Genetic Transformation And Functional Analysis Of SlBZR1Gene In Tomato

BZR1(Brassinazole-resistant1) is a key transcription factor involved in BR (Brassinosteroid) signal transduction, which palys an important role in the regulation of plant growth and development, as well as responses to biotic/abiotic stresses. Ectopic overexpression of AtBZRl-1D, a gene encoding the constitutively active form of AtBZR1protein, caused non-uniformity coloring, enhanced lycopene accumulation and increased SIGLK2expression level in tomato (Solarium lycopersicum) fruit. Powell et al (2012) suggest that no uniformity coloring is mainly attributed to a pair of alleles, U (SIGLK2) and u (slglk2), which can influence the development and distribution of chloroplast, and increase the content of lycopene during ripening.Tomato cultivar M82in u background was used as experimental material for further elucidation of the hypothesis that BZR1could enhance the content of lycopene via regulation of SIGLK2expression. SIBZR1gene was indentified through bioinformatic tools and cloned through PCR. We constructed both SIBZR1overexpression and RNA interference vectors, and got the transgentic tomato plants by Agrobacterium tumefaciens transformation.1. A Phylogenetic tree of BZR1was constructed and we figure out two genes, LAT61and SlBES1-Likel, showing highly homologous to AtBZRl. LAT61was named as SlBZR1with62.76%homology to AtBZRl. Results on the transient expression experiment suggest S1BZR1was located in nucleus to perform its biological function.2. From the performance of the electrophoretic mobility shift assays (EMSAs) using biotin-labeled DNA fragments of SIGLK2promoter and bacterially expressed AtBZR1proteins fused to the maltose binding protein (MBP) and SlBZR1proteins fused to the His (His), results indicate that both MBP-AtBZR1and His-SlBZR1fusion proteins can bind to the SIGLK2promoter.3. Tomato cultivar M82in u background was used to perform Agrobacterium tumefaciens mediated transformation. Transgenic plants were screened by PCR amplification. RT-PCR was used to measure the mRNA levels of SIBZR1. Date on one overexpression line and one RNA interference line show significant up-regulation and down-regulation of SIBZR1, respectively. Transgenic fruits show different expression levels of SlBZR1at different mature stages with the peak at pink stage.4. Experimental results of T1germination show that overexpression of SIBZR1shortens the time of seed germination without affecting the final germination rate and seedling phenotype. The interference of SIBZR1delays germination time point and final germination rate decreases. However, at the seedling stage, silenced plants show reduced height and chlorophyll content in leafs. These results demonstrate that SIBZR1plays an important role in the germination and plant morphogenesis.5. Overexpression of SIBZR1results on fruits with the obvious’mastoid’. The expression of SIGGPS and SIPSY1increase at the mature red stage, whereas the contents of lycopene, lutein and β-carotene does not change. The interference lines have nearly circular fruits with the reduced content of lutein and β-carotene and lycopene level is rarely affected at pink and mature red stage. Briefly, both overexpression and RNA interference of SIBZR1in M82do not increase the content of lycopene.These results indicating that BZR1might enhance lycopene accumulation by promoting SIGLK2expression is in agreement with Liu et al.(2014) overexpressing AtBZR1in tomato cultivar ZS4(Zhong shu si hao, U background) with significant improvement of the lycopene content.