The Cloning Of Cassava Cold Resistance Genes CBF1、CBF2 And Analysising The Different Of Expression

This study was taken the hardy variety of cassava RS01 and the non cold resistant variety SC205 as the material, By using RT-PCR and nested PCR technology, cloned the cassava cold resistance gene CBF1 and CBF2, Using fluorescence quantitative PCR technique and bioinformatics method, analysised the gene differential expression and functional. At the same time, we studied the physiological characteristics of different varieties of cassava. The main results are as follows:1. According to the conserved sequence of the homologous CBF1 gene, we designed the primers, using RT-PCR and nested PCR technique, first cloned CBF1 gene sequence in the cassava varieties RS01 and SC205, the total length were 1000 bp and amino acid coding has 238, molecular weight 616.51 kDa. By sequence comparison analysis, it is found that SC205 encodes only 129 amino acids, which makes the protein structure of SC205 is not complete, so we predicted it is the reason that the ability to respond to low temperature stress was poor.2. By bioinformatics analysised the amino acid sequence of the CBF1 gene expressed by the cold resistant and non resistant varieties of cassava, it found that RS01 showed strong hydrophobicity in the 119th amino acid positions, while SC205 showed strong performance at the tenth amino acid positions. The isoelectric point of RS01 CBF1 gene was 6.11, and the isoelectric point of SC205 was 4.36. Through CBF1 gene sequence of protein two and three structure prediction, found that the target gene and transcription factor (PDB ID: 3GCC) is very similar to the 63 amino acids and a series of amino acid composition. The analysising to the CBF2 gene for protein structure we get its HLH conservative structural domain, and it screw-ring-screw structure having important effects on function.3. Through analysis the different varieties of cassava, rubber tree, populus tomentosa CBF1 gene sequence and its evolutionary tree, the comparison between the cassava and the rubber tree reached 84%, and the homology of the populus tomentosa was 60%.4. Through detecting the cloned CBF1 gene by real-time fluorescence quantitative PCR, we found the gene in different cold resistant varieties of expression are different, at 5℃, the difference was the most significant, the RS01 expression is SC205’s 4 times, verify the CBF1 gene is associated with plant cold resistance gene.5. The cassava under 5℃,10℃ and 25 ℃ treatment 7 days, the cold hardiness indexes are measured in different cassava varieties. The results showed that the same cassava varieties at a low temperature, content of proline and soluble sugar than normal temperature is higher, and with a linear dependence on temperature, temperature is lower in the higher. MDA in SC205 showed a trend of increasing first and then decreasing. The SOD and CAT activity in RS01 is higher than in SC205, meanwhile the difference is obvious.