The Function Of Drought-induced Gene TaDlea3 And Temporal And Spatial Expression In Wheat During Different Developmental Stages

Abiotic stresses such as drought, high-salt, and low-temperature are major constraints to growth and yield of crops. In response to various stresses, plants accumulate a range of proteins to protect cell metabolism. Late embryogenesis abundant protein(LEA) is closely related to environmental stresses. Based on sequence similarity and conserved domains, LEA proteins are classified into at least seven groups, among which group 3 LEA proteins are characterized by highly conserved 11-amino-acid repeat motif of “TAQAAKEKAGE”, forming an amphiphilic helical structure. They can protect biological macromolecules and reduce the damage to the plant under abiotic stresses. However, the protective mechanisms of LEA3 proteins remain poorly understood.In the present study, a novel group 3 LEA gene namely Ta Dlea3 was cloned from wheat(Triticum aestivum L.).The function of Ta Dlea3 gene was evaluated by subcellular localization, protection of LDH in vitro and its responding to abiotic stresses. Moreover, the resistance ability of drought stress in transgenic Arabidopsis was also evaluated. We further investigated Ta Dlea3 transcript and protein expression of two different drought-tolerant varieties wheat in response to drought stress and rehydration during four different developmental stages(seedling, tillering, jointing and flowering stage). The main results are as follows:1. A novel group LEA3gene(named Ta Dlea3) and four new Ta WSI18 genes which were homologous to LEA3 gene, were obtained by silico cloning and RT-PCR in wheat shaanhe 6. Bioinformatics analysis showed that Ta Dlea3 and four Ta WSI18 proteins were highly hydrophilic, located in the cytoplasm, and contained no transmembrane domain. The Ta Dlea3 and Ta WSI18 protein are homologous to LEA3 proteins of other species. The prokaryotic expression and purification analysis of the Ta Dlea3 protein was conducted to obtain the high specificity of polyclonal antibody.2. The subcellular localization vector of Ta Dlea3 gene was transformed into Agrobacterium tumefaciens and was injected into tobacco leaf epidermal cells. We also prepared the protoplasts. The results showed that the Ta Dlea3 protein was located in the cytoplasm. Under high and low temperature stress, Ta Dlea3 protein has protective effect on LDH. The Ta Dlea3 gene in two wheat cultivars responded differently to various adversity stress. The Ta Dlea3 gene in wheat shaanhe 6 significant responded to drought stress more significantly, while the Ta Dlea3 gene in wheat zhengyin 1 responded to ABA stress more significantly.3. Transgenic Arabidopsis plants were obtained by the floral dip method. No clear difference in phenotype and physiological indices were observed between the transgenic and wild-type plants under normal conditions. After water drought stress treatment, the wild-type plants showed extreme wilting, whereas the transgenic lines remained turgid. The results of changes in several antioxidant enzymes and drought-resistance indicators showed that under drought-stress conditions, the contents of MDA and relative electrical conductivity in transgenic plants were lower than those in wild-type plants, while the contents of proline and three antioxidant enzyme activity in transgenic plants were siginificantly higher than those in wild-type plants. Ta Dlea3 overexpression improved the tolerance capability of Arabidopsis to drought stress.4. We conducted quantitative RT-PCR and Western blot analysis of the Ta Dlea3 transcript, and protein expression of two different drought-tolerant varieties wheat shaanhe6(drought tolerance type) and zhengyin1(drought sensitive type) in response to drought stress and rehydration during four different developmental stages. The results showed that the tendency of Ta Dlea3 protein accumulation was also consistent with the Ta Dlea3 transcription levels during all the developmental stages with drought stress treatment. The Ta Dlea3 expression and Ta Dlea3 accumulation were increased largely in severe drought stress, while decreased after rehydration. Lower levels of Ta Dlea3-gene expression were observed in the flowering period along with weaker protein signals after rehydration than the three other growth periods. The change of MDA, relative electrolyte leakage, proline and chlorophyll content of two wheat cultivars under drought stress were consistent. The results showed that drought stress at seedling and tillering stages had little effect on wheat yield, while drought stress at jointing and flowering stages had a strong impact on wheat yield, demonstratig that Ta Dlea3 gene and protein expression were related to drought stress.