Construction of a chalcone reductase expression vector and transformation of soybean plants

Abstract

The present study aimed to clone the soybean chalcone reductase 3 (CHR3) and create a recombinant expression vector pCAMBIA3300-CHR3 containing Bar resistance gene as a selection marker, and then obtain transgenic soybean plants using Agrobacterium infection. The plant expression vector pCAMBIA3300-CHR3 was transferred into soybean receptor plants, Jinong 17 and Jilin 30. Polymerase chain reaction (PCR) and Southern blotting were used to confirm the positive transgenic plants. Additionally, reverse transcription-quantitative PCR (RT-qPCR) was used to detect CHR3 expression and isoliquiritigenin content was measured using high-performance liquid chromatography (HPLC) in the transgenic offspring. Soybean CHR3 (932 bp fragment) was successfully cloned into the plant expression vector pCAMBIA3300-CHR3, which was subsequently transferred into soybean receptor plants. In the T1 generation positive plants were validated by PCR analysis, including eight Jinong 17 and five Jilin 30 transgenic plants; Southern blotting demonstrated that the functional components of the pCAMBIA3300-CHR3 vector had been integrated into the soybean genome; RT-qPCR results demonstrated that the expression of CHR3 mRNA was increased by 2 to 20-fold in the transgenic plants compared with the non-transgenic soybean plants. Furthermore, the isoliquiritigenin content was increased by 8.56% in the transgenic Jinong 17, compared with control plants, as detected by HPLC. The CHR3 gene can produce isoliquiritigenin, a precursor of daidzein, which in turn can improve the ability of soybean to resist phytophthora root rot.