Arabidopsis Cbf3 Alleles The Atcrap2 The Cloning And Cold Functions

Low temperature is one of the significant limiting factors that affect crops output. In our lab, with 32 core accessions of Arabidopsis, association analysis of CBF3 single nucleotide polymorphism (SNP) showed that the haplotype of 203AV accession's CBF3 allele was related to cold-resistance. In this paper, the CBF3 allele was amplified from 203AV accession; the structure of the CBF3 allele was analyzed, and its functions were also studied. Our experimental results were shown as following:1. AtCRAP2 (DQ415923) was amplified which has 1094bp and encode 150 amino acids. There is one site mutation existing in AtCRAP2, which causes terminal code ahead of 198bp compared with CBF3 of 186AV accession. Compared with CBF3 encoding protein, AtCRAP2 encoding protein lacks C-terminal 66 amino acids.2. Two vectors harboring AtCRAP2 and CBF3 gene respectively driven by CaMV (cauliflower mosaic virus) 35S promoter. The two constructs were transformed into Arabidopsis Clombia accession 186AV. Transgenic plants were obtained by PCR analysis and kanamycin screening, T3 seeds of three homozygous lines for each kind of transgenic plants were used in this study.3. There was no phenotype difference between AtCRAP2 transgenic plants and wild-type Arabidopsis. However, CBF3-overexpression plants displayed strongly phenotypic alterations, including stunted plants, reduced weight and growth retardation. When plants had four leaves, the average root length of AtCRAP2 transgenic plants was 3. 70cm, and that of wild-type plants was 3. 75cm, while that of CBF3-overexpression plants was only 2. 75cm, which was much shorter than the forenamed ones. Comparing plant fresh weight between the wild-type plants and transgenic Arabidopsis at 12-leaf stage, the fresh weight of CBF3-overexpression plants (0. 13g) was about 50% of that of AtCRAP2 transgenic plants(0.26g) or wild type plants(0.27g). Moreover, the florescence of AtCRAP2 transgenic plants similar to wild-type plants was 12 daysearlier than that of CBF3-overexpression plants. Using differential interference contrast microscope assay, it was found that there was on difference for the volume of the epidermal cell between AtCRAP2 transgenic plants and wild-type plants; but the epidermal cell of CBF3-overexpression plants was much smaller than that of AtCRAP2 transgenic plants or wild-type plants.4. Using the electrolyte leakage analysis, freezing tolerance was measured with transgenic and the wild-type Arabidopsis. It was found that treated for 4 hours at -6°C, the percentage of electrolyte leakage of the wild-type was 66 ± 23%, and that of AtCRAP2 transgenic plants was 49 + 14%. After treated for 6 hours at -6°C, the surviving percentage of AtCRAP2 transgenic plants was 94%, but that of wild-type plants was only 17%. Interestingly, the surviving percentage of CBF3- overexpression plants was similar to that of the AtCRAP2 transgenic plants.5. During 4 °C treatment, the transcript levels of COR genes in AtCRAP2 transgenic plants were higher than those of wild-type plants with semi-quantity RT-PCR analysis. However, the transcript levels of COR genes in AtCRAP2 transgenic plants were similar to that of CBF3-overexpression plants.These results further suggest that AtCRAP2 gene may be useful for cold-tolerant transgenic engineering.