Study On Transformation Of Gene Tri101into Wheat Mature Seed

Wheat is one of the main food crops, in the world’s total output in second, and the acreage of wheat is in the first, second only to corn. In recent years, Scab is an important diseases affecting on production of wheat, which is often outbreak in the Northwest area and the yellow Huaihai River of wheat production, resulting in great losses. At present, in terms of breeding failed to obtain a breakthrough. Studies have showed that the Tri101gene is a structural gene encoding a trichothecene enzyme3-O-acetyl-invertase, which can make single spore enzymes end into similar single spore olefin C-3-hydroxy variant acetyl derivative, in order to reduce its toxicity. In this study, by constructing pCAMBIA3304-Tro101plasmid, according the plant genetic engineering methods, to try the Tri101gene into wheat genetic stably inherited and specific expression, in order to reduce the incidence of wheat scab. This experiment is on the basis of previous research. Innovation point is that the use of scratching germinating embryo and vacuum infiltration process assisted Agrobacterium-mediated transformation of wheat mature embryo. The research results showed that scratching germinating embryo and vacuum penetration have some effect in improving the wheat transgenic conversion rate. To explore a more simple and economic, high efficiency of wheat transgenic method, this experiment provides a scientific reference. The main research contents and research results were as follows:1. The research of influence of scratching embryos on the wheat genetic transformationRespectively,dip the embryo,discussing of the influence of zoned embryos into Wheat germinating seed. GUS staining was done, the results showed, the Paddling immature embryos, there were in the radicle, coleoptile and growing point, which were dyed blue. While not paddling were only in the radicle and growing point of cells of epidermis, therefore, the experiments showed that paddling embryos which can secrete phenol compounds, under the action of Acetosyringone, using Agrobacterium-mediated the wound of action, as liquid into the wheat germ cells of growing point, which could help improve the transformation frequency of wheat mature embryos.2. Scratching embryo assisted Agrobacterium-mediated the Tri101into wheat mature embryosFirstly, exploring the influence of scratching embryo and scratching way on the genetic transformation of wheat, discussing the factors affecting transformation of wheat, including the selection period of embryo, bacteial concentration, genetype, preliminary established the transformation system of germinating embryo. GUS histochemical staining and the reportergene of Gfp were done, the results showed, which were preliminary proof of the foreign gene has been inserted into the plant genome, but whether the Tri101gene could stably expression in wheat, need to do further research at the molecular level.3. Vacuum penetration mediated Tri101gene transformation of wheat mature embryoUsing Agrobacterium-mediated transformation of wheat germinating embryo transformation system, which has been established initially a new transformation system, discussing the factors affecting Vacuum penetration transformation of wheat, as intensity of the Vacuum, Vacuum infiltration time, bacteria liquid concentration, the results indicated, cut part of the transformation of plant seedling for GUS histochemical detection, with wheat germ as the statistics, GUS gene transient expression rate was2%. Preliminary evidence that exogenous gene tri101has been integrated into the genome of wheat chromosomes, which confirmed that Vacuuming penetration can improve the transformation frequency of wheat germinating embryo, in order to obtain the real heritable transgenic plants providing a scientific basis.