Studies On The Transfer Of Oryzacystatin Geneinto Mulberry

Insect pests of mulberry are one of the main factors which cause the damage of mulberry leaves and loss of cocoon. It is a future in agricultural practice that applying plant anti-insect genetic engineering controls insect pests of mulberry. Currently, The gene used for plant anti-insect genetic engineering mainly is Bacillus thuringiensis (Bt) insecticidal crystal protein and proteinase inhibitor (PI) genes in Oryza sativa (Called as Oryzacystatin). Bt insecticidal crystal protein and PI is toxic to lepidopterous insects, while silkworm belong to their species. Therefore, Bt insecticidal crystal protein gene and the majorities of PI gene are not used for plant anti-insect genetic engineering. Oryzacystatin gene itself is a natural anti-insect gene, which strongly inhibit coleoptera insects in a lot of reports but not in lepidopterous insects., There are several successful cases applied in plants on the side. Consequently, we selected Oryzacystatin gene as target gene to introduce it into mulberry tissue by Agrobacterium-mediated and biolistic methods and primarily achieved goal.1. the results,which compared effect of phytohormone and antibiotic on the differentiation of adventitious buds in test-tube seedling of Morus alba, show that the concentration ratio of 6-BA to NAA from 20:1 to 30:1 promotes the differentiation of adventitious buds; the cultures containing abundant fructose promote ability of adventitious buds to grow strongly after leaves differentiated; the effect of a variety of antibiotic on the differentiation of adventitious buds within a suitable concentration is not prominent.2 genetic transformation system was improved and perfected in studying on Oryzacystatin gene transformed into mulberry by Agrobacterium-mediated method. The experiment of the effect on tissue culture of mulberry and transformation conditions demonstrates that affinity of mulberry receivers to OC gene or vectors containing OC gene is poor, transformation efficiency of adventitious buds which was infected by Agrobacterium on selected cultures is lower, about 5%., It was confirmed firstly that the OC gene in a few transgenic plants was transformed into the tissue cells of mulberry and integrated into genome of regenerative plant by PCR identification, DNA blot hybridization and southern blotting hybridization.3 the results researching bombarded parameter of boilistic method and transformation efficiency display that peak transformation efficiency occurred when mulberry explants selected from tip of young stems, buds in the winter, axillary buds and calli of middle size were bombarded with 500 gold particles coated with OC gene each times under the pressure of 135 psi at 7 cm distance.4. The result which Oryzacystatin gene was introduced into cell tissue of mulberry by biolistic method was confirmed by PCR identification, DNA blot hybridization and southern blotting hybridization as well as RNA blot hybridization, northern blotting hybridization. At the same time, it is certain that not only have foreign genes been incorporated into the genome, but also have expressed at RNA level. We still found that the amount of RNA expressed among different transgenic plants is different, even in different organs of a transgenic plant.