Cloning And Functional Study Of A New Drought-tolerant Gene TaDRLea3-2 In Wheat

Wheat is the important grain resource to the survival of mankind, but it often encounter all sorts of adversity stresses such as drought, salinity, extreme temperatures, heavy metal pollution and so on. These Abiotic stresses are serious threats to agriculture, as they adversely affect the crop growth and yield. In order to survive, the plants form a series of defensive signaling pathways, activate the expression of stress-inducible genes, and thus to minimize plant cells damage. The Late Embryogenesis Abundant Protein(LEA) is a kind of important stress-tolerant protein, which may function in protection and damage repair of plants. On the basis of their hydrophilic coefficient and conserved motifs, LEA proteins are divided into seven families. LEA3 protein is the third member of the family. There are many reports have proved that group 3 LEA protein is associated with plant drought resistance ability.In this study, we isolated a novel LEA3 gene TaDRLea3-2 in wheat, analyzed by bioinformatics software and used real-time quantitative PCR to detect the expression of TaDRLea3-2 gene in different tissues and under various abiotic stresses, and also observed the subcellular localization of TaDRLea3-2 with GFP as report gene. Based on these experiments, we have a preliminary understanding of this LEA3 protein, which can lay a foundation for elucidating its protection mechanism under adversity conditions in wheat. Main experiment results are as follows:1. Using the method of homological cloning, LEA3 gene TaDRLea3-2 from wheat "Zhengyin No.1" was cloned and the sequence was analyzed by bioinformatics software. The full length of TaDRLea3-2 is 668 bp, whose ORF is 570 bp, encoding 189 amino acids, consists of 9 conserved 11-mer repeating motifs, and belongs to 3A subgroup of LEA3 family; Protein prediction showed that the protein is highly hydrophilic and located in the cytoplasm, with three phosphorylation sites and has no transmembrane domain or signal peptide; Secondary structure analysis indicated that the main structure is alpha helix; Multiple sequence alignment and evolution analysis were carried out by BLAST and MEGA5.1.2. Real-time quantitative PCR detection results showed that, TaDRLea3-2 was expressed at the highest level in leaves, followed by stems and at the lowest level in roots. The expression level of Ta DRLea3-2 was also examined by qRT-PCR under various abiotic stresses. Expression analyses revealed that TaDRLea3-2 gene was induced by drought, low-temperature, high-salt stress, moreover, can be induced by ABA treatment. These results suggested that TaDRLea3-2 gene is an ABA-dependent LEA3 gene, and participates in Wheat abiotic stress response process via different mechanisms.3. The transient expression vector pCAMBIA1300-TaDRLea3-2 was successfully constructed and transformed into agrobacterium EHA105. Then the recombinant vector was imported into tobacco protoplast by agrobacterium mediated method and used confocal laser scanning microscope to observe the location of the fusion protein(TaDRLea3-2 : GFP) in plant cell. Results indicated that the fusion protein was located in the cytoplasm, consistent with software PSORT forecast result, and implied that TaDRLea3-2 protein may play its protective role in the cytoplasm.