| Temperature was one of important factors influencing plant growth and distribution. Most of plants would be suffered from chilling or freezing injury under low-temperature stress. Every year, the loss led by cold in crops was very heavy in the world. Therefore, it was theoretically and practically significant to study and enhance the cold-tolerance in plants.With the development and wide application of biotechnology, some of cold-tolerant genes were isolated and identified from plants in recent years, and the evidences showed that a part of them played important roles in resisting cold stress in plants. Moreover, some hypotheses and models about low-temperature signal transduction and regulatory mechanisms were put forward. These works provided a new way for cultivating cold-resistant species by genetic engineering technique.Highland barley (Hordeum vulgare L. var. nudum HK. f.), belonging to the genus Hordeum in the family Gramineae, distributed mainly in the Qinghai-Tibet Plateau, China. It has stronger cold-tolerance than other cereals, and was a potential resource of cold-resistant genes. Therefore, highland barley was used as materials in this study, the cold-induced genes cloned from it were studied on the basis of established high efficient regeneration system. The results obtained were as follows:I. In vitro protocol for efficient plant regeneration has been established from mature embryo-derived calli of highland barley. Embryos with part of (endosperm; E) or without endosperm (non-endosperm; NE) were excised from mature seeds and cultured on MS medium supplemented with 2, 4-D (1～5 mg/L) for callus induction. Percentage of callus induction from E explar ts was significantly lower than that from NE. The highest frequency (93.1%) of callus induction was obtained from NE explants on MS medium containing 3 mg/L 2, 4-D. The viable calli were subcultured at reduced concentration of 2, 4-D (2 mg/L), and then transferred to MS medium supplemented with 6-BA (1～5 mg/L) and 500 mg/L CH for shoot regeneration. Contrarily, percentage of plant regeneration from E explant-derived calli was higher than that from NE. The highest frequency of plant regeneration (65.2%) was obtained from E explant-derived calli on MS medium containing 2 mg/L 6-BA. The regenerated shoots were multiplied and rooted on half-strength MS medium without growth regulators. The rooted shoots were transplanted to soil with 100% survival. This method could be employed for the genetic manipulation studies.II. A cold-induced gene was cloned from highland barley by PCR, named hblt14.2, which exhibited 98.9% homology to bltl4.2 of barley, and there were 2 amino acids difference between hblt14.2 and blt14.2 proteins. The length of hblt14.2 was 470 bp, containing a 249 bp ORF, 69 bp 5' untranslated region (UTR) and 152 bp 3' UTR. Hblt14.2 gene encoded a protein of 82 amino acids with a calculated molecular weight of 7.71 kDa and a isoelectric point of 7.03. Hblt14.2 protein was a highly hydrophilic small protein with rich Gly(17.07%), Ala(15.85%), Leu(13.41%) and Val(9.76%) amino acids. The secondary structure analysis showed that hblt14.2 protein was rich in alpha helix and random coil, which were similar to other cold-induced genes.Hblt14.2 expression patterns under cold and other abiotic stresses were analyzed by RT-PCR. The results showed that the expression of the gene could only be induced by low temperature, while there were not expression under salt stress and ABA treatment. Thus, it was speculated that hblt14.2 was possibly a low temperature-specific gene. Furthermore, the level of hblt14.2 expression was higher in the leaves than in roots when plants were exposed to 4℃, which suggested that hblt14.2 expression had probably tissue specificity.Hblt14.2 gene linked with CaMV 35S promoter and terminator were inserted into multiple clone site in plasmid CAMBIA1301, which constructed the plant expression vector pCAMBIA-BI-hblt. Then, the target gene was transformed into Nicotiana tabacum by Agrobacterium tumefacien LBA4404 as vector. PCR and Southern examination showed that hblt14.2 had been inserted into tobacco genome. Hblt14.2 expression pattern in tobacco under cold stress was analyzed by RT-PCR. The results indicated that the gene could be transcribed correctly in transgenic tobacco.The mesophyll protoplasts were isolated through enzyme digestion from transgenic tobacco plants and the controls. Subsequently, they were cultivated under 4℃stress. The results showed that the survival rates of protoplasts from transgenic plants were higher slightly than that of the controls. After 35 days under cold stress, the survival rate of protoplasts from transgenic plants was 56.7%, whereas the controls was 43.2%. First cell division was observed in transgenic tobacco protoplasts after 1 week of culture, but no division was observed in the controls. Moreover, the contents of proline were analyzed in leaves of transgenic tobaccos and the controls under 4℃stress. The results showed that the proline contents were lower in transgenic plants than that in the controls. After 72 hours under cold stress, the proline content was 1.22 mg/g in the controls, while in transgenic plants was 0.57 mg/g. It was speculated that the cold-tolerance of tobaccos was possibly enhanced partly owing to hblt14.2 gene overexpression. As a result, other cold-resistant pathways such as proline synthesis pathway might be weakened. The results as stated above suggested that hblt14.2 was related to cold resistance in plant.III. Cold-induced genes of highland barley were studied by using suppression subtractive hybridization (SSH) technique, in which the cDNA from the materials treated with 4℃was used as "tester" and the cDNA from the materials growing in green house as "driver". A subtractive library of highland barley including 240 cDNA clones was constructed in the study. Enzyme digestion of 32 clones chosen randomly from the library indicated that there were inserts in 87.5% of them. The cDNA inserts of 16 clones were sequenced and blast searches were performed. Blast analysis showed that these cDNA were homology to the following genes: metallothionein, protein kinase, ethylene signal transcription factor, bZIP transcription factor, zing finger transcription factor, ribulose-1, 5-bisphosphate carboxylase, leucine rich protein, fructose-bisphosphate aldolase, copper/zinc superoxide dismutase, ribosomal protein, sodium: hydrogen antiporter, catalase, NADPH-cytochrome reductase, ascorbate peroxidase, DNA binding protein and sugar transporter-like protein. The results indicated that the cDNA clones in the library were related to cold-induced genes. It was suggested that the cold-tolerait mechanism of highland barley might be a complicated, interactive system involving multiple approaches and multiple genes. Construction of subtractive cDNA library provided a advantage for further studies to isolate and clone cold-induced genes in higriland barley.
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