| Hydrogen sulfide?H2S?,as the third gasotransmitter following nitric oxide?NO?and carbon monoxide?CO?,has been developing a hot topic in recent years.With the intensive research on H2S,the mechanism regulating the life activities of animals,promoting the growth and development of plants,and enhancing stress resistance of plants,has been gradually elucidated that the enzymes involved in catalysis of endogenous H2S production in plants and animals have been systematically reported,as well.In animals,cystathionine?-synthase?CBS?,cystathionine?-lyase?CSE?,and 3-mercaptopyruvate sulphurtransferase?3-MST?are involved in the production of H2S.While,there are four major classes of enzymes that catalyze the production of endogenous H2S in plants:cysteine desulphydrases?CDes?,cysteine desulfurases,?-cyano-alanine synthase?CAS?,and O-acetylserine?thiol?lyase?OASTL?.In the research of physiological roles and signaling pathways of H2S,mutants of L-cysteine desulphydrase?LCD?and L-cysteine desulfhydrase 1?DES1?in CDes were also applied wildly.In addition,Cystathionine?-lyase?CBL?plays a crucial role in the synthesis of methionine via cysteine in plants.In other words,cystathionine can be cleaved by CBL to form homocysteine,and then the methionine synthetase catalyzes homocysteine to form methionine,this is one of the common functions of CBL.The relationship between CBL and CSE in animal cells is very close,the sequence identity reaches 43%,the similarity reaches 64%,but whether CBL in plants has the function similar to CSE in animals can catalyze qH2S production,currently,No one has reported yet.This article mainly uses the prokaryotic expression to analyze the relationship between CBL and endogenous H2S production in the model plant Arabidopsis thaliana.Results are shown as follows:1.Construction of expression vector:The Arabidopsis CBL coding gene?AtCBL,At3g57050?was cloned into the prokaryotic expression vector pET28a.After successful sequencing,the recombinant pET28a-AtCBL plasmid was transformed into E.coli BL21?DE3?.The cells are named BL21?DE3?/pET28a-AtCBL.At the same time,pET28a plasmid was transferred into BL21?DE3?competent cells and named BL21?DE3?/pET28a.2.Optimum conditions for inducing protein expression:The expression E.coli BL21?DE3?/pET28a-AtCBL protein was induced by isopropyl-?-D thiogalactoside?IPTG?,and the optimized final concentration of IPTG was0.05 mol·l-1,incubate at 20°C for 20 h.At the same time,expression of the total protein of BL21?DE3?/pET28a and BL21?DE3?was induced under the same conditions,and a control group without IPTG but equivalent double distilled water was set.Under the induction of IPTG,AtCBL protein was expressed in BL21?DE3?/pET28a-AtCBL,but no expression of this protein was observed in other E.coli strains.3.Determination of production rate of H2S:Using L-cysteine as substrate,determine the H2S production rate of soluble proteins in bacterial lysates from BL21?DE3?/pET28a-AtCBL,BL21?DE3?/pET28a and BL21?DE3?induced by IPTG or without the induction of IPTG,respectively.The results showed that the H2S production rate of soluble protein in BL21?DE3?/pET28a-AtCBL lysate induced by IPTG was significantly higher than that of other E.coli strains,demonstrating that the recombinant AtCBL protein has the activity of catalyzing H2S production.In summary,this study demonstrated that AtCBL protein has a novel function of catalyzing the production of H2S using L-cysteine as a substrate through prokaryotic expression and a series of control experiments. |
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