Function Evaluation Of Antifreeze Protein Gene AnAFP From Ammopiptanthus Nanus

Antifreeze proteins are a class of polypeptides produced by certain animals, plants and fungi that permit their survival under the subzero environments. Ammopiptanthus nanus is the unique evergreen broadleaf bush endemic to the Mid-Asia deserts. It survives at the west edge of the Tarim Basin from the disappearance of the ancient Mediterranean in the Tertiary Period. Its distribution region is characterized by the arid climate and extreme temperatures, ranging from-30℃ to 40℃.In this study, the antifreeze protein gene AnAFP was cloned from Ammopiptanthus nanus. After bioinformatics analysis for its possible function, the antifreeze protein gene AnAFP was used to construct prokaryotic and plant expression vectors, and transform Escherichia coli and tobacco(Nicotiana benthamiana). The function of the AnAFP gene was evaluated by the difference of the survival rate between the transformaned E. coli strains and the wild-type control, and of the cold tolerance between the transformed tobacco lines and the wild-type control, under cold stress.The results were summarized as follows:1. Bioinformatics analysis showed that AnAFP is homologous to many stress-responsive proteins in the legume family (Fabaceae). The highest identity (68%) was found with a cold-induced protein from Ammopiptanthus mongolicus (GenBank accession no. AAT01285.1). The percentage of glycineis is as high as 23.2%, similar to that (23.7%) found in winter-collected bittersweet nightshade (Solanum dulcamara). AnAFP is a hydrophilic and stable protein, without signal peptide. This characteristic indicates that the AnAFP is neither a transmembrane protein nor a secreted protein, functioning in the cytoplasm. The percentage of random coils is predicted to be 72.91%. This is a characteristic of the type Ⅱ AFP. The above analysis revealed that the putative protein of the submitted cDNA sequence functions as AnAFP, and its encoding sequence should be nominated as AnAFP gene.2. The AnAFP gene was inserted into plasmid pET-32a to construct prokaryotic expression vector pET-32a-AnAFP, and used to transforme E.coli strain Rosetta. Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the total protein expressed under the induction of isopropy1 β-D-thiogalactoside (ITPG). The result showed an obvious additional band about 42 kDa in the lines transformed by the pET-32a-AnAFP vector, indicating the heterologous overexpression of the AnAFP gene in E.coli.3. The transformaned E. coli lines as well as the control were treated at 0℃, and counted for survival rate by plate colonycount. The survival rate of the IPTG-induced transformaned lines were significantly higher than the uninduced transformaned lines, as well as the transformed line by the empty vector and the untrsnformed wild line, demonstrating the obvious enhancement of cold tolerance.4. Plant expression vector pRI201-GUS-AnAFP was constructed, and used to transform tobacco(Nicotiana benthamiana) variety’ Wisconsin38’ by the mediation of Agrobacterium tumefaciens. More than 100 To plantlets were regenerated after kanamycin selection. One third of them were identified as positive by specific PCR amplification, and three of them (plantlets 17,35 and 76) showed positive in GUS staining. The bagged-self pollinated T1 lines were detected by specific PCR amplification again, and by Southern blotting. The result showed that these three T1 lines were derived from independent transformation events.5. The differential expression of the AnAFP gene in the T2 transformed tobacco lines under low temperature (-3℃) stress was detected by real-time fluorescence quantitative PCR. After a decline at the beginning of the low temperature stress, the relative expression level of the AnAFP gene increased gradually, and reached the peak values at 4-12 h of the stress, although there was a little range among the different transformed lines. This result indicates that the heterologous expression of the AnAFP gene in the transformed tobacco lines, in response to low temperature stress.6. The relative electrical conductivity of the tobacco leaf was detected at the different times of low temperature (-3℃) treatment. An obvious increase was found for all the transformed tobacco lines and the untransformed wild-type control. However, the increased ranges of the transformed tobacco lines were significantly less than that of the untransformed wild-type control. This result indicates that the cytoplasmic membrane permeability of the transformed lines was more stable under the low temperature stress, probably conducive to the enhancement of cold tolerance.7. The transformed tobacco lines as well as the untransformed wild-type control were treated with low temperature (-3℃) for 16 h, and then transferred to room temperature for thawing. The whole plants of the untransformed wild-type control withered quickly, whereas the plants of the transformed lines kept erect, with only the lower leaves withered.In conclusion, the antifreeze protein gene AnAFP from Ammopiptanthus nanus shows a strong antifreeze function. Its heterologous expression in E. coli and tobacco increases their cold tolerance. This gene is hopeful to be applied for transgenic research of crops for cold tolerance, after further evaluation for function and safety.