The Modification Of PPA Gene,Vector Construction And Transformation

Wheat is a widely grown food crops all over the world. Its total production rankssecond in the world, only less than corn. As an important food crops, the quality and yieldof wheat have been a major concern nowadays. China is a big country of wheat planting,but the wheat quality and yield decreased year by year due to the influence of pests. Weoften adopt chemical insecticides to kill the Aphids; however, the method may cause aseries of environmental troubles. How to avoid these troubles? Scientists gradually beginto pay attention to the restraining insect breeding. We integrate resistant insect PPA geneinto the plant genome by using Modern Molecular Biology Techniques.Finally, we obtaina new varieties of restraining insect wheat.Experimentally, we modified the PPA gene in order to enhance PPA gene expressionin plants. Main strategy was introducing the Kozak sequence in the front of purpose gene.The program was designed to improve the transcription rate and translation efficiency.PPA Gene and modified PPA gene were constructed into the plant expression vector pCAMBIA1380. The plant expression vectors also need to be constructed with thepromoter CAMV35S and GUS report gene before introducing the purpose genes. Finally,we used the method of restriction endonuclease digestion to identify the recombinationvector.The correct expression vectors were transfered into Agrobacterium tumefaciensGV3101. We make use of Agrobacterium tumefaciens, which carrying plant expressionvector, to infect COL wild-type Arabidopsis thaliana. Then, we sieved the homozygouslines of Arabidopsis thaliana. Three homozygous lines were selected by randomly fromtwo plasmids transformed homozygous lines. Following, we extracted their RNA and usedsemi-quantitative PCR method to assay PPA expression level. The experiment resultsdemonstrate that the expression of modified PPA gene was higher than that of PPA gene.The pCAMBIA1380-35SKPG plasmid was transfered into Agrobacteriumtumefaciens GV3101and EHA105, and then we integrated the target fragment into thegenome of wheat applying Agrobacterium-mediated transformation. After the co-culture,recovery culture, screening culture, differentiation, strong seedlings and a series ofprocesses, we got13differentiation seedlings. Two positive seedling was identifiedthrough the detection of DNA level.