Physiological Response Of Exogenous Ca²⁺ To Nitraria Tangutorum Bobr. Under Drought Stress,Cloning And Functional Analysis Of NtCDPK1 And NtCIPK5

Nitraria tangutorum Bobr.is a perennial deciduous shrub of the genus Zygophyllaceae.It has strong resistance to drought and salt stress,and is of great significance for controlling soil desertification and salinization.In the process of plant response to stress signals,calcium play an important role as a second messenger.There are few reports on the effects of exogenous calcium on relieving drought stress on the growth of Nitraria tangutorum Bobr..In this paper,15%,20% and 25% PEG-6000 were used to simulate different degrees of drought stress.The changes of physiological indexes of Nitraria tangutorum Bobr.under drought stress were detected by external application of 5 m M,10 m M and 15 m M Ca Cl2.The regulation of exogenous calcium ions on drought tolerance of plants was investigated.At the same time,two calcium-binding protein genes were cloned from Nitraria tangutorum Bobr.,which are calcium-dependent protein kinase NtCDPK1 and calcineurin B-like protein interaction kinase NtCIPK5.The eukaryotic expression vector was constructed and transferred into Arabidopsis thaliana to analyze the function of the gene under abiotic stress.In order to further explore the regulation of Ca²⁺ signaling pathway on plant response to abiotic stress and the molecular mechanism of tolerance to environmental stress in Nitraria tangutorum Bobr.The main findings are as follows:1.Drought stress inhibited the growth of Nitraria tangutorum Bobr.,and the application of certain concentration of Ca Cl2 could increase the relative water content and chlorophyll content of leaves under drought stress,reduce stomatal density,increase proline content,and increase SOD,POD and CAT activities.The content of H2O2 and MDA decreased,which enhanced the tolerance of Nitraria tangutorum Bobr.to drought.2.Bioinformatics analysis indicated that the ORF of NtCDPK1 is 1665 bp in length and encodes 554 amino acids.The ORF of NtCIPK5 gene is 1359 bp in length and encodes 452 amino acids.Both proteins have transmembrane structure,ATP binding site and serine/threonine protein kinase active site.NtCDPK1 has four EF-hand domains that bind to Ca²⁺,and has the highest homology with Citrus Clementina CPK10.NtCIPK5 has a NAF domain that binds to CBL and is closely related to Cephalotus follicularis.3.The results of qRT-PCR analysis showed that the expression of NtCDPK1 and NtCIPK5 could be induced by Na Cl,PEG,ABA and Ca Cl2,and EGTA inhibited its expression.Furthermore,the expression level of NtCDPK1 was up-regulated under low temperature stress.4.The pPZP221-NtCDPK1 and pPZP221-NtCIPK5 eukaryotic expression vectors were constructed to transform Arabidopsis thaliana.The wild-type and T3 transgenic Arabidopsis thaliana were subjected to drought,salt and low temperature stress.The growth of transgenic lines was significantly better than that of wild type,root length,number of lateral roots,fresh weight,chlorophyll content,antioxidant enzyme activity and proline content were significantly higher than wild type,H2O2 and MDA contents were significantly lower than wild type.These results indicated that overexpression of NtCDPK1 and NtCIPK5 could increase the antioxidant enzyme activity of transgenic Arabidopsis thaliana and reduce the degree of cell membrane damage,thereby reducing the effects of environmental stress on plant growth,development and photosynthesis,and enhancing the tolerance of plants to environmental stress.5.qRT-PCR was used to detect the expression of stress-related genes in NtCDPK1 and NtCIPK5 transgenic Arabidopsis thaliana under drought,salt and low temperature stress.The results showed that compared with wild type,the antioxidant enzyme genes At SOD1,At POD1,At APX1,ion transport related gene At SOS1,ABA synthesis related gene At NCED3,other stress-resistant genes At RD29 A,At DREB2 A,At RD22 and At AZI1 were all significantly induced in the transgenic lines.These results indicated that NtCDPK1 and NtCIPK5 could induce the expression of anti-reverse genes in transgenic plants and affect the ABA signaling pathway,thereby regulating the oxidative balance,osmotic balance,ion balance of plants under abiotic stress,and improving plant tolerance.