Characterization And Anti-freezing Function Of Dehydrin Members In Loquat(Eriobotrya Japonica)

Dehydrins (DHNs) are a family of plant proteins typically induced in response to stress conditions that cause cellular dehydration, such as low temperatures, high salinity, and drought. Loquat(Eriobotrya japonica) is a perennial fruit crop, it usually suffered unfavorable environment during its growth and development process. Especially it blossoms during winter. Loquat fruitlets are frequently injured by freezing. A reduction or cessation of growth frequently takes place during the winter. This dramatically reduces its yield and quality. In this work, we studied the characteristics of different EjDHNs, their expression patterns, as well as the relationship between their expression levels and the freezing resistance ability of loquat, tried to reveal the response mechanisms and the possible functions of different EjDHNs members.The main results are as follows:1. Seven EjDHNs, showing four different structure types, were obtained from loquat by RT-PCR and RACE, including YnSKn type of EjDHNl (Y2SK3), SKn type of EJDHN2(SK3), EjDHN3(SK9), EjDHN5(SK3), EjDHN1(SK3), Kn type of EjDHN4(K4), and YnKn type of EjDHN6(YK3). These DHN proteins differ significantly in size and in biochemical properties. The expression results indicated that EjDHN5was high expressed in different tissues, EjDHN4was mainly expressed in flower, while EjDHN1, EjDHN3, EjDHN6and EjDHN7were expressed especially high in stem. The variation of expression levels showed that all these members except EjDHN3may play an important role in loquat seed development, and EjDHN1, EjDHN2, EjDHN5may also be related to pulp development. It was found that the expression pattern of EjDHN members was rather similar during the seasonal variations. Their highest transcript levels all appeared in the coldest month, which indicated that these genes were all response to low temperature stress. The transcript levels of all seven genes were significantly increased in the drought treated leaves, and the abundance of the EJDHN6transcript was higher than that of the other six EjDHNs. Results also showed that EjDHN6was most sensitive to ABA100μM treatment, and EjDHN2, EjDHN5, EjDHN7were partly induced by ABA. It suggested that EjDHN6was played a main role in response to drought and ABA stress.2. To evaluate the role of the EjDHNs in freezing resistance in loquat fruitlets, two cultivars of loquat, the freezing-sensitive ’Ninghaibai’(NHB) and the freezing-tolerant ’Jiajiao’(JJ), were analyzed under induced-3℃freezing stress. Freezing stress led to obvious accumulation of reactive oxygen species and considerable lipid peroxidation in membranes during the treatment period. Both these phenomena were more pronounced in ’NHB’ than in ’JJ.’ Freezing treatment resulted in up-regulation of the expression levels of all seven EjDHNs, regardless of structure type. The accumulation of the transcripts of these EjDHN genes was much more pronounced in ’JJ’ than in ’NHB.’Immunogold labeling of dehydrin protein was performed. DHN proteins were found to be concentrated mainly in the vicinity of the plasma membrane, and the density of the immunogold labeling was significantly higher after freezing treatment, especially in the more freezing-tolerant cultivar ’JJ.’ Altogether, this study provides evidence that EjDHNs are involved in the cryoprotection of the plasma membrane during freeze-induced dehydration in loquat fruitlets. The severe membrane damage observed in ’NHB’ plants after freezing treatment can be partially ascribed to its lower ability to accumulate DHN proteins.3. To examine the function of the EjDHN in response to low temperature stress in loquat, EjDHN1and EjDHN5were transformed into tobacco. As a result,7lines of EjDHNl and22lines of EjDHN5were got. Among them, two lines of each gene, which produced relatively higher transcripts levels of the target gene, were choses for functional characterization. The effect of EjDHNl and EjDHN5transgene on the tolerance to cold and oxidative in tobacco was determined. The results showed that the survival rate were significantly higher in the14d old transgenic lines than the wide-type tobacco. The plant growth condition of the transgenic lines was better than the WT after recovering from0℃cold stress for2weeks. Cold stress led to the reduction of the leave PSII activity, and the membrane damage, however, these happened more seriously in WT than transgenic lines. To examine oxidative stress tolerance, plants were imposed to different concentrations of MV. The result also showed that plant growth was inhibited both in WT and transgenic lines. At10days after being exposed to2.0μM MV, the WT plants had significantly lower survival rate and much worse growth condition. The results indicated that the transgenic plants overexpressing EjDHNl and EjDHN5showed increased tolerance to cold and oxidative stresses compared to WT plants. It also suggested that EjDHN protein may be involved in the response to cold and oxidative stresses.