The Clone Of A AP2 Transcription Factor Gene Of Maize And The Construction Of Prokaryotic Expression Vector

Plant growth and development process, often subject to high humidity, low temperature, drought, heat stress and other abiotic factors, light to plant growth and development delay, severe cases, lead to plant death. Under stress conditions, plants usually occurs within the body a series of physiological and biochemical reactions, also form a certain stress response mechanism. After a series of signal transmission, transcription factors was activated to combine with the cis-element, and then stimulated to form a transcription complex of RNA polymeraseⅡ, which initiates gene transcription. Therefore, transcription factors play vital roles in stress signal transduction, gene expression modulation and phase transition during plant development.AP2/DREB transcription factor family are widely present in plants, involved in plant cell cycle, growth and development, and biological and abiotic stress-related gene expression. ERF family of transcription factors, which are a class of stress-related transcription factors play an important role in plant defense responses. According to reports, ERF transcription factors can be combined with GCC-box cis-acting element involved in plant of high humidity, low temperature, drought, high temperature and other abiotic stress response process. The intention of this paper is to isolate genes of AP2/DREB transcription factors by methods of biochemistry and molecular biology, and can help to further reveal regulation mechanisms of DREB family, to develop maize stress-tolcrent breeding, to clarify the stress-tolerant molecular mechanism.Progresses of this research as follows:In this paper, taking advantage of UniGene database, using the conserved sequence of Arabidopsis transcription factors AP2/DREB superfamily as information probe, a transcription factor BT039300.1 of B2 subfamily of ERF family was isolated. A transcription factor of AP2/DREB species was cloned By PCR and RT-PCR from the Preharvest sprouting plant of A318 maize inbred line. The Sequence analysis showed a high genetic similarity between the BT039300.1 was cloned and Electronic cloning of transcription factor gene, only having a different amino acid sites. This study make a more comprehensive analysis from the similarity of amino acid sequence, composition, physicochemical properties, hydrophobicity, hydrophilicity, sequence alignment, phylogenetic tree, protein functional domain, secondary and tertiary structure prediction and other aspects. The results showed that the full length of BT039300.1 is 1350bp, containing a complete open reading frame of 1023 bp, encoding 340 amino acids, and having a very conserved ERF domain. The phylogenetic tree which was constructed indicates that, BT039300.1 belongs to AP2/EREBP subfamily, and the relationship of heredity is quite close to the gene RAP2.12 belonging to B2 subfamily of ERF family. BT039300.1 is a hydrophilic protein, the tertiary structure of proteins is similar to AtERFl. The result of real-time PCR demonstrate that BT039300.1 expresses highest in cob of PHS plants after 15 days of pollination, expresses highest in endosperm of CK plants after 15 days of pollination, expresses highest in stem of PHS plants after 20 days of pollination, expresses highest in endosperm of CK plants after 20 days of pollination In addition, expresses highest in stem of PHS and CK plants after 25 days of pollination.In addition, the gene was constructed into a prokaryotic overexpression vector, providing a foundation for further studying the function of BT039300.1 in regulation of stress tolerance in maize.