Cloning And Functional Analysis Of Rutin Degrading Enzyme Gene Promoter In Tartary Buckwheat

Rutin, as one of the most important flavonoids in tartary buckwheat, has been shown the ability of anti-hyperglycemia, anti-hypertension and anti-oxidation. Rutin degrading enzyme is one of the most critical enzymes of decomposition and metabolism of rutin, which can degrade rutin and result in low rutin content of product. The promoter region of FtRDE were cloned, the cis-acting elements in the promoter region of FtRDE were analyzed by bioinformacics and identified by transient expression in tartary buckwheat protoplast, and the regulation mechanism of the response to exogenous signal substances were studied, which revealed the regulation mechanism of FtRDE and laid a foundation for studing the biological function and regulation mechanism of secondary metabolites of tartary buckwheat. The following conclusions have been made in this paper:(1) The optimal enzyme solution for mesophyll protoplast isolation in tartary buckwheat was 1.5% cellulase R-10+0.5% macerozyme R-10+0.5 mol/L mannitol+20 mmol/L MES+20 mmol/L KC1+10 mmol/L CaCl2+0.1% BSA. The true leaves, which were removed the lower epidermal layer with tape, were incubated in enzyme solution for 4 h at 25 ℃ in dark, and centrifugal speeds was 90×g for protoplast collection. The protoplast yield amounted to 6×106/g fresh weight and the vitality was up to 90%.(2)Genome walking was used to clone the promoter region of FtRDE, whose lengh was 1907 bp. The cis-acting elements in the promoter region of FtRDE were predicted by bioinformacics, including TATA-box(-60 bp), CAAT-box(-131 bp), transcriptional start site (-30 bp), TGACG-motif(MeJA-responsive element,-443 bp), ABRE(ABA-responsive element,-438 bp、-535 bp、-1145 bp) and RY repeat(seed-specific element,-94 bp).(3)The recombinant vectors of stepwise truncation of 5’upstream region of the FtRDE were construsted, and the cis-acting elements in the promoter region of FtRDE were analyzed by transient expression in tartary buckwheat protoplast, indicating that the region from -1077 bp to -1829 bp might contain an inhibitory cis-acting element. The result of real time RT-PCR suggested that the expression of FtRDE was up-regulated by exogenous signal substances such as MeJA and ABA, indicating that the TGACG-motif(MeJA-responsive element) and ABRE(ABA-responsive element) were functional.The result of semi-quantitative RT-PCR showed that in inmature seed, the expression of FtRDE gene was stronger than other organs during grain filling stage, indicating that the RY repeat were functional.(4) The sense fragment and antisense fragment of FtRDE and overexpression fragment of FtRDE were inserted into pTCK303 respectively, showing that the FtRDE RNAi vector and overexpression vector were constructed successfully, which laid a foundation for studing the biological function of FtRDE.