| Temperature is one of the major environmental factors that limit plant growth, development and geographical distribution. The biologic membrane was regarded as the position which was firstly suffered damage under temperature stresses. The content of unsaturated fatty acid in plant membrane lipids was higher, the temperature of transition of membrane lipids was lower and the chilling tolerance of plants was higher. Contrarily, the temperature of transition of membrane lipids was higher and the thermal tolerance of plants was higher. The omega-3 fatty acid desaturase (FAD3) is located in the endoplasmic reticulum (ER) and is the key enzyme which catalyze 18:2 (9, 12) to form 18:3 (9, 12, 15) in the membrane glycerolipids with phospholipid excluding the inner membrane of plastid and non-photosynthetic tissues. In this research, a full length cDNA of endoplasmic reticulumω-3 fatty acid desaturase gene was isolated from tomato using homological clone. The expression pattern and functional analysis showed that expression of the gene was induced by low temperature stress, whereas it was inhibited by heat stress. Interestingly, overexpression of the gene can enhance the chilling tolerance of tomato plants, and inhibition of this gene can improve the thermal tolerance of tomato plants. The main results are as follows:1. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from tomato leaves according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. Then the 5′and 3′of the cDNA were isolated by 5′and 3′RACE. The clone contains 1184 bp nucleotides with an open reading frame (ORF) of 1134 bp, comprising 377 amino acid residues with the predicted molecular mass of 41 kD. Then these sequence data has been submitted to the GenBank databases under accession number EU251190 (address is as follows: http://www.ncbi. nlm.nih.gov).2. The deduced amino acid sequence of tomato endoplasmic reticulumω-3 fatty acid desaturase showed high identities with endoplasmic reticulumω-3 fatty acid desaturase from Nicotiana tabacum, Elaeis guineensis, Glycine max and Brassica napus by homologous sequences blast. The phylogenetic analysis of variousω-3 fatty acid desaturases indicated that the gene was clustered to the endoplasmic reticulum FAD3 type, so it was named after LeFAD3. At the same time, the homology analysis indicated that the amino acid sequences had four conserved motifs. The last three motifs were rich in histidine and necessary for maintaining the membrane-bound desaturase enzymatic activity.3. Southern blot analysis showed that LeFAD3 gene was a single copy in tomato genome and a number of which encoded theω-3 fatty acid desaturase of tomato genome. Northern blot analysis showed that LeFAD3 gene was expressed constitutively in all the organs of tomato. The transcripts were markedly abundant in the root tissue and poor in the leaf tissue. Meanwhile, the expression of LeFAD3 gene was obviously induced by low temperature while markedly inhibited by high temperature stress.4. The full-length LeFAD3 cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense and antisense constructs. Then the gene was introduced into the tomato by A. tumefaciens-mediated leaf disk transformation. The transgenic plants were selected by 5‰kanamycin. Then the kanamycin-resistant transgenic tomato plants were further verified by PCR and northern hybridization. It was indicated that the LeFAD3 gene had been recombined into the genome of tomato in parts of the transgenic tomato plants. It resulted in the increase of unsaturated level of membrane lipids in sense transgenic plants, with increase of 18:3 and decrease of 18:2 correspondingly. However, decreased level of 18:3 was accompanied by a concomitant increase in the level of 18:2 by inhibiting the expression of LeFAD3 in the tomato genome. 5. A recombinant of prokaryotic expression vector pET-LeFAD3 was constructed and transformed to E.Coli. BL21. The strong induced fusion protein bands were collected into PBS solution and used to immunize white mice to obtain antiserum. The value of antibody reaches 1:500. Western blot analysis revealed the presence of the strong positive protein signals corresponding to LeFAD3 in sense transgenic plants.6. The sense transgenic plants had higher chilling tolerance than WT plants. After germination and cultivation for 30 days at 16℃/8℃(day/night temperature regime), the fresh weight of the sense transgenic plants was higher than that of WT plants. The relative electronic conductance of roots and leaves increased in both WT and