Effects Of Calcium On Physiological And Biochemical Characteristics Of Maize Under Drought Stress And Cloning And Expression Analysis Of CIPK

Maize is the first grain crop in utilization of heterosis and sensitive to drought stress.The yield of maize is limited by serious drought in the main maize-producing areas throughout China.Previous research on plant drought resistance showed that calcium ions could promote the growth and development of plants under drought stress,because it could regulate the physiological changes in plants as the second intracellular messenger.In this study,maize hybrid Zhengdan 958,its maternal inbred line Zheng 58 and paternal inbred line Chang 7-2 were used as experimental materials to study the effect of Ca²⁺ on the physiological and biochemical characteristics and yields of maize hybrids and their parental inbred lines.On this basis,a protein kinase gene?Calcineurin B-like interacting protein kinases,CIPK?related to calcium signal transduction was cloned,and its tissue and cell specific expression was initially studied by semi-quantitative RT-PCR and transient expression.The main results are as follows:1.The effects of Ca²⁺ on the physiological and biochemical characteristics of maize seedlings under polyethylene glycol?PEG 6000?simulated drought stress were studied using Zhengdan 958 and Zheng 58 as experimental materials.The results showed that the biomass,relative water content and chlorophyll content of maize seedlings were significantly decreased.CaCl₂ solution could reduce the effect of drought stress on the above indexes of maize seedling,promote the accumulation of proline,and the effect of CaCl₂ was the highest at 20 mmol/L.2.The effects of exogenous CaCl₂ on physiological and biochemical indexes and yield of maize through the crop cycle were studied by potted water control method with Zhengdan 958?Zheng 58 and Chang 7-2 as experimental materials.The results showed that the content of malondialdehyde?MDA?,the activity of three antioxidant enzymes?POD,CAT and SOD?and the content of soluble protein were significantly increased under drought stress treatment at seedling,jointing and tasseling stage.CaCl₂ pretreatment of maize seeds,jointing and tasseling stage maize could reduce the malondialdehyde content,increase the activity of the above three antioxidants,keep the protein content at a high level in maize leaves so as to improve the drought resistance of maize.Drought stress in different growth stages will significantly reduce the yield of maize,and CaCl₂ pretreatment could reduce the decline of yield.Among them,the effect on maize yield of tasseling stage water deficient was the most serious,and the yield of Zhengdan 958,Zheng 58 and Chang 7-2 decreased by 33.43%,52.86%and 60.55%,respectively.In this period,CaCl₂ pretreatment increased the yield of the above lines by 9.8%,26.12%and 18.42%,respectively.Thus it can be seen that the hybrid Zhengdan 958 is more resistant to drought stress than its parental inbred lines,while the latter is more sensitive to exogenous Ca²⁺ responses.3.The expression of ZmCIPK gene in leaves and roots was analyzed by semi-quantitative RT-PCR using maize seedlings treated with PEG,LaCl₂,Ca²⁺ chelator?EGTA?and calcium channel blockers?LaCl3?.The results showed that the expression of ZmCIPK in maize leaves increased continuously within 24 h under drought stress,while the expression of ZmCIPK in maize roots reached the peak at 1 h and then decreased,but still significantly higher than that of control.The results of EGTA and LaCl3 showed that the expression of ZmCIPK in maize leaves and roots was inhibited by EGTA and LaCl3,indicating that calcium ion was involved in PEG-induced drought stress signal transduction process.Addition of CaCl₂ could increase the expression of ZmCIPK in leaves,which was similar to that of the effect of PEG-induced drought stress.However,exogenous CaCl₂ could decrease the expression of ZmCIPK in maize roots,and the detailed mechanism was to be further studied.4.The total RNA was extracted from maize leaves and ZmCIPK gene of maize was cloned by RT-PCR.Bioinformatics analysis showed that the full length of the gene was 1554 bp,encoded 518 amino acids,the molecular weight was 57.27 kDa,the theoretical isoelectric point was 8.84,contained a conserved NAF domain of the CIPKs protein family,had the highest similarity with sorghum CIPK,and subcellular localization prediction is endoplasmic reticulum.5.The pCAMBIA1303-35S-ZmCIPK-GFP transient expression vector was constructed by double-sticking.The agrobacterium containing the expression vector was injected into tobacco leaves by injection method.The fluorescence observation showed that the fusion protein ZmCIPK-GFP could be expressed normally in tobacco leaves,and the specific subcellular localization was to be further studied.