Elite Germplasms Selection With High Isoflavone Content In Soybean And Expression Analysis Of Related Genes From Huanghuaihai Ecotype Region

Soybean is an important economic crop widely planted in many countries. Its seedsconsist of rich protein, fat and many physiological active substances such as isoflavone.Isoflavone is a kind of biological flavonoids, which is only synthesized in plants notanimals. To analyze the genetic variability and select elite germplasms with high content oftotal isoflavone and its components in soybean seed, accessions originating fromHuangHuaiHai ecological region were tested by high performance liquid chromatography(HPLC) technique. After that, the expression patterns of related genes to soybeanisoflavone metabolic pathway in different varieties were analyzed by using the selectedelite germplasms through real-time quantitative PCR. And also some transgenic soybeanplants with isoflavone metabolic related genes were obtained. The main results were asfollows:1. A precision and accurate HPLC method to determine soybean seeds isoflavone contentwas constructed. The precision test results showed that relative standard deviation(RSD) were0.96%,1.53%and1.31%, respectively. The RSD of repeatability testwere1.89%,1.85%and1.65%, respectively. The RSD of stability test were1.76%,0.89%and1.27%in the same day, and0.98%,1.28%,1.02%by the day, respectively.The spike recovery rate of daidzin, glycitin and genistin were98.91%,99.93%and101.46%, and the corresponding RSD were1.42%,1.89%and1.73%. The results alsoshowed that the linear range of isoflavone components were all between2.5~80.0μg/mL.2. The analysis results of213accessions, including181cultivated and32wild soybeansoriginating from HuangHuaiHai ecological region, showed that a large geneticvariation existed in both total isoflavone and its components in all soybeans, and theaverage content of total isoflavone in wild soybean seed is much higher than that ofcultivated seed. The range of total isoflavone in cultivated soybean was1463~6116μg/g (4.2-fold variation), with an average of3558μg/g, while the total isoflavone inwild soybean was3896~7440μg/g (1.9-fold variation), with an average of5182μg/g.From the accessions, four elite germplasms with high isoflavone content (>6000μg/g)were screened out, and one elite accession content was above7000μg/g. 3. To select the pivotal genes in soybean isoflavone synthesis pathway, the expressionpatterns of eight related genes in different varieties were studied by using thereal-time quantitative PCR method. The results showed that expression levels of PAL,C4H and4CL in Zhongdou27(high seed isoflavone content variety) weresignificantly higher than that in Chuxiu (low seed isoflavone content variety) duringthe R2growing period. Meanwhile, the expression levels of CHS and IFS inZhongdou27were also significantly higher than that in Chuxiu during the R8growing period. While the expression levels of CPR and F3H in Chuxiu weresignificantly higher than that in Zhongdou27during the R7and R5period,respectively. Thus, the different expression levels of related genes in Zhongdou27and Chuxiu may be the reasons to explain the seed isoflavone contents in these twovarieties.4. The RNAi vector of pCAMBIA3301-ASF3H was successfully constructed, and wastransformed into different soybean varieties by using the soybean pollen-tube pathwaytechnique. And some transgenic soybean plants were obtained through the PCR andherbicide resistance detection methods.