Screening And Expression Analysis Of Imazethapyr-Resistant Gene

Imazethapyr, which belongs to imidazolinone herbicide, is mainly used in soybeans and other field crops, for controlling annual and perennial grasses, broadleaf weeds, sedges weeds, etc. It also belongs to ALS inhibitor herbicides, because of the advantages of its high activity, strong selectivity, broad spectrum ability and low toxicity, it has become an important and efficient herbicide and a extensive focus of attention, at the same time also became more widely used herbicide in the field; However, due to its long residual herbicide, because its used heavily and repeated, caused crop phytotoxicity,influence the crop production, and even had no harvest, destroyed the soil ecosystem and the sustainable use of resources. Therefore, how to deal with the phytotoxicity from long residual herbicide to later sensitive crops has became the problem. So far, there are two solutions:1. Ease soil environment by excessive pollution by microbiology which can degrade the herbicide residual in the field.2. Ease the damage of residual herbicide to next later crops by cultivate genetically modified crops which has herbicide resistance.The study first screened Bacillus megaterium which has imazethapyr-resistant, while built it’s genomic library, and through the expression of candidate gene, probed the structure properties of the protein of candidate gene. The main findings are as follows:1. Successfully screen an Imazethapyr-resistant (7500ppm) strain T-1, which was isolated from sludge of Imazethapyr factory mud. By analysis the phylogenetic tree, T-1was identified as Bacillus megaterium. The logarithmic phase of the strain is12-24h in the liquid culture medium, the greatest growth temperature was36℃, the optimum pH value is6.0.2. Enzyme digested of genomic DNA and connected pACYC184vector to construct genomic library. Caculated based on the the library’s average insertion fragment (Approximately2.5kb) and total capacity (about8900clones), this library covers the genome sequences of about2225Mb, in accordance with Bacillus megaterium genome4Mb, this library covers Bacillus megaterium genome556times. This library is up to standard, that can be used to screen resistantl gene.3.When the library screened to imazethapyr concentration of15000ppm, obtain a candidate gene BM-1, length of1293bp, after digestion and sequenced of the fragment, re-transferred to BL21, successfully induced the expression of the protein; The protein expression is45kDa, imazethapyr-resistance functional validation result of Escherichia coli BL21containing the candidate gene from the original7500ppm to15000ppm, much higher than the empty vector-negative (7500ppm) controls, indicating that candidate gene could successfully improve the E. coli resistance to imazethapyr.4. Analysis and forecast the amino acid which is encoded by candidate gene, that research shows that:BM-1encoded430amino acids, that has1293bp length, and its leucine (Leu) content of up to19.3%, followed by proline (Phe) and tryptophan (Ser) were8.1%, the minimum content of methionine (Met), only0.9%; Formula is C2322H3566N624O610S15, he molecular weight is50.4645kDa, GC content of53%; And the pI is8.60, tThe hydropathy turns0.062>0, in this way, it is a hydrophilic protein. By secondary structure prediction with coil domain, found that the protein has347a-helix spirals,the helicity was20.3%,396β-folding, the folding rate was22.5%; through the tertiary structure prediction it found each domain by tertiary structure prediction results center has2β-folds, which is surrounded by several a-helix.