Chilling Tolerance Identification Of Transgenic Maize With Antifreeze Protein Gene(AnAFP)of Ammopiptanthus Nanus And Chilling Tolerance Mechanism

Low temperature is an environmental stress that seriously affects maize growth and development,yield,quality,sowing area and sowing time.Chilling injury results in maize growth and development retardation,plant deformity,and affects yield.Freezing injury directly leads to plant death.In order to make better use of light and heat resources in early spring,high latitude and high altitude,there is an urgent need for maize varieties with chill and freeze resistance.Although there have been many reports on candidate genes for chilling tolerance in maize so far,they have not been used in variety breeding,which is limited to theoretical research.Antifreeze protein(AFP)is a functional protein form Ammopiptanthus nanus.It inhibits the growth of ice crystals by binding with small ice crystals in the cell fluid and prevents death caused by th formation of large ice crystals in cells under freezing stress.In our previous study,we transferred AnAFP gene form Ammopiptanthus nanus into prokaryotic cells and tobacco to improve the antifreeze tolerance,and transfected it into18-599 red maize inbred line in Southwest China by Agrobacterium tumefaciens.A strain transfected with AnAFP gene maize was obtained.1.The phenomenon of"gene separation"did not occur in self-intersecting and intersecting offspring.Three insertion sites were found on chromosome 1 from 13 T₁ transgenic maize plants by TAIL-PCR.Homozygous line 1 and homozygous line2 were found in T₁ generation by three primers,and homozygous line 3 was found in T₂ generation.Line 1 and line 2 are head-tail tandem duplicates.2.Exogenous genes were heterologously expressed in three lines at the level of gene and protein,but the expression levels of Line 1 and Line 3 were higher than those of Line 2.T-DNA insertion in line 3 resulted in an increase in gene expression near the insertion site,while T-DNA insertion in line 1 and line 2 did not affect gene expression near the insertion site.RNA-seq showed that there were four common DEGs which were down-regulated exptession between the three lines and the non- transgenic control under normal growth conditions and 24 h treatment at 4?.3.Histochemical staining and electrolyte leakage of transgenic lines 1 and 2 after five days of low temperature stress at 4-8?showed significant improvement in cold resistance.The survival rate of lines 1 and 2 treated at-2 C for 5 h was significantly higher than that of non-transgenic control.In the field experiment of 2018,the survival rate of GM maize was significantly higher than that of non-GM maize under-1.8?short-term freezing in late spring.The growth of GM maize was better than that of non-GM control under long-term low temperature stress and short-term freezing stress.4.The promoter analysis of the antifreeze protein gene of Ammopiptanthus nanus showed that there were several abiotic stress-induced cis-acting elements. Evolutionary tree analysis revealed that AnAFP belonged to KnS type dehydration protein.Homologous comparison analysis revealed that there were K domain,NLS domain,S domain of dehydration protein and possible ice binding domain of antifreeze protein.Phosphorylation prediction analysis revealed that there were multiple phosphorylation sites in the S domain of AnAFP that could be phosphorylated by CKII kinase.5.AnICE1 and AnCBF genes were cloned from Ammopiptanthus nanus and homology comparison revealed that AnICE1 conservative HLH domain and ACT_UUR_ACR_LIKE domain and AnCBF conservative AP2 domain.qRT-PCR manalysis of AnAFP,AnICE1 and AnCBF genes in Ammopiptanthus nanus seedlings under cold,ABA and dehydration stress showed that the three genes had the samechange trend.The results of Y1H showed that AnCBF could bind DRE cis-acting elements in the promoter of AnAFP gene to activate downstream reporter gene expression in yeast.6.Domain deletion mutation of AnAFP Gene by Overlapping PCR,prokaryotic expression and purification of domain mutant proteins.Phosphorylation in vitro showed that S domain could be phosphorylated by CKII protein kinase,and the phosphorylation could be inhibited by TBB.The purified domain mutant protein can increase the activity of lactate dehydrogenase more than three times,and the activity is independent of the domain.7.The subunit beta of CKII protein kinase was cloned from maize.Subcellular localization revealed that subunits beta 2,3 and 4 were located in cytoplasm and nucleus,while subunit beta 1 was only located in nucleus.Biomolecular fluorescence complementation experiments showed that beta 1,3,4 could interact with AnAFP gene,and the occurrence of this interaction requires the participation of other components.Subcellular localization of AnAFP showed that AnAFP was in the mixed state of phosphorylation and non-phosphorylation in normal growth cells and was located in the nucleus and cytoplasm of cells.When CK II phosphokinase inhibitor was added,AnAFP was only localized in the nucleus and existed only in the cytoplasm under low temperature stress.That is,the phosphorylation of AnAFP by CK II was transferred from the nucleus to the cytoplasm.Give full play to its functions.In this study,we have developed a method for identifying transgenic homozygous lines in T₁ generation of transgenic plants,and the principle of this method will be applicable to any diploid transgenic plants.It was also found that AnAFP is not only an antifreeze protein but also a KnS dehydrated protein.Under normal growth conditions,the mixture of phosphorylation and non-phosphorylation is located in the cytoplasm and nucleus.Under low temperature stress,CK II further phosphorylates AnAFP,leading to the transport of AnAFP to the cytoplasm.Antifreeze proteins in cytoplasm can improve the cold tolerance of transgenic maize by improving the activity of enzymes in cytosol and the stability of cell membrane.Under freezing stress,AnAFP can improve the freezing resistance of transgenic maize by reducing the freezing point of cytosol and inhibiting the aggregation of small ice crystals.Pot and field experiments showed that the transgenic lines 1 and 2 were significantly better than the non-transgenic ones under long-term low temperature,oligoirradiation and short freezing stress.In view of the serious impact of low temperature and freezing injury on maize,the mechanism of freezing resistance and cold tolerance of AnAFP studied in this paper will provide theoretical guidance for the research of cold tolerant maize.Meanwhile,the transformed AnAFP maize studied in this paper will provide cold tolerant maize resources for conventional breeding.