Prokaryotic Expression And Expression Pattern Analysis Of TaATQ In Wheat

Drought and high salt stresses can significantly decrease crop yield, so it is important to find genes against these stresses. Aldehyde dehydrogenase (ALDH) is a protein superfamily, which is widely found in bacteria, plants, animals and insects, and the members of the superfamily can catalyzed a variety of endogenous and exogenous aldehydes to generate the corresponding carboxylic acids which are dependent on NAD(P)+. In vivo, ALDH involved in some import biological process, such as cell detoxification, intermediary metabolism, and protect from osmotic stress by generating osmoprotectants. ALDH7 (ATQ)is a subfamily of ALDH superfamily. Amino acid sequence among members of this subfamily from different species is highly conserved, in order to reflect the "antique" feature of these proteins, We call them "Antiquitin".In order to elucidate the biochemical function and the roles of ALDH7 in wheat(TaATQ) in the process of metabolism, the following studies were carried out:1. Bioinformatic analysis of TaATQ. TaATQ shares 91.7% similarity with ATQs in rice,and 78.1% in pea.Comparing to nematode and Drosophila melanogaster,the similarity of ATQs are 58.1% and 52.5%.Like the other family members,it may exist as an oligomer, each monomer is compoased of three typical domain, a NAD+-binding domain, a catalytic domain and an oligomerization domain,whose molecular weight is 54.35kD and pI is 5.9.2. The encoding sequence of ATQ protein from wheat was digested by EcoRⅠand NotⅠ,then it was cloned into pET-21a-MBP, pET-28a and pET-41a vector. We named the three recombined vectors pET-21a-MBP-TaATQ, pET-28a-TaATQ and pET-41a-TaATQ. Fusion proteins were expressed in T7 Express Competent E. Coli strain.However,the activity of aldehyde dehydrogenase was not detected by the systems reported before.Changes of condition of prokaryotic expression may be needed,or it is related to the characteristic feature of ATQ protein from wheat.3. By Semi-quantitative RT-PCR,the expression levels of TaATQ under NaCl treatment and PEG treatment were detected. The expression levels of TaATQ in both leaf and root were increased significantly during the application of these two treatments.Under NaCl treatment, We observed the obvious response in leaf 3 hours after the application of treatment and the expression level of TaATQ in root increased significantly after 12 hours. Under PEG treatment, The expression levels of TaATQ increased significantly after 6 in leaf hours and 3 hours in root. To gain further insight into the expression levels of TaATQ, fluorescence quantitative PCR was carried on. The results show that the expression levels of TaATQ in flower and seed were higher than the other tissues. The expression levels of TaATQ in both leaf and root were increased significantly during the application of high salt. Under drought stress, the transcripts of TaATQ was elevated only in leaf.TaATQ has typical structure features of ALDH protein family, it plays a important role during the process of stress response. TaATQ can be used as a target gene for crop genetic improvement.