Medicago Sativa Plant Defensins Gene (MsDef1) Genetic Transformation Of Sugarcane And Scaning Of Disease-resistant Transgenic Plants

Sugarcane (Saccharum officinarum L.) is a species of important producing sugar and energy cash crop. But some diseases have severely affected the sustainable and stable development of sugarcane industry. Sugarcane smut, caused by Ustilago scitaminea Syd., is one of the severe diseases in sugarcane. Planting resistant varieties to replace susceptible varieties is the most cost-effective measures for the prevention and treatment of sugarcane smut. Since sugarcane is an asexually propagated plant and its genetic background is very complex, to improve its disease resistance is very difficult with conventional cross-breeding. Therefore, transgenic technology is an important way of sugarcane breeding for disease resistance.MsDefl is one kind of plant defensins divided from Medicago sativa. It has a broad-spectrum antimicrobial activity in vitro. It inhibits the growth of plant pathogens, including fungi and bacteria. It inhibits the filamentous fungi strongly, but it is harmless to human, animals and plant cells. In this study, we transferred MsDefl/KDEL gene with the endoplasmic reticulum positioning protein sequence into sugarcane genome through genetic engineering. So that it can be effectively expressed in sugarcane cell. We expect to get a sugarcane genetically modified new variety which has a good resistance to sugarcane smut pathogen and other pathogenic bacterias.This study constructed a plant expression vector pADKNB of MsDefl/KDEL gene screened by bar gene. Then it was introduced into Agrobacterium tumefaciens EHA105through freezing and thawing method. It was introduced into sugarcane by Agrobatorium-mediated transformation.41PPT-resistant plants were obtained. After detection of PCR, Southern blot and RT-PCR of the target gene,25of sugarcane genetically modified plants were obtained. The target gene had been expressed at the transcriptional level in these plants. Having a testing of leaf crude protein antibacterial in vitro of these25plants,7of sugarcane genetically modified plants were obtained. They showed a strong inhibition of Fusarium graminearum and smut fungi. These seven sugarcane transgenic plants show resistance in the field inoculation test of smut resistance. The levels of disease resistance of four plants were grade2, and the other three were grade3. These results showed that MsDefl/KDEL gene had been integrated into the sugarcane genome and seven sugarcane transgenic plants have expressed active defensins and have enhanced their resistance to smut.