The Construction Of Genetic Transformation System For Sisal And Hevein Genetic Transformation For Zebra Disease Resistance

Sisal (Agave sisalana L.) is the most important fibre crop in the south subtropical and tropical regions in China and the world. It possesses multiple usages, broad application prospects and high integrated values. Unfortunately, a kind of disease, named Zebra Disease, is one of the most common and serious disease of sisal. Caused by Phytophthora nicotianae, the Zebra Disease seriously affected the growth of sisal, led to the reduction of seedling in the early growth stage and eventually caused critical damage to yield of sisal. To improve resistibility of sisal against ZebraDisease, following steps have been taken in this research:1. We construct a plant expression vector pCAMBIA3301-Hevein of Hevein gene screened by bar gene. Then it is introduced into Agrobacterium tumefaciens EHA105through freeze-thaw method.2. The vector pCAMBIA3301-Hevein was introduced into150sisal callus by Agrobatorium-mediated transformation to get thirty-seven PPT-resistant plants, and the transformation rate is about24.7%. After detection of PCR, ten PPT-resistant plants contain the Hevein genes. Seven sisal transgenic plants have hybridization signals through dot hybridization and Southern blot. One of the seven plants even has single copy. These results show that Hevein gene have been integrated into the genome of sisal. Then seven sisal transgenic plants are tested positive by RT-PCR. It shows that the target gene has been expressed at the transcriptional level in these plants, and the positive rate is about18.9%.3. An antibacterial testing in vitro of blade crude proteins abstracted from the seven plants, five genetically modified plants show strong inhibition of Phytophthora nicotianae. Two sisal transgenic plants of them express resistibility through the artificial inoculation test of Phytophthora nicotianae. The level of disease resistance of No.T8and No.T6are respectively grade2and grade3. No.T5and No.T9show moderate resistance. Levels of disease resistance for all of them are grade5.All in all, we have transformed the Hevein gene into sisal and expressed successfully. The transgenic sisal plant obtains better zebra disease resistance, and it has laid a solid foundation for the transgene sisal of disease-resistant and high yielding variety.