMBIA3301and driven by promoter CaMV35S. Thus, the binary expression vector pCA3301-p35-iap was created.Totally,52transgenic maize plants were obtained by transferring the genes p35and iap into two inbred maize lines A188and Z31using Agrobacuterium-mediated transformation. Nested PCR analyses showed that19transgenic maize plants appeared PCR positive. It is likely that the p35and iap were integrated into the genome of the19transgenic maize plants.We tested disease resistance of the transgenic maize plants. Leaves from19transgenic plants were inoculated with R. solani kuhn. We found6transgenic lines are highly resistant to the R. solani kuhn. But, the resistence to R. solani kuhn between transgic lines of transferring fusion gene expression vector and those transferring binary expression vector had no significant difference.