Agrobacterium-mediated transformation of B. juncea reveal BjuLKP2 functions in plant yellowing

A stable and efficient transform system was necessary to verification gene functions in plant. To construct an agrobacterium-mediated transform system in B. juncea, explants, as well as the combination and concentration of hormones, the concentration and timing of infection were optimized. Eventually, a stable transgenic system was established and two BjuLKP2 OE lines were obtained. Phenotypic observation found that both two BjuLKP2 OE lines displayed yellowing cotyledon, shoot tips, leaves, flower buds and reduction total Chl content. qRT-PCR assays indicated that five Chl synthesis genes were significantly up-regulated and one gene was down-regulated in BjuLKP2 OE line. Additionally, antioxidant capacity assays revealed decreased activities of APX, CAT and SOD, while POD activity was up-regulated in BjuLKP2 OE line. Further chlorophyll fluorescence induction kinetic determination results indicated a reduction in photosynthetic ability in BjuLKP2 OE line. GUS assays results showed that BjuLKP2 was expressed in various tissues, including root, hypocotyl, cotyledon and vascular tissues of leaves. It was also expressed in trichome, sepals, petals, filaments, styles and the stigma base, but did not detect in seeds. Scanning electron microscope observed found that chloroplasts ultra-structure in both sponge and palisade tissues were changed. All the results demonstrated that BjuLKP2 functions in plant yellowing by reduction in Chl content and changing chloroplasts structure.