Cloning And Functional Analysis Of ALS And The Toxicity Of Its Inhibitors To A. Azotica

Anabaena azotica and higher plants have similar photosyntheticsystem and ALS. As natural resources, A. azotica are going extinctionin filed with the extensive application of herbicides.Bensulfuron-methyl is one of the sulfonylurea herbicides, which are usedwidely all over the world, especially in China. Bensulfuron-methyl dono harm to humans and animals who do not have the target enzyme.However, environmental effect of ALS inhibitors on non-target organismshas drawn increasing attention resently. The study on its environmentalsafety, residual analysis and ALS should be done at present and in thelong future. There are few researches on the toxicity of ALS inhibitors tocyanobacteria. We adopt a great deal of biotechnology in Ecotoxicology,Molecular Biology, Bioinformatics and Computer Simulation on A.azotica, ALS and ALS inhibitors studies. Our experiments involve: theeffect of different doses of Bensulfuron-methyl on the growth rate,protein synthesis and photosynthetic pigments of A. azotica; the study oncloning, expression, structure and function of ALS gene; the active siteand the mode of action between ALS and its inhibitor; the construction of resistant mutants by PCR and so on. The results as follows:The168h-NOEC of acetone upon A. azotica was0.1%; acetone hadno effect on the components of pigments in A. azotica. The effect on thedensity of pigments was same to the effect on the growth rate, whichindicated that A. azotica was a little sensitive to acetone to an extent.Bensulfuron-methyl had a significant influence on the growth,protein content and photosynthetic pigments of A. azotica; the toxicity ofbensulfuron-methyl to the A. azotica was consistent with Hormesis,which refers low-dose stimulation high-dose inhibition.The change of light had a significant influence on the growth of A.azotica, the growth rate, cell number, dry weight and protein content. Theinteractions effect of light and bensulfuron-methyl on A. azoticastrengthened as the extension of time. Bensulfuron-methyl had toxiceffects in the general concentration and the toxic effects would beenhanced with the stronger natural light.The gene ALS encoding the acetolactate synthase from an industrialstrain of A. azotica was isolated by PCR. The nucleotide sequence of theALS gene consists of an open reading frame (ORF) of1,644bp, codingfor a547aa protein with a Mr of59.2kDa. The results were analysed bya BLAST similarity search of the GenBank database, which revealed theamino acid sequence was similiar to ALS derived from Anabaena sp.PCC7120. The ALS gene was subcloned into the plasmid pET28a(+) and was expressed in E. coli BL21(DE3) by IPTG induction. The bestconcentration of IPTG was0.6mmol/L and the best induced time was5h.The recombinant protein was purified to electrophoretic homogeneity andsubsequently characterized. The best elution concentration of imidazolewas100mmol/L. Conserved amino acid sequence and the predictedstructure of a monomer of ALS were consistent with those observed forthe bacterial enzyme in similar experiments which would provided cluesabout the differential effects of mutations in the active site tunnel onvarious inhibitors.The three-dimensional (3D) model of ALS, which was onlyresponsible for acetolactate producing activity, was constructed basedon the crystal structure of1OZG by using SWISS-Model/Homologymodule. With the aid of the molecular interaction analysis between thenatural substrate and inhibitors,3D model of the complex ALS-HE3,ALS-TPP, ALS-TB were developed by molecular docking program, andthe result may be helpful for further experimental investigations and thenew mutant designs as well. In addition, the key binding-site residueswere A381, G433, G434, D459, G461, G463, L464, and Y530, whichplayed an important role in the catalysis of ALS, was in consistent withexperimental observation from1T9A. The inhibitor tribenuron methylwas docked to ALS. Our results also showed that F276、S277、K279wereimportant in inhibition. Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which was a fast andsaving method, and it may be helpful for the future inhibitor study.