Cloning And Expression Analysis Of Stress Induced Gene ZmGST23in Maize (Zea Mays L.)

Maize (Zea mays L.) is the major food, feed and industrial materials which playsan important role in the world. China is the second largest maize producing countryonly to the United States, owning a sown area of35,029,800hm2in2012. But variousbiotic and abiotic stresses such as drought, salinity, high temperature, low temperature,water-logging and disease can seriously affect the growth and yield formation of maize.However, the development of plant molecular biology, genomics and functionalgenomics pave the way for the breeding and improvement of stress tolerance cropvarieties.Plant Glutathione S-transferases(GSTs, E.C.2.5.1.18) are a superfamily ofmultifunctional enzymes which catalyse the conjugation of electrophilic xenobioticsubstrates with GSH, play an important role in stress defense system. GSTs catalyze thebinding of various xenobiotics (including numerous pesticides) and their electrophilicmetabolites with GSH to produce less toxic and more water-soluble conjugates. Variousabiotic stresses are powerful inducers of GST activity in plants, the research on themechanism of how GSTs functioning in the response to various stress can supply us atheoretical basis for functional gene based crop improvement.Current research on plant GSTs were mainly involved in the detoxication ofherbicide, litter research had focused on the function of stress tolerance in plantespecially in maize. What is inspiring, a study on plant resistance to three maize leavediseases (southern leaf blight, gray leaf spot and northern leaf blight) had showed thatZmGST23was associated with all three diseases. But little is know about the role ofZmGST23in various abiotic stress responses. To this end, the relative expression levelof ZmGST23in six abotic stress were detected using the Real-time Quantitative PCRassay, the full-length CDS region were cloned from the leave of an elite maize inbredline F83and a prokaryotic expression assay was conducted with the aim to verify thegene expression activity. The main results were as follows:1. The Real-time Quantitative PCR assay revealed that ZmGST23in leaves can beinduced by Salt, ABA, Dehydration, Cold, Heat and herbicides. The relative expressionlevel of ZmGST23gene reached a peak in3hours after the ABA and NaCl treatment,6hours after the low temperature and30%glyphosate treatment,1hours after the high temperature and24hours after natural dehydration. ZmGST23has a higher expressionlevel in leaves than roots and maybe a leaf-specific gene involved in the ROS cleaningprocess.2. The full-length CDS region was cloned using specific primers GST23F/GST23R, the sequence alignment results showed that ZmGST23contains an ORF of669bp，which encoded a protein of222protein. The cloned sequence has conformity of99.40%compared with the original sequence, and the translated protein has an identityof99.09%compared with the sequence in Uniprot. Bioinformatics analysis showed thatZmGST23encoded protein with24.84kDa molecular weight and5.98of isoelectricpoint.3. We connected the cloned fragment to the prokaryotic expression vectorpEASY-E1, obtained a prokaryotic expression vector pEASY-E1-GST23. Theprokaryotic expression assay showed that the cloned ZmGST23has a high abundanceinduced by0.5mM IPTG after1h to4h at37℃culture condition. The specificexpression band of ZmGST23was approximately30kDa.