Cold-resistance Function Analysis Of Transcription Activation Factors CmICE1 In Chrysanthemum Morifolium 'Huoyan'

Chrysanthemum morifolium is one of the major garden flower species in the Northeast China.There are many varieties of Chrysanthemum morifolium that are easy to grow and manage,have economic value,and have good drought tolerance,cold resistance,and salt-tolerance.The flowering period of most chrysanthemum is concentrated from September to October.It has entered the fall in the northeast and the temperature is relatively low,which affects the economic value of the chrysanthemum.Therefore,it is of great significance to study the cold-resistance mechanism of Chrysanthemum morifolium,to improve the cold-resistance of the existing chrysanthemum,and to develop a cultivar with a stronger cold-resistance ability.In this study,the key genes CmICEl,CmDREBa and CmDREBb in ICE-CBF/DREB pathway of plants were cloned and analyzed genes using bioinformatics-related software;we analyzed the expression pattern of cold-resistant genes by qRT-PCR method;we constructed a plant expression vector pCAMBIA1301-PMI-CmICE1 with a safety marker gene and transformed the Chrysanthemum morifolium 'Huoyan' by Agrobacterium-mediated leaf infection.The transgenic plant was obtained and the cold resistance of the transgenic plant was identified.The conclusions are as follows:(1)Three cold-resistant genes CmICEl,CmDREBa and CmDREBb were cloned,of which CmDREBa and CmDREBb were downstream genes of CmICEl;the CDS region of CmICE1 gene is 1410bp and encodes 470 amino acids,CmDREBa gene is 579bp and encodes 193 amino acids,CmDREBb is 558bp and encodes 186 amino acids;Compared with other species homologous genes,CmICEl protein has the same bHLH domain,serine-rich region,and ACT domain at the C-terminus;The CmDREBs protein has the same AP2 domain as other species CBF/DREB proteins.Phylogenetic tree analysis showed that CmICE1 protein was closely related to Chrysanthemum dichroum CdICEl protein;CmDREBs protein was the closest relative to Eupatorium adenophorum CBF protein followed by the same family.(2)Using the tissue parts of the "Huoyan" as experimental material,qRT-PCR showed that the expression levels of CmICEl,CmDREBa and CmDREBb genes were the highest in leaves;Under long-day conditions,the expression of CmICEl,CmDREBa and CmDREBb genes in leaves was detected and found that the expression of CmDREBa and CmDREBb genes was related to light and showed rhythmic changes within 24 hours,while the expression of CmICEl gene was not related to light,and did not show periodic changes;Under 4?treatment,the expression of Cm/CE1 rapidly increased and then decreased rapidly with the prolonged treatment time.The expression of CmDREBa and CmDREBb increased with the treatment time.(3)The plant expression vector pCAMBIA1301-PMI-CMICE1 with the marker gene was successfully constructed,and the Chrysanthemum morifolium 'Huoyan' was transformed by Agrobacterium tumefaciens-mediated leaf infestation method.Twelve transgenic plants were obtained and the genetic transformation efficiency was 5.9‰;The expression of CmICEl gene in transgenic plants was higher than that in wild-type plants under normal growth conditions.After treatment at 4?,the expression levels of CmICE1,CmDREBa,and CmDREBb in the transgenic plants were higher than those in the wild type plants;After 10 days of treatment at 4?,the leaves of the wild-type plants were yellow,and the leaves of the transgenic plants were tender green.