Expression,Purification And Crystal Screening Of A New Gibberellin Synthase In Arabidopsis Thaliana

Gibberellin acid?GA?is a type of plant hormone belonging to tetracyclic diterpene.Though there are more than 130 kinds of gibberellins in nature,only a few are active in plant,such as GA₁,GA₃,GA_4,GA₇ and so forth.These active GA molecules regulate various stages of plant growth and development,including promoting seed germination,elongation of main root,unfolding of the leaf and foundation of flower meristem.Both GA and ABA are important plant hormones which control seed germination.Previous studies have suggested that GA enhances seed germination,plant flowering and fruit ripening while ABA inhibits seed development.GAS1?gain function of ABA insensitive 1?is a mutant plant formerly screened in the laboratory and proved to be insensitive to ABA.Previous studies have indicated that the seed of GAS1 featuring over expression show resistance to ABA,the deficient mutant is characterized by a reduced germination rate.Further studies have found that GAS1 encodes an oxidoreductase which catalyzes GA₁₂ into a new GA molecule called GA_x.However,the detailed reaction mechanism generating GA_x remains unclear,and it is also unknown whether GA_x possesses biological activity,just like GA.Because no structural analysis of GA-related oxidoreductase has been conducted according to the findings of the earlier studies,we attempt to carry out expression and purification of GAS1 proteins and analysis of crystal structure,expecting to figure out how GAS1 perform biological function.This study practiced expression of protein with utilizing multiple prokaryotic expression vectors in order to attach different tags to proteins.Due to the poor expression level of GAS1with GST tag,we mainly expressed GAS1 with 6xHis tag for further purification.By activating strains in appropriate duration and measuring cell growth,this study proved that to collect strains when the value of OD₆₀₀ being between 0.6-0.8 is the optimal choice.The next step was to purify GAS1 protein by means of broken,ultrasonic disruption,Ni column affinity chromatography,ion-exchange chromatography and gel-filtration chromatography.Western Blot experiment was conducted to test the expression of protein.The crystallization of GAS1 protein was preliminarily screened by using 10 crystal screening kits with 480 pools.But the initial results that being not satisfactory for the full-length GAS1 crystal was not found in the 480 crystal screening reagents.In order to increase the stability of the protein,we used GA_12,the protein substrate,to deal with the protein to mix the substrate with the protein.However,due to high concentration of the substrate on the first try,a similar to GA crystal was harvested.After that,we add different concentration gradients of the substrate to the protein respectively,aiming to find the optimum concentration of the substrate which is conducive to crystal growth.But we still only got the salt crystal.According to the results of protein absorption peak of ion-exchange chromatography and gel-filtration chromatography,we assumed that the failure of crystallization resulted from the heterogeneity caused by GAS1degradation.Through combining of the results of bioinformatics software and the N-terminal sequencing of the full length protein,we decided to build truncated clones of GAS1 protein,GAS1?N62 and GAS1?N56,and still took pET-28a as prokaryotic expression vector.After getting the recombinant of clone,we worked on expression,purification and optimization of GAS1?N62 and screening of crystals.Meanwhile,we used molecular docking software Auto Dock to simulate the interaction between GAS1 and its substrate to predict GAS1 role in the GAs signal transduction,and found that GAS1 can interplay with GA₁₂ through some interaction sites.These amino acids may play key roles in GAS1 functions of recognizing and catalyzing.At the same time,we also simulated whether GA_x interacts with the gibberellin receptor GID1A,and found that some interaction sites,these results also showed that GID1A may recognize and binding to GA_x to play certain biological functions.