Study On The Construction And In Planta Transformation Of Different Types Of RNAi Vectors Target To Maize SBEⅡb

As an important crop, corn(zea mays.) is widely applied. With the rapid development of corn processing industry, which put forward higher requirement in quality of maize. For conventional breeding has difficult to meet the development of modern farming industry, the combination between transgene technology and conventional breeding method can accelerate breeding process greatly. However,all of the existent transformation method has its liability, which have become one of the backward in applying transgene technology in maize. Two types of starch branching enzyme interference vector were constrcted which used to improve amylose content in corn endosperm. The successful experience of in planta transformation in other plants was attempted to apply to maize transformation, and the effect of applying in planta transformation in maize transformation was discussed. The major results are as following:1.Double fragment expression vector p1300(bar)+HMW+2F(16+20) and single fragment expression vector p1300(bar)+HMW+2F(16) were successfully constructed. These vectors contain endosperm specific promoter and herbicide resistance gene(bar gene) that will enhance gene expression specificity and facilitate large scale selection of corn transformant in farm land, respectively. The constructed interference vectors were transformed to agrobacterium Tumefaciens LBA4404 by protocol of direct transformation. The agrobacterium transformant can be used in corn transformation.2. The suitable PPT screening concentration for maize seedling (11~12d) is 75μg/mL which was confirmed by spraying different concentration PPT on untransformed seedlings. The large scale selection system for maize transformants was established. With this concentration, transgenic plants was selected and most of non-transgenic plants were killed3.Three kind of method-the method of applying bacteria in maize silk with tampon,the method of removal of maize silk and applying bacteria,and the method of removal of maize silk and applying plus dripping bacteria solution-used in zea mays. was probed. The results indicated that the last method is most effective in transformation, the transformation rate of the method with one disposal reach 3.78‰.The method suitable to maize in planta transformation was selected preliminarily.4.By analyzing every factor of the method of removal of maize silk and applying plus dripping bacteria solution, it is believed temporarily that the suitable dispose culture medium and method is as follows: 200μmol/L AS+1/5MSabundance ingredients+MS other ingredients+2,4-D1mg/L+10mmol/L fructose+10mmol/L glucose+50g/L sucrose+0.01% Tween 20,5 hours inducement,every ears disposed twice in two days。5.Determination of amylose content in endosperm and PCR amplification of part of the maize T1 generation transformants showed that all the genome of endosperm whose amylose content higher than 38% indicate PCR positive result. Some of corn transformant obtained with double fragment expression vector p1300(bar)+HMW+2F(16+20) and single fragment expression vector p1300(bar)+HMW+2F(16), whose amylose content in endosperm are higher than that of untransformed seeds. However, there is no difference between effect of the two vectors.To sum up, two types of interference vectors were constructed and transformed successfully to agrobacterium Tumefaciens LBA4404. The method and transformation system suitable to maize in planta transformation was established preliminarily by probe. There is no difference between effect of the two vectors via determination of amylose content in endosperm of the maize T1 generation transformants.