Cloning,characterization And Transformation Of Cold Response Gene ZmCyp40 In Maize (Zea Mays L.)

Maize(Zea mays L.)is the largest food crop in China.Chilling injury has become the main abiotic stress which threatens the early growth and development of maize and restricts its geographical distribution.Therefore,it is necessary to study the cold resistance of maize and understand the physiological and biochemical and molecular genetic mechanism of maize under low temperature stress,so as to improve the cold resistance of maize through genetic improvement.For this purpose,this study has carried out the following research work:1.The ZmCyp40 gene(maize cyclophilin 40 gene)was successfully cloned from the cold maize inbred line W9816 with a fragment length of 1396 bp.The open reading frame analysis showed that the complete reading frame of the ZmCyp40 gene contained 1170 bp,encoding 389 amino acids.Chromosome mapping analysis revealed that the ZmCyp40 gene was located on chromosome 6 and covered 8454 bp,containing 8 exons.The conserved domain prediction showed that the ZmCyp40 protein contained PPIase and TPR conserved domains,indicating that it is a member of the typical Cyp40 protein family.Multiple sequence alignment and phylogenetic tree analysis showed that the ZmCyp40 protein was highly conserved,with the highest homology with the gene in sorghum.Protein structure prediction implied that the N-terminus of the ZmCyp40 protein was formed by a ?-fold beta barrel,upstream and downstream connected to an ?-screw composition,and C-terminus was formed by a series of ?-screw,both ends connected through the flexible ring.2.The expression of ZmCyp40 in roots,stems and leaves of W9816 inbred maize which treated at 4 ° C for 0 h,3 h,6 h,12 h,24 h and 48 h,was analyzed by q RT-PCR.The ZmCyp40 expression of cold response in different parts of maize was different,but it was induced by cold.3.The overexpression vector p CAMBIA-3301-35s-ZmCyp40 was constructed and the ZmCyp40 overexpression Arabidopsis thaliana was obtained.The analysis of Arabidopsis thaliana seed germination showed that ZmCyp40 overexpression arabidopsis was insensitive to 4 ? treatment,indicating that ZmCyp40 overexpression Arabidopsis thaliana had higher chilling tolerance than mutant and wild type arabidopsis at germination stage.Frozen resistance analysis showed that overexpression Arabidopsis had stronger frozen resistance compared to wild type and mutant Arabidopsis.The results of cold-related physiological indexes showed that overexpression of ZmCyp40 gene increased the tolerance of Arabidopsis thaliana to cold stress.4.Genetic transformation of ZmCyp40 gene was carried out by using the genetic transformation system of maize inbred line Y423.A total of 1401 regenerated plants were obtained by Agrobacterium tumefaciens-mediated transformation and 82 were positive tested by PCR.The expression of ZmCyp40 gene in the leaves of ZmCyp40 positive plant C1 was significantly increased by q RT-PCR analysis.5.The Agrobacterium tumefaciens-mediated genetic transformation system of maize inbred line Y423 was optimized.Compared with the control,42? heat shock treatment and glucose addition made the positive rate of regenerated plants increase from 5.1% to 5.9%,4.4% to 5.2% respectively.