Cloning Of AtCBF3and ThNHXl Genes And Construction Of Marker-free Plant Expression Vectors

Agricultural production, plant growth and development are subject to various environmental factors such as stress, cold, drought, salinity and others as a major stress factors. This type of CBF3（CRT/DRE-binding factor） protein that can regulate the temperature-related genes are important transcription factor. CBF3genes are cloned and identified in Arabidopsis thaliana, specific binding of cis-regulatory elements in DNA on the DRE, DRE contains the COR gene cluster, through the continuous over-expression CBF3induced COR gene expression.Promoter Rd29A affected by drought, low temperature, high salinity and other factors in the induction, so that a large number of downstream target gene expression. Construction of stress-inducible promoter-driven regulation of plant gene expression vector, the gene in plants for efficient and selective expression has important practical significance. CBF3gene and Rd29A gene can regulation of COR gene expression in drought, low temperature and high salt stress conditions, and plays an important role in the signal transduction.Many, too many, species have been used to examine salinity response physiology, but the names associated with genetic or molecular genetic studies in salinity stress research are few. The recent discovery of the halophytic plant species, Thellungiella halophila, which is native to the seashore saline soils, meets all the criteria for being a genetic model system. Thellungiella halophila which is a relative of the Arabidopsis has small size, short life cycle, self-pollination, and high seed number, and favorable genetic traits such as self-fertilization, a small genome, efficient transformation, and mutagenesis. So, Thellungiella halophila becomes the prospective salinity tolerance model. Concerns have been raised that the Presence of selectable marker genes such as antibiotic or herbicide resistant genes might be unpredictable hazard to the ecosystem as well as to human health, the presence of marker genes serious impediment to the commercialization of transgenic plants the effectiveness of the process and conversion technologies, Marker-free is a rapidly developed strategy that offers a new approach for the elimination of public concerns caused by the selectable marker genes conferring antibiotic or herbicide resistance and so on.. Therefore, how to cultivate marker-free transgenic plants in recent years, plant genetic engineering has become the focus of the studyThe test material for the study oi Arabidopsis thaliana, the genes and promoter Rd29A, CBF3study results and the study of Thellungiella halophila, the ThNHXl study results are as follows:1. According to the sequence of the Arabidopsis thaliana by PCR, cloned from the Arabidopsis thaliana genes related to cold-induced CBT3and the stress inducible promoter Rd29A. Sequencing results showed that the cloned CBFi gene has750bps and encodes216amino acids, and has100%identity with the sequence published on GeneBank. The methods of bioinformatics are applied to analyze the similarity of amino acid sequences and phylogenetic tree; the promoter Rd29A has1425bps, and has100%identity with the already published promoter sequence.2. By molecular cloning techniques, the use of PMD18-T vector, successfully Rd29A promoter、CBF3gene and constructed into binary plant expression vector pCAMBIA1301, Successfully constructed pCAMBIA1301-Rd29A-CBT3and introduced into Agrobacterium tumefaciens LBA4404by freeze-thaw method.3. According to the sequence of the Arabidopsis thaliana by PCR, cloned from the Thellungiella halophila genes related to ThNHXl. Sequencing results showed that the cloned ThNHX1gene has1761bps and encodes545amino acids, And the known Arabidopsis thaliana Na+/H+antiporter protein （GenBank registration number: NM₁22597） with94.4%similarity, And with other plants of the Na+/H+antiporter also has relatively high similarity.4. By molecular cloning techniques, no selectable marker gene of plant expression vector pCAMBIA1301was successfully constructed. And with cloned YMD18T-CBF3, PMD18T-ThNHXl digestion and ligation, Co-transformation vector pCAMBIA1301-NOhyg-CBF3and pCAMBIA1301-NOhyg-ThNHX1been successfully constructed, Agrobacterium tumefaciens LBA4404was transformed into tobacco in order to obtain transgenic plants.