Cloning And Functional Analysis Of O-acetylserine (Thiol) Lyase (Oastl) Family Genes In Foxtail Millet

O-Acetylserine?thiol?lyase?OASTL?is a class of enzymes that well known as the cysteine synthase,which catalyze the final step of the biosynthesis of cysteine.In recent years,more and more evidence has shown that the O-Acetylserine?thiol?lyase?OASTL?had multiple roles apart from its ability to catalyze the biosynthesis of cysteine.It has been shown that OASTLs were also involved in the biogenesis of H₂S,S-sulfocysteine synthase,as well as the detoxification of cyanide.At present,detailed studies about this enzyme have only been found from the Arabidopsis thaliana,though sporadic work on OASTL was also found from other dicots,such as tomato and rape.However,few reports about the function of OASTLs have ever been found from monocots.Foxtail millet is a kind of crop with great commercial value in the production of agriculture.In China,foxtail millet is mainly grown in the northwest,as it is drought-tolerant,adaptable,and gives high and stable yields.Nowadays,with the completion of its genomic sequencing,foxtail millet has become a new model plant for the study of monocot.In this study,three Si-OASTLs were cloned and studied from multiple levels.And our results provide a theoretical basis for further understanding of the function and mechanism of the Si-OASTL family proteins.The followings are the main results of this study:1.Gene cloning and sequence analysis:Three OASTL family genes were amplified from the cDNAs of foxtail millet by PCR and named as Si-OASTL7,Si-OASTL9 and Si-OASTL10.The coding regions of Si-OASTL7,Si-OASTL9and Si-OASTL10 were 807bp,858bp and 855bp,and the encoded proteins were 268aa,285aa and 284aa,respectively.Furthermore,the result of the multiple protein sequence alignment showed that all the three proteins consist of the highly conserved regions of the OASTL proteins.However,the phylogenetic analysis showed that none of the three Si-OASTLs belonged to any published subfamily of the OASTL protein family.The three Si-OASTLs constituted a new subfamily specific to the OASTL proteins from monocots with proteins form Oryza sativa,Zea mays and sorghum.2.Exogeneous expression of the three Si-OASTLs:For the construction of the recombinants,the cds sequences of the three genes were firstly ligated into the p-Cold expression vector and transformed into the E.coli strain DH5?;then the recombinant vectors were identified through bacterial PCR and double enzyme digestion,finally the three recombinant plasmids were transformed into BL21?DE3?.For expression induction of the proteins,isopropyl-?-D-thiogalactoside?IPTG?was added to the medium,and the optimized induction condition was 0.2 mmol/L IPTG at 16?overnight.Then the proteins were purified through Ni affinity chromatography,and the optimum imidazole concentrations for the elution of the hybridprotein and the target protein were 200 mmol/L and 500 mmol/L,respectively.3.Enzymatic analysis of the protein:The enzyme activities of the three proteins purified above were eventually analyzed.The results showed that Si-OASTL9 was the only protein among the three that possessed the abilities to catalyze the biosynthesis of cysteine and S-sulfocysteine,while Si-OASTL7 and Si-OASTL10 could not perform as a catalyst during the biosynthesis of cysteine and S-sulfocysteine,or the generation of H₂S.4.The expression analysis of three genes:First,we analyzed the expression patterns of Si-OASTL7,Si-OASTL9 and Si-OASTL10 by qRT-PCR,and the results showed that all the three genes were broadly expressed among the whole tissues of the foxtail millet with a preference in the leaves.The changes of expression levels were also detected when the plants were treated with salt,drought and cadmium?heavy metal?,and the results showed that the expression levels of the three genes were significantly up-regulated under drought and salt stresses.However,under Cd stress,only the expression of Si-OASTL9 was significantly up-regulated,while the RNA levels of the other genes did not change.In summary,in this paper,three Si-OASTL genes were cloned and characterized through sequence analysis,enzymatic analysis,as well as the expression analysis.This study provides more information on studying the function of the OASTL family genes in plants,especially in monocots.