transgenic plants under chilling stress (4oC) for 0, 3, 6, 9 and 12 h. But it increased more quickly in WT plants than that in sense transgenic plants. Compared with WT plants, the cell membrane system ultrastructure of chloroplas in leaf cell and all the subcellular organelles in root tips of sense transgenic plants kept membrane more intact and the sense transgenic plants alleviated membrane damage during chilling stress for having higher unsaturated fatty acid content of total lipids. Under chilling stress, the O2-|- evolution rates and Fv/Fm in WT and transgenic plants obviously decreased. Those decrease was more obvious in WT than those in transgenic plants. Under chilling stress for 12 h, the O2-|- evolution rates of WT, T1-16, T1-20 and T1-6 lines decreased to about 22.9 %, 32%, 32.2 % and 38.3 %, respectively. Fv/Fm of WT, T1-16, T1-20 and T1-6 lines decreased 47.6 %, 33.5%, 29.7% and 14.8%, respectively. The respiration rate increased at first and then declined under chilling stress both in WT and transgenic plants, but the wild type plants decreased more quickly than that in sense transgenic plants. These results indicated that the increase of 18:3 of thylakoid membranes had a role in protecting the photosynthetic apparatus from chilling stress.The SOD and APX activities of WT and sense transgenic plants increased at beginning of chilling stress and then decreased, and SOD and APX activities of sense transgenic plants were higher than those of WT under chilling stress for 12 h. The contents of O 2 and H2O2 in WT increased after chilling stress, while the contents of O 2 and H2O2 of sense transgenic plants increased only after 6 h chilling stress. Both O 2 and H2O2 contents increased more markedly in WT plants than that in sense transgenic plants. At the end of chilling stress, O 2 content in leaves of WT, T1-16, T1-20 and T1-6 plants increased about 53% , 47%, 32% and 19% of initial values, respectively, and H2O2 content of WT, T1-16, T1-20 and T1-6 increased about 79%, 43%, 35% and 28% of initial values, respectively,and MDA content of WT , T1-16, T1-20 and T1-6 increased also 146%,114%,100% and 87%, respectively.7. The antisense transgenic plants had higher thermal tolerance than WT plants. After germination and cultivation for 30 days at 40oC, the fresh weight of the antisense transgenic plants was higher than that of WT plants. The relative electronic conductance of roots and leaves increased in both WT and antisense transgenic plants under 40oC stress for 0, 3, 6, 9 and 12 h. But it increased more quickly in WT plants than that in antisense transgenic plants. Under heat stress for 12 h, the chloroplast in WT plants became round and it appeared peroxisome in which there appeared diamond structure crystals. There were lipid droplets in both leaves and roots in WT plants. The cell membrane system of roots were slightly damaged and could kept intact stucture in the antisense transgenic plants. Under heat stress, the O2 evolution rates and Fv/Fm in WT and antisense transgenic plants obviously decreased. This decrease was more obvious in WT than that in antisense transgenic plants. Under heat stress for 12 h, the O2 evolution rates of WT, T1-10, T1-8 and T1-5 lines decreased to about 21%, 33.5%,36.2% and 45.5%, respectively. Under heat stress for 12 h, Fv/Fm of WT, T1-10, T1-8 and T1-5 lines decreased 33%, 28%, 21% and 8.9%, respectively. Under heat stress, the respiration rate increased at first and then declined both in WT and antisense transgenic plants, but WT plants decreased more quickly than that in antisense transgenic plants.At beginning of heat stress, the SOD and APX activities of WT and antisense transgenic plants increased and then decreased. But SOD and APX activities of antisense transgenic plants were higher than those of WT during heat stress. The contents of O 2 and H2O2 in WT increased under heat stress for 3 h, while the contents of O 2 and H2O2 of antisense transgenic plants increased only after heat stress for 6 h. Both O 2 and H2O2 contents increased more markedly in WT plants than those in the antisense transgenic plants. At the end of heat stress, O 2 content in leaves of WT , T1-10, T1-8 and T1-5 plants increased about 78%,68%, 63% and 14% of initial values, respectively, and H2O2 content of WT , T1-10, T1-8 and T1-5 increased about 70%, 55.6%, 48% and 22.8% of initial values, respectively. The MDA contents increased in both WT and antisense transgenic plants under chilling stress, but it increased more quickly in WT plants.
|
PDF Full Text Request