Functional Analysis And Genetic Transformation Of Cold Response Gene ZmNAP1 In Maize(Zea Mays L.)

Maize（Zea Mays L.）is a thermophilic crop,but due to the high latitudes in the northeastern region of China’s important food production area,chilling injury occurs frequently,which seriously threatens China’s food security^[1].Therefore,it is very important to study the cold-resistance related genes and cold response mechanism in maize.For this purpose,this study carried out cold-resistant gene cloning,functional verification,and related research on genetic transformation to lay the foundation for breeding new varieties of maize germplasm.The specific results are as follows:1.Based on the proteomic analysis of the cold-resistant maize inbred line W9816,ZmNAP1（maize nucleosome assembly protein）under cold stress was cloned and its fragment length was 1292 bp.ZmNAP1 protein was found to have high conservatism.2.The maize inbred line W9816 was treated at a low temperature of 4°C.The results showed that the expression level of ZmNAP1 gene was different in roots,stems,and leaves,and the response speed to cold stress was different,but all were induced by cold induction.3.The ZmNAP1 gene overexpression vector pCAMBIA-3301-35s-ZmNAP1 was constructed and the T₃ overexpression Arabidopsis line was obtained by Agrobacterium-mediated method.The function of the gene was identified by low temperature germination and NaCl treatment.The results showed that overexpression of ZmNAP1 could improve the tolerance of Arabidopsis to cold stress and salt,and it also showed higher frost resistance.The results of physiological and biochemical indicators such as proline content,POD and SOD activity showed that overexpression of ZmNAP1 gene improved the cold tolerance of Arabidopsis plants.4.Optimization of Agrobacterium-mediated transformation system,somatic embryos was subjected to hyperosmotic culture for 0h,4h,8h before infection.Compared with the control,the positive rate of regenerated plants after hyperosmotic culture increased from 2.4%to 6.3%.5.According to the optimized conditions of maize transformation system,ZmNAP1 gene overexpression vector was used to genetically transform maize inbred line Y423 to obtain 151 positive plants.