Effects Of Slbri1 Phosphorylation Site THR-825 On Agronomic Traits Of Tomato

Brassinosteroids（BRs）play important roles in regulating plant growth and development.Tomato is one of the most widely cultivated vegetables in the world.As a BRs receptor,Sl BRI1（Brassinosteroid Insensitive 1）transmits signals downstream through the phosphorylation of the kinase region after sensing BRs.Therefore,exploring the phosphorylation sites and functions of Sl BRI1 are very important for regulating BR signals and enriching the research of BR in tomato molecular breeding in the future.In this study,the biological function of Thr-825 phosphorylation site of Sl BRI1 and its effect on agronomic traits were explored by mutation of Thr-825 site（from Thr to Ala to mimic non-phosphorylation）.By constructing a prokaryotic expression vector for expression in E.coli,the effect of Thr-825 on the phosphorylation level of Sl BRI1 was studied.By constructing a plant overexpression vector(P_{Sl BRI1}::Sl BRI1-T825A-GFP-p BI121)and genetic transformation of tomato,a tomato transgenic line cu3^-abs1::Sl BRI1-T825A-GFP（T825A-GFP）was obtained.Compared with unmutated Sl BRI1 overexpressing transgenic line cu3^-abs1::Sl BRI1-GFP（Sl BRI1-GFP）,the plant architecture,morphologies of floral and leaf organs,and the yield and quality of fruit were analyzed to study the biological function of Sl BRI1（Thr-825）and its impact on agronomic traits.The main results are as follows:1.Plant overexpression vector P_S1BRI1::T825A-GFP were generated,and tomato transgenic line T825A-GFP of cu3^-abs1（weak BRI1 mutant）background were obtained through genetic transform.2.Prokaryotic expression vectors p Flag-MAC-Sl BRI1-T825A-CD and p Flag-MAC-Sl BRI1-CD-T825D were generated.An in vitro phosphorylation assay was conducted and found that Thr-825 negative regulates the phosphorylaton of BRI1.3.q RT-PCR analysis found that the expression level of Sl BRI1 in T825A-GFP and Sl BRI1-GFP transgenic lines were slimilar and significantly higher than that of in cu3^-abs1,while the transcription levels of Sl CPD and Sl DWARF are also comparable and significantly less than cu3^-abs1,which indicating that both of the transgenic lines properly restored the BR signaling pathway of cu3^-abs1.4.The hypocotyl elongation test suggesting that the transgenic line T825A-GFP is more sensitive to BR than Sl BRI1-GFP,and the BR signaling pathway is stronger.5.Measureing and comparing the agronomic traits of tomato transgene found that compared with Sl BRI1-GFP,T825A-GFP plant is taller and has a thicker stem,larger flowers and leaves,and has a larger size of fruits and a larger weght of single fruit,which resulting in higher yields.