Mutation Identification Of Glutathion S-transferase Gene In Drought-resistant Maize Mutant 18-599M

Maize originates from the tropical zone of high temperature and humidity in Central and Southern America,so that it has poor tolerance to drought.With the worsening of ecological conditions in the global areas,drought has been becoming the major limitative factor to maize production in many countries and regions.Breeding and extending drought tolerant varieties is the most effective and economical method for reducing the loss of drought.Therefore,extending germplasm resources,creating germplasm material and especially innovation and utilization of excellent anti-stress material are important in maize breeding. vGlutathione S-transferases(GST) is a general and universal soluble detoxification enzyme which has variety of functions.GST catalyses the mercapto-group of glutathione(GSH) integrating with electrophilic group of endogenous and exogenous injurants,and makes injurants degradation or dissolving and discharging from organism.GST can make the stress resistance strong by excessive expressing in biotic and abiotic stress conditions.So,studing on GST' sequence and function will be of great significance to better understand stress resistant mechanism and improve the resistance of plants in adversities.In former studies,the calluses of excellent inbred line 18-599 were treated by 60Coγ-ray and NaN3 respectively,and drought tolerant mutations were screened at high osmotic potential culture of 1.0%NaCl.The drought tolerance of inbred lines of mutant with together wild was identified under plastic rain shelter with Anthering and Silking Interval(ASI) and per plant kernel yield as indices.The result showed that the drought tolerance of 18-599M is significantly stronger than wild 18-599.SSR analysis indicated that the difference of GST exist between 18-599 and 18-599M.Based on the submitted sequence of GST cDNA of maize(EC2.5.1.18) from GenBank,one pair of gene specific primers were designed and GST was amplification through RT-PCR.The sequence homology analysis indicated GST-B exhibited 98%identity respectively with GST-1,and as the same as GST-Y.And the homology of amino acid is up to 98%.The GST-B cDNA containing an open reading flame of 645 bp and encoding 214 amino acids,flanked by 52 bp 5' UTR and 202 bp 3' UTR;The GST-Y containing an open reading frame of 645 bp and encoding 214 amino acids,flanked by 77 bp 5' UTR and 193 bp 3' UTR.Compared with GST-B, GST-Y has seven point mutations in the ORF,insertion mutation of five AGGAG in the 5'UTR and four point mutations and a lack of TGACGAAAG mutation in the 3'UTR.18-599M has three amino acid site changes,In order to determine the gene sequences have the activity of expression,GST-B and GST-Y were cloned into pET-32a to constructed the prokaryotic expression vector for prokaryotic expression.SAS-PAGE of induced protein of pET-B and pET-Y strain which were induced by 0.4 mmol/L IPTG at 30℃for 4h,showed that there was a strengthened protein band in 35 kD protein Marker.So the cloned genes have complete open reading frame.For further determine the three amino acid changes whether or not have an impact on GST activity,GST activity was measured on different time treated with drought.The result shows that GST activity of 18-599M is significantly higher than that of 18-599 under water stress.So mutations increased the activity of enzyme in 18-599M.Transcriptional regulation of the enzyme and the stability of the cDNA are affected by insertion mutation,point mutation and deletion mutation in the 5'and 3' UTR.