Physiological Effects Of Exogenous Ca²⁺ On Nelumbo Nucifera Gaertn Under Salt Stress, Cloning And Expression Analysis Of Ca²⁺-dependent Protein Genes-NnCDPKs

Lotus root (Nelumbo nucifera Gaertn), a member of the family Nymphaeaceae, is an aquatic herb vegetable, and it has been widely cultivated in China for multiple purposes. The products of lotus root, lotus seeds and lotus root starch are very popular in the daily diet because of its richness in nutrients. Calcium-dependent protein kinase(CDPK) is an important signal mediator in regulating numerous growth and developmental processes and biotic and abiotic stress responses. It also regulates metabolism of plant and the expression of downstream genes and transmembrane transport of ion and water. The physiological effects of exogenous Ca2+ on lotus root under salt stress, and cloning, expression analysis, vector construction of NnCDPKl 3 were studied in this paper1. Lotus root is used as material and treated with NaCl and Ca (NO3)2, the physiological effects of exogenous Ca2+ on lotus root under salt stress was studied. The results show that chlorophyll and soluble protein concentration and SOD activity of the material are decreased and electrolyte leakage and MDA and proline content get increased with time of salt stress. Applying appropriate calcium can be able to increase the chlorophyll and soluble protein concentration and SOD activity, and the proline content increased more than before, and electrolyte leakage and MDA content are reduced. It suggested that appropriate calcium can effectively alleviate the salt stress damage to lotus root, and the alleviating effect was not obvious under high salt stress, even showing inhibition to plant.2. Full length of NnCDPKs are amplified with RACE-PCR method, where are between 1000bp-2000bp.NnCDPK11, NnCDPK21 and NnCDPK22 are meta-alkalescence, and others are partial acid protease. There are 14 stable protein, but the others are not in all of the NnCDPKs. Most of NnCDPKs are activated upon Ca2+ binding to four EF hands in the calmodulin-like domain except NnCDPK17 with none and NnCDPK9 with five. All of NnCDPKs are hydrophilic proteins.3. Semi RT-PCR and qPCR method were carried out to evaluate the expression of NnCDPKs with β-actin as internal standard. The results show that expression of NnCDPKs is significantly increased in 3h to 6h, then decreased. Contrary to other NnCDPKs, the expression of NnCDPK26 is decreased. The expression of NnCDPK17 has no significant change.4. Full-length cDNA of NnCDPK13 was ligated into a binary vector (pAMBIA1301) 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. Plating medium with antibiotics is used to screen seeds, and chose the Arabidopsis thaliana seedlings which can grow up normally, then Preliminary identify them as genetically engineered seeds.