| The sensitivity of plants to low temperature is closely related to their initial origins. Plants from temperate regions have the capacity of cold acclimatation, that is, to develop increased freezing tolerance after being exposed to low nonfreezing temperature, whereas species from tropical and subtropical areas nearly do not have it. Phaseolus lunatus L. belonging to legumes origins from tropical regions.Large amounts of experiments have demonstrated that the plant resistance to cold is quite related to UFAI (Unsaturated Fatty Acid Index) of cell membrane. The cell membrane of cold-resistant plant has higher UFAI than cold-sensitive ones. Fatty acid compositions and UFAI of membrane phospholipids are directly determined by the type and quantity of fatty acid desaturases (FAD). The ability to adjust membrane lipid fluidity by changing levels of unsaturated fatty acids is a feature of cold acclimating plants, which is mainly regulated by activity and categories of FADs. The growth critical temperature of cold-resistant Phaseolus lunatus L. was identified and three FAD genes were cloned from its leaf in this study to help understanding the mechanisms of cold-sensitivity. The results are summarized as follows.1. Seedings of Phaseolus lunatus L. could resist 8±1℃according to phenotypic observation and Arrhenius plot of REC (Relative Electric Conductivity). Dynamic changes of various indexes in leaves of plant treated under 8±1℃indicated that the seedlings could truly resist low temperature of 8±1℃through increasing activities of oxidase system.2. Primers were designed according to relative sequences on NCBI. Constant fragments of three FAD were isolated from leave of Phaseolus lunatus L. by RT-PCR. The full-lenth of their cDNAs were acquired by 5'and 3'RACE. The sequence of SAD, FAD3 and FAD2 had 1777 bp,1510 bp,1652 bp respectively with ORF of 1317 bp, 1116 bp and 1059 bp, encoding a 439,372 and 353 amino acid protein. Putative protein sequence alignments and phylogenetic analysis of three genes showed that three genes shared the highest identity of 86%,85% and 91% respectively with their counterpart species. The full-lenth DNAs of SAD and FAD3 contained 1 and 7 introns respectively, while FAD2 had no introns.3. Tissue-specific expression analysis revealed that SAD and FAD2 expressed highly in leaves, but nearly did not express in root and sterm; while FAD3 expressed in all three tissues, especially high in leaves.4. Expression pattern of each gene under different stresses including 8±1℃, 42℃,0.15 mmol/L NaCl and 15% PEG by RT-PCR showed that FAD2 could be induced by the stresses, while high temperature and salt somewhat inhibited the expression of SAD and FAD3. The expression patterns of three genes were different under low temperature. SAD was down-regulated in first 12 h and reached its lowest level, then returned in the following 12 h-dark till its maximum. Expression of FAD3 reached its maximum at the time point of 3 h and maintained to 12 h, then decreased. Expression of FAD2 was up-regulated from 1 h to 3 h, then decreased. To resist chilling temperature, the expression of FAD3,FAD2 in Phaseolus lunatus L. might enhance the amount of poly-unsaturated fatty acid to change the fluidity of cell membrane.
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