Maize Transformation Of Antifreeze Protein Gene(AnAFP)from Ammopiptanthus Nanus

Maize is the first important grain crop in China and in the world,with the largest cultivated area and the most total yield.Along with the continuous growing of the,it is important for the food safety to increase the cultivated area of maize and give full play to the high yield advantages of the C4 plant.However,maize originated in the tropoics,and it is sensitive to low temperature.This limits the further expansion of maize cultivated area.It is an effective approach to improve maize for cold resistance by transgenic transformation of antifreeze genes of other species.Antifreeze proteins are a class of active protein peptides that are generated in animals and plants living in cold regions to adapt to the environment.Antifreeze proteins can inhibit ice crystal growth,reduce lower the freezing point in the form of non-colligative,but have little effect on the melting point.Ammopiptanthus nanus is the unique evergreen broadleaf bush endemic to the northwest deserts.It mainly survives between the west Tianshan and Kunlun Mountains.Its distribution region is characterized by the arid climate and extreme temperatures,ranging from-30 ℃ to 40 ℃.In this study,the AnAFP gene cloned and functional validated from Ammopiptanthus nanus in our previous study was inserted into pTF101.1 plasmid and generated monocot expression vector pTU-AnAFP.Then pTU-AnAFP was used to transform maize immature embryos and callus by the mediation of Agrobacterium tumefaciens.Regenerated plants were detected of molecular and identified of antifreeze phenotype.The results were summarized as follows:1.The antifreeze protein gene(AnAFP)was inserted into plasmid of pTF-101.1-Ubi-Tnos to generate plant expression vector pTU-AnAFP.2.The immature embryo of maize inbred 18-599 were collected and inducted into callus,then used for transforming the expression vector pTU-AnAFP by the mediation of Agrobacterium tumefaciens.After screening,differentiation,regeneration and PCR amplification,we obtained a positive To plant.3.After PCR amplification and Southern bloting in T1 lines,the results indicate that the AnAFP gene may insert into two non-alleles of homologous chromosomes in maize performing T1 lines is homozygous.4.T1 lines and the control seedlings were treated at 0 ℃ for 24 h,recoveried at room temperature and observed phenotype.The WT plants exhibited leaf wilting and severely dehydrate while the positive transgenic lines were normal.5.The results of real-time quantitative PCR showed thatAnAFP gene was heterologous expression in T1 lines and responsive to low temperature stress.These results indicate that the AnAFP gene from Ammopiptanthus nanus is a excellent candidate gene functions as enhancing cold tolerance.Heterologous expression of AnAFP in maize could significantly enhance cold toleranceof transgenic line.