Expression,Purification,Crystal Growth And Interaction Protein Analysis Of Stem Color Related Gene RcGST-F11 In Castor

Glutathione-S-transferases(GST)is a multifunctional protein widely existing in plants,which has many functions,such as detoxification of heterologous substances,transport of anthocyanins and natural hormones,protection of cells from oxidation and resistance to abiotic stress.In the previous study,we screened the differential expression of RcGST-F11 in red-stem and green-stem castor,and speculated that it was related to the biosynthesis and metabolism of anthocyanin in castor.In this paper,the RcGST-F11 gene cloning,the establishment of the prokaryotic expression purification system,the screening of crystal growth conditions to obtain protein crystal,the use of Surface Plasmon Resonance(SPR)and other proteomics techniques to screen the interaction protein of RcGST-F11 from the total protein of castor,and the combination of Yeast Two-Hybrid(Y2H)point-to-point verifivation of the interaction between proteins,the aims to lay a theoretical and practical foundation for the function of RcGST-F11 protein,the molecular mechanism of anthocyanin biosynthesis and metabolism in castor,and stress-resistance breeding of castor.A series of prokaryotic recombinant protein expression systems such as pETM 13-RcGST-F11 and pHAT2-RcGST-F11 were constructed.E.coli BL21(DE3)was used as host,and IPTG was used to induce the expression of recombinant proteins.It was found that when the induction conditions were 0.1 mM IPTG,37℃ and 220 rpm for 5 h,the recombinant proteins were insoluble.We chose to increase the solubility of His-RcGST-F11 protein through renaturation,and found that When 8 M urea(pH 8.0)solution was used as denaturant and 20 mM Tris-HCl,pH 8.0,500 mM NaCl solution was used as renaturation buffer,the solubility of the recombinant protein was higher by dilution renaturation.The protein was further purified by anion exchange chromatography and molecular sieve chromatography,and the recombinant protein was found to exist in polymeric form and was resistant to low salts.Finally,high purity recombinant protein was successfully obtained.The crystal growth test of His-RcGST-F11 protein was carried out by using the crystal growth screening kit.When the volume of crystal growth pool solution is 100 μL,the concentration of protein is 17.31 mg/mL,the volume of sample is 2 μL,the ratio of pool solution to protein is 50%:50%and 25%:75%,the precipitant is 20%PEG 3350,and the buffer is 0.2 M ammonium sulfate(pH 6.0),His-RcGST-F11 produces protein crystals which are different from the salt crystals in the pool solution and have a good growth state,showing microcrystalline and polycrystalline state in the sample pool.The SPR was used to screen the protein interacts with the RcGST-F11 protein,and the RcGST-F11 protein was used as the bait protein to screen the total protein of castor.A total of 22 interacting proteins were obtained.Y2H analysis showed that when RcGST-F11 interacts with RcRBCL,blue colonies could be clearly observed on the four-deficiency medium QDO/X/A(SD/-Leu/-Trp/-His/-Ade/X-α-Gal/AbA),which proves that RcGST-F11 and RcRBCL have direct interaction.It is tentatively assumed that the combination of RcGST-F11 and RcRBCL affects the accumulation of sugar in castor through 3-PGA,which in turn affects the accumulation of anthocyanins in castor.The above results provide a new perspective and experimental basis for explaining the molecular mechanism of stem color development of castor and stress-resistant breeding of castor.