Molecular Cloning,Expression Analysis And Function Identification Of NnDREB2A From Lotus Root(Nelumbo Nucifera Gaertn)

Lotus root （Nelumbo nucifera Gaertn.）, a member of the family Nymphaeaceae, is an aquatic herb vegetable. Lotus root which originated from China and India has been widely cultivated in China for multiple purposes. The products of lotus root and lotus seeds are very popular in the daily diet because of its richness in nutrients. The area of land in coastal regions has become larger due to sea water retreating in China recently, and how to fully utilize these stalinization soils is a challenge. Dehydration responsive element binding factor （DREB） is believed as a cis-acting element to regulate down-stream gene expression when plant is subjected to salt stress. In this study, an NnDREB2 was isolated with RT-PCR and RACE method and gene expression and possible function was also analyzed with aim to identify gene function in lotus root based on salt-tolerated evaluation of lotus resources.1. Evaluation of lotus resources. In this study,7 lotus varieties were treated with 0.6%NaCl, and we found that the difference was significant in index of salt injury, chlorophyll content, MDA content, antioxidant enzyme activity and root activity. Among which, the permeability of cell membrane in H8 leaf was the lowest than that of other species, and while highest root activity and lowest salt damage index in H5 was found after salt treatment. Comprehensive evaluation of the 7 lotus varieties to salt tolerance using subordinate function method was carried out, and we find that the ability of salt tolerance was as follows: H5>H8>E2>E4>E1>E3>H2.2. Cloning of NnDREB2A and analysis. A full length of DREB2A gene was amplified with RACE-PCR method, which was found to be 1146 bp consisting of a 63 bp of 5’non encoding region, and a 162 bp of 3’non encoding region. NnDREB2A contained a single open reading frame which encoded a putative polypeptide of 306 amino acids. The gene was composed of two exon regions and a intron region. The molecular weight of deduced protein was 34.75 kD, and isoelectric point was 5.70. When compared against NCBI database, this gene showevd 96%, 78%, and 73% sequence similarity with the DREB2A from Nelumbo nucifera （XM₀06353357.1）, Arabidopsis thaliana （NM₁20623.2）, and potato （XM₀06353357.1） respectively. On the basis of this similarity, we designated it as NnDREB2A.3. Expression of NnDREB2A gene in lotus root. Semi RT-PCR method was carried out to evaluate the expression of NnDREB2A with fi-actin as internal standard. Total RNA was extracted from different organs including leaf, leaf stalk, rhizome and stem tip at different time intervals after 0.6% NaCl treatment to study the mRNA accumulation of NnDREB2A. Results showed that NnDREB2A expression was induced by NaCl treatment in various organs, especially in rhizome and stem tip, the expression was significantly increased after exposure to NaCl for 3 h. The expression of NnDREB2A showed highest level in petiole and leaf after 18 h of salt treatment. NnDREB2A expression to exogenous ABA （100 μM）, mannitol （300 mM） and low temperature （4℃） was also analyzed. Exogenous （100 μmol/L） ABA significantly induced the NnDREB2A expression in root tip after 12 h treatment under normal growth conditions. However, no significant change was found to low temperature and manninol within 24 h.4. NnDREB2A function identification. Full-length NnDREB2A cDNA was ligated into a binary vector （pSN1301） with GUS reporter gene under the control of CaMV 35S promoter, and then inserted into Agrobacterium tumefaciens strain EHA105. Arabidopsis transformation was carried out using the floral dip method. Three positive plant, such as PC43, PC44 and PC48 was obtain after selection. Seed germination was investigated on MS medium containing 200 mM NaCl.72%,72% and 60% germination rates was found in PC43, PC44, PC48, and whereas only 20% germination rates for wild type plant after 10 d exposing to salt stress. The seeds were planted in MS medium containing 100 mM NaCl, root length was investigated after growth for 20 d. The result showed that root length of PC43, PC44 and PC48 was 4.64,6.54 and 6.01 cm respectively, and wild type plant was 3.88 cm. For NaCl stress treatment, transgenic plants and wild type plants were treated with 250 mM NaCl for about 10 days. Results clearly showed that the survival rates of three lines of transgenic plant were 57%,39% and 62%, and only 12% survival rates of plants were found in wild type plants after NaCl treatment, suggesting transgenic plants with overexpression of NnDREB2A enhanced salt tolerance. To identify the function of NnDREB2A on the growth of Arabidopsis plants under drought stress, water was withheld from six leaf old Arabidopsis, and plant survival rates was recorded after 15 d. We found that overexpresssion of NnDREB2A leaded to a decrease for survival rates in Arabidopsis. In addition, the root growth of three 35S::NnDREB2A lines of Arabidopsis plants was worse than that of the non-transformed plants in the medium supplemented with 300 mM mannitol. To evaluate above three transgenic lines survival rates under lower temperature condition and water-logged condition. It was evidence that survival rates of wild type plants showed 1.9 and 13.7 fold than transgenic plants, suggesting that constitutive expression of NnDREB2A decreased low temperature and water-logged survival ability. The expression of two genes （PIP1-2,PIP2-5 and PIP2-7） was enhanced in transgenic plants compared with that of wild type plants. At the same time, four genes （COR47, PIP1-4, PIPl-5 and PIP2-2） was found to decrease mRNA level in transgenic plant, and expression of other sixteen genes （COR15, RD29A, RD29B, RD28, P5CS, ADH, PDC1, PDC2, PIP1-3, PIP2-1, PIP2-3, PIP2-4, PIP2-4, PIP2-6 and PIP2-8） was not significantly changed after transformed with NnDREB2.