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 (932bp 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.