Isolation And Characteirzation Of A Sirohydrochloirn Ferrochelatase Gene From Maize

Siroheme is the most simply functional tetrapyrrole, as the prosthetic group of sulfiteand nitrite reductases, involving in the assimilation of nitrogen and sulphur in plant. Thebiosynthesis of siroheme is promoted in plastids and contains three steps, methylation,oxidation, and ferrochelation reactions. Sirohydrochlorin ferrochelatase is a key enzyme inbiosynthesis of siroheme, one of the second classes of chelatases, ATP-independent.Currently, there are only relevant researches about sirohydrochlorin ferrochelatase inArabidopsis thaliana. This paper has initially researched the overexpression andpurification of ZmSFC, the mutant ZmSFCM₁and ZmSFCM₂in E.coli. ZmSFC and themutant ZmSFCM₁protein were analyzed by UV-visible spectra scanning and EPRexperiment. The expression of ZmSFC treated with different concentrations of sulfite andnitrite were analyzed by Real-time PCR analysis. As following results：1. Extract the total RNA from the leaves of Inbred Line B73, the mature gene ofZmSFCuse was amplified with the template of cDNA, inserted it into the pMF-Dute-1vector. We added0.5mmol/L IPTG, induced it12h at28℃, purified ZmSFC byNi-NTA, the colour of the protein was coffee. The purified proteins showed a mainband on SDS-PAGE and the subunit molecular weight was estimated to be18kDa.2. Site-directed mutate primers of ZmSFC were designed. Only one amino acid residue（C60A） was mutated in the first round, which was named ZmSFCM₁.Then two newmutant sites （E42I, P43A） were added on the basis of ZmSFCM₁, which was namedZmSFCM₂（C60A, E42I, P43A）. Both mutants are overexpressed and purified.SDS-PAGE indicated that the mutants were the same with the ZmSFC protein.3. The UV-visible spectra scanning of purified ZmSFC and ZmSFCM₁displayed3absorption peaks of315nm,415nm and459nm; the absorption peaks are the typicalpeak of Fe-S cluster, indicating that the purified proteins may contain the Fe-S cluster.Further, the EPR experiment suggested the proteins comprise the [2Fe-2S] cluster.4. The ZmSFCM₁and ZmSFCM₂was concentrated to12mg/ml, with the solution of20mmol/L Tris-HCl,100mmol/L NaCl, pH8.0and. Crystallization of ZmSFCM₁andZmSFCM₂was acquired at10°C using sitting-drop vapor diffusion in98-welltrays（Hampton Screen Ⅰ&Ⅱ）. The crystals were appeared after1week in the17thwells of ScreenⅠat10°C, is sticked shape. The17th well is:0.2M Li2SO4,0.1MTris-HCl, pH8.5,30%PEG4000. 5. The leaves of maize were treated with different concentrate NaNO₃and K2SO44hoursat25℃, and then extract the total RNA of leaves, analysis expression of ZmSFC byReal Time PCR, the results show that: the expression of ZmSFC in leaves is the highestwith the treating of20mmol/L NaNO₃and50mmol/L K2SO4.Consequently, this paper cloned the mature gene encoding ZmSFC, obtained twomutanted proteins ZmSFCM₁（C60A） and ZmSFCM₂（C60A, E42I, P43A）, analyzed theiroverexpression in E.coli, and purified by Ni-NTA. UV-visible spectra scanning and EPRsuggested the purified proteins contain the Fe-S cluster. The ZmSFCM₁and ZmSFCM₂crystals were appeared after1week in the17th wells of ScreenⅠat10°C. Real-time PCRindicating that leaves were treated with NaNO₃and K2SO4at the low concentrate, theexpression of ZmSFC in leaves is the higher, when use the high concentrate, the expressionof the genes were indicate. The study is proved a foundation for further researching theactivity of sirohydrochlorin ferrochelatase.