Study On Functional Domains Of Antifreeze Protein In Ammopiptanthus Nanus

Temperature stress is acommon type of abiotic stress.Whether it is low temperature stress or high temperature stress,it will ultimately affect the yield and quality of crops,causing huge economic loss.Antifreeze protein(AFP)is a class of protein compounds with the ability to improve biological antifreeze ability.In the previous studies,it was found that ectopic expression of the antifreeze protein gene(AnAFP)of Ammopiptanthus nanus improved cold resistance of Escherichia coli,tobacco and maize.Sequence analysis showed that the amino acid sequence of AnAFP protein shared the same conserved domains with the members of the Kn S type subfamily of dehydrated proteins of the second abundant protein family in the late embryonic development of plants.According its functional domain prediction,the full length coding sequence(CDS)of the AnAFP gene was truncated to obtain four mutated sequences AnAFP?A,AnAFP?K,AnAFP?N and AnAFP?S,which were respectively deleted for the ice binding domain A,dehydration protein domain K,dehydration protein nuclear localization domain N and dehydration protein domain S.In the present study,the full-length CDS of the AnAFP gene and these four deletion mutated sequences were transformed into ordinary and cold sensitive strains of Escherichia coli,and Arabidopsis mutant with KS type dehydrating protein gene mutation,respectively.The resistance phenotype was identified under low and high temperature stress.Along with subcellular localization,interaction protein screening and comformation,and real-time quantitative PCR detection,the thermal stability function of these four domains was analyzed to explain the mechanism of the AnAFP protein.The main results are as follows:(1)By homologous recombination,the CDS of the AnAFP gene and its four deletion mutated sequences AnAFP?A,AnAFP?K,AnAFP?N and AnAFP?S were constructed into prokaryotic expression vectors p ET28a and cold-sensititive expression vectors p IN?,and transformed into ordinary strain BL21(DE3)Ply Ss and cold sensitive strain BX04 of E.coli.After high temperature treatment of 50?for 30 min,the average survival rate of BL21(DE3)Ply Ss strain transformed with full-length CDS was significantly higher than that of the blank control.The average survival rate of the BL21(DE3)Ply Ss strain transformed by the mutated sequences AnAFP?K?AnAFP?N and AnAFP?S were not significantly different from the BL21(DE3)Ply Ss strain transformed by the full-length CDS.However,the average survival rate of the BL21(DE3)Ply Ss strain transformed by the mutated sequence without A domain(AnAFP?A)was not significantly different from the negative control transformed by the empty vector.After low temperature treatment of 17?for9 days,the average multiplication multiples of BX04 strain transformed with full-length CDS was significantly higher than that of blank control.The average multiplication multiples of BX04 strain transformed by the mutated sequences AnAFP?K?AnAFP?N and AnAFP?S were not significantly different from the BX04strain transformed by the full-length CDS.However,the average multiplication multiple of the BX04 strain transformed by the mutated sequence without A domain(AnAFP?A)was not significantly different from the negative control transformed by the empty vector.These results indicated that the ice crystal binding domain A domain is a key functional domain for AnAFP heterologous to improve the heat and cold tolerance of E.coli.(2)All the T₃ lines of Arabidopsis Kn S type dehydration protein(AT1G54410)gene mutant athird11 transformed by the mutated sequences deleting for the AnAFP?A,AnAFP?K,AnAFP?N and AnAFP?S domains died after heat shock treatment at 46?for 3 h and recovery for 2 weeks,showing no difference from the untransformed control.However,a few plants of the T₃ lines transformed the full-length CDS of the AnAFP gene survived.This result indicated that the ectopic expression of the AnAFP gene improve the heat tolerance of the Arabidopsis to a certain extent.All of the four domains of the AnAFP protein are related to its heat-resistant function.The deletion of any of the A,K,N and S domains will lead to the loss of heat-resistant function.(3)The result of subcellular localization showed that the deletion mutants of the A and S domains were located in both the cytoplasm and nucleus as the full-length AnAFP protein.However,the deletion mutatns of the K and N domains were only located in the nucleus and the cytoplasm,respectively.This result showed that the deletion of K and N domains changes the subcellular localization of the AnAFP protein.(4)The Y2H result showed that the full-length AnAFP protein and its deletion mutants of the A,N,and S domains did not interact with the AnICE1 protein.Only the AnAFP?K mutant sequence lacking the K domain interacted with the AnICE1protein.This result showed that the dehydration protein domain K of AnAFP protein hinders its interaction with AnICE1 protein.(5)The result of RT-qPCR showed that the expression of AnICE1,AnCBF and AnAFP genes responded to high-temperature stress with a similar trend,all of which increased first and then decreased and then increased.The result of cDNA library screening and Y2H showed AnAFP may also interact with downstream glyceraldehyde 3-phosphate dehydrogenase A subunit AtGAPA.These results indicate that the up-regulated expression of AnAFP gene under high temperature stress is likely to be also regulated by the ICE1-CBF-COR signal transduction pathway,regulating various physiological and biochemical processes downstream.In summary,the heterologous expression of AnAFP can improve the heat and cold tolerance of E.coli and the cold tolerance of Arabidopsis,and the A domain is a key functional domain that can resist temperature stress,and the functions of other structural domains are also indispensable.