The Cold Tolerance Of Transgenic Populus Alba×Populus Glandulossa 84K With Fatty Acid Desaturase Genes PtFAD2 And PtFAD3

Low temperature is one of the major environmental factors that limits plant growth. Lipids play an important role as the structural constituents of most of the cellular membranes. The increased production of polyunsaturated fatty acids is a response connected with cold acclimation of higher plants and is thought to protected plant cells against cold damage. Fatty acid desaturases are important enzymes involved in the vast majority of polyunsaturated fatty acids synthesis. Both of the endoplasmic reticulum 18:1 desaturase (FAD2) and the endoplasmic reticulum 18:2 desaturase (FAD3) are among of them.The endoplasmic reticulum 18:1 desaturase (FAD2) is to provide 18:2 polyunsaturated fatty acids; the endoplasmic reticulum 18:2 fatty acid desaturase (FAD3) is to provide 18:3 fatty acids, which required for the correct assembly of cellular membranes throughout the plant. We succeed in cloning PtFAD2 (GenBank accession number: DQ316788) and PtFAD3 (GenBank accession number: DQ354393) full-length coding sequence cDNA in Populus tomentosa using in silico and molecular cloning for the first time. PtFAD2 gene is composed of 1276bp and the open reading frame encodes a peptide of 388 amino acids; PtFAD3 is 1187 bp in length and the open reading frame encodes a peptide of 383 amino acids.Overexpression vectors of pBin438-PtFAD2 and pBI121-PtFAD3 were constructed, and RNAi vectors of pBI121-PtFAD2-RNAi and pBI121-PtFAD3-RNAi were constructed. These vectors transformed into GV3101 used for transformation. Poplar was transformed via the leaf-disc method. The kanamycin-resistant seedlings were subjected to further analyses of real-time PCR and southern blotting. Southem hybridization of genomic DNA was then carried out to confirm that the foreign genes have been integrated into the genome of transformates.The transcription level of FAD2 in both over-expression and RNAi inhibition transgenic lines were analyzed by real-time PCR. 109% and 74% increase were observed in over-expression lines F2-100 and F2-35, comparing to the non-transgenic controls. In contrast, 64% and 46% reduction were obtained in the RNAi inhibition line 2R-11 and 2R-31. Similarly, 60% and 150% increase in the transcription level of FAD3 were observed in over-expression lines F3-17 and F3-62, comparing to the non-transgenic controls. In contrast, 60% and 54% reduction were obtained in the RNAi inhibition lines 3R-14 and 3R-20.To test the change of expression would lead to the change of fatty acid compostion; we measured the compositon of fatty acids in leaves. The content of 18:3 fatty acids increased by 7.1% and 5.7% in over-expression lines F2-100 and F2-35, respectively. As expected, decresed by 19.3% and 14.7% in RNAi lines 2R-11 and 2R-31, respectively. Similarly, the content of 18:3 fatty acids increased by more than 8.5% in F3-17 and F3-62, but decreased more than 5.9% in RNAi lines 3R-14 and 3R-20.When plants exposed to 4℃for 1d and then back to 25℃, seedlings about 70cm high, over-expression lines F3-17,F3-62,F2-35 and F2-100 survived at higher frequency rate than wild-type plants and the RNAi line 3R-14,3R-20,2R-11 and 2R-31.Percentage of uninjured plants for F3-17,F3-62,F2-35,F2-100 and wide type plants are 71.4％,75％, 100％,80％and 25％,respectively;the RNAi line plants were all injured.For the number of samples used in cold treatment were small,the result indicated that overexpression lines probably are more resistant to cold than wide-typle plants and the RNAi transgenic lines.When plants exposed to - 4℃for 3 h back to 25℃,the seedlings with 3 or 5 leaves, overexpression lines F3-17,F3-62,F2-35 and F2-100 survived at high as 70±&5.7％,73.3±5.7％,56.6±3.3%％and 60±5.7％,while wild-type plants at 36.6±3.3％,the RNAi lines 3R-14,3R-20,2R-11 and 2R-31 survived at 10±10％,10±10％,20±0％and 10±10％.Statistical result indicated that overexpression lines F3-17,F3-62,F2-35 and F2-100 survival frequency were significantly higher than wild-type plants and the RNAi lines 3R-14,3R-20,2R-11 and 2R-31.In cornection to the membrane leakage,membrane permeability was measured for overexpression transgenic lines and the wide-type plants.It indicated that membrane permeability of overexpression transgenic lines were significantly lower than wide-type plants.As expected,cold tolerance of overexpression transgenic lines wss higher than that of wide-type plants.The fatty acid competent can be modificated through overexpression and RNA interference fatty acid desaturase genes in poplar.This study indicates that poplar with increased polyunsaturated fatty acids especially the component of tfienoic fatty acid can enhance the ability to resistant cold through genetic modification on FADs.The manipulation of fatty acid desaturase genes provides a new method to enhance the cold tolerance of trees in tree breeding.