Gene Expression Pattern Analysis And Identification Of Low Temperature Tolerance Function Of ICE In Ammopiptanthus Nanus

Low temperature stress affects the geographical distribution and potential yield of plants worldwide.As one of the conditions of abiotic stress,low temperature chilling severely endangers plant growth,causing plant dehydration and membrane phase changes in cells.Ammopiptanthus nanus which grows in a bad environment all the year round,has strong cold resistance and can resist low temperature below-30 ?,so it is an ideal material to study the mechanism of plant cold resistance.A systematic study on the cold resistance genes of Ammopiptanthus nanus can lay a foundation for the molecular mechanism of cold resistance and the utilization of cold resistance gene resources.The transcription factor ICE is inactivated at normal temperature,and combined with the promoter of the CBF gene at low temperature,activates the expression of the CBF gene,thereby regulating the expression of the cold resistance COR gene downstream of the CBF,and conferring higher cold resistance to plants.In the study,the cold resistance transcriptional activator ICE gene and its downstream transcriptional factor CBF gene were used as the research material,and the expression patterns of ICE gene and its downstream transcriptional factor CBF gene were analyzed.Meanwhile,the cold resistance function of ICE gene was identified.The specific research results are as follows:1.According to the An ICE1 gene sequence obtained and the An ICE1 and An ICE2 gene sequence cloned by the research group,sequence alignment and molecular evolution analysis were carried out.The results showed that An ICE1 was consistent with other plant ICE proteins with high homology,including S-rich region(serine rich region),ACT-like domain,SUMO site and highly conserved b HLH-ZIP domain.The results of evolutionary analysis showed that An ICE1 amino acid sequence was closely related to soybean ICE protein.In the phylogenetic tree of plant CBF protein,An CBF1 and An CBF2 were found to be closely related to soybean CBF protein and Ammopiptanthus mongolicus CBF protein,and they shared an AP2 / ERF domain.2.After low temperature stress and exogenous spraying with ABA,Me JA,GA,SA and BR,Analysis of the expression patterns of the three genes An ICE1,An CBF1 and An CBF2 by q RT-PCR showed that all three genes were expressed under low temperature and exogenous ABA,and the expression levels of Me JA,GA and BR were increased.Under the treatment,An ICE1 significantly activated the transcriptional activation of An CBF2.However,the expression of three genes in SA treatment had no significant change.3.In this study,p CAMBIA1302-An ICE1-GFP recombinant plasmid was constructed,and the recombinant plasmid vector was transferred into tobacco using transient expressionmethod.The subcellular localization of An ICE1 protein was studied.Tobacco leaves transferred into the p CAMBIA1302-An ICE1-GFP recombinant plasmid were observed with a confocal microscope.The results showed that An ICE1 protein was localized on the cell membrane.4.Using stable genetic transformation technology,the constructed p CAMBIA1302-An ICE1-GFP recombinant plasmid was over-expressed into Arabidopsis plants,and the over-expressing plants were identified for phenotype and physiological and biochemical indicators.The results showed that the over-expressing An ICE1 plants were more wild Compared with type,it has stronger cold resistance.In order to further verify that An ICE1 can improve the ability of Ammopiptanthus nanus to protect itself from cold,a transient genetic transformation technology was used to transiently transform the p CAMBIA1302-An ICE1-GFP recombinant plasmid into Ammopiptanthus nanus,and the physiological and biochemical plant protection assays were performed for the transient transformation of An ICE1.The physiological and biochemical results of the transiently transformed An ICE1 plants under low temperature stress corresponded to the stable transformed over-expressed ICE1 plants in Arabidopsis.These results indicate that An ICE1,as a cold-resistant transcription activator gene,plays an important role in the cold resistance of Ammopiptanthus nanus,and lays a theoretical foundation for further utilizing the transgenic method to improve the cold resistance of other plants.