Study On Genetic Transformation Of Sweet Protein Gene Brazzein In Fragaria Vesca

Strawberry has wide cultivation area and large amount of cultivation.It has become a highlight of fruit production in China.Strawberry is rich in nutritional value,which is rich in many kind of amino acids,fructose,citric acid,malic acid,pectin,carotene,vitamin C,vitamin B1,B2,niacin and minerals like calcium,magnesium,phosphorus,potassium,iron etc.These nutrient elements have a good role in promoting growth and physical development.Sweet protein has the characteristics of high sweetness,low calorie and natural safety.As a new type of sweetener,it has gradually entered people's field of vision.The greatest feature of sweet protein is the high sweetness,which is about several hundred times or even thousand times than the same concentration of sucrose,and the low calorie content of sweet protein is in line with the consumers' demand for low-calorie foods.In recent years,with the development of plant molecular biotechnology,sweet protein gene has been introduced into crops to improve the taste of crops with the characteristics of high sweetness.Great achievements have been made in improving the taste of crops.The edible part of the strawberry comes from its receptacle.It was found that FBP7,a receptacle-specific promoter,can also drive gene expression in strawberry(receptacle).In this study,sweet protein gene Brazzein expression vector driven by FBP7 was constructed.The vector mediated by Agrobacterium tumefaciens was transferred into the ecotype Ruegen of Fragaria vesca to obtain transformed plants expressing sweet protein gene Brazzein in the receptacle.The transgenic strawberry plants with positive Brazzein gene were obtained by screening kanamycin,GUS staining and common PCR.Using GAPDH gene as an internal reference,the relative expression of sweet protein gene Brazzein in transgenic Fragaria vesca fruits was determined by fluorescence quantitative PCR.The soluble solids in the transgenic fruits were determined by refractometry.The protein content was determined by the Coomassie brilliant blue method.The content of soluble sugar was determined by HPLC(high performance liquid chromatography).The main results are as follows:1.Expression vector of the sweet protein gene Brazzein driven by the receptacle-specific promoter FBP7 was constructed.The sweet protein gene Brazzein was specifically expressed in strawberry receptacle.The constructed vector also contains a GUS reporter gene and a kanamycin resistance gene driven by the 35S promoter for using in the process of strawberry transformation to obtain plants for preliminary screening and validation.2.Using the leaves of ecotype diploid Fragaria vesca Ruegen as explants,genetic transformation of strawberry leaves was carried out by Agrobacterium tumefaciens.18 Brazzein-transgenic Fragaria vesca lines were screened by antibiotic screening,GUS staining and common PCR.3.Transgenic plantlets were subjected to acclimatization,transplanting and growth management in greenhouse.The mature red fruit of strawberry fruit was harvested 2 days after pink fruiting period and subjected to quantitative PCR,protein content,soluble solids,sweetness,and tasting experiments.In the 16 transgenic lines detected by real-time quantitative PCR,the sweet protein gene Brazzein of them was expressed.The protein content of 13 Brazzein-transgenic strawberry fruits was determined,and their content was significantly higher than that of the wild type.The soluble solids content of 9 transgenic lines was measured,and the soluble solids content in 9 lines was significantly higher than that in the wild type.The sweetness of 9 transgenic lines' fruit increased compared with wild type through tasting.It can be inferred that the sweet protein gene Brazzein is expressed in the strawberry fruit of Brazzein-transgenic strawberry.We also determined the soluble sugar content of 9 transgenic lines and there was no significant difference in the soluble sugar content between the 7 lines and the wild type.The soluble sugar content of the two lines was higher than that of the wild type.Our research provides a reference for the use of molecular breeding methods to improve the sweetness of strawberries and increase the diversity of fruit varieties.