Isolation And Expression Of The Genes Involved In Tolerance To Low Temperature In Maize

Maize is a kind of important crop in the world and is significant to national food supplies. Chilling temperature is the important environmental factors to limit the distribution and decrease the productivity of plant chilling-sensitive species. To improve maize production by studying on the mechanism of chilling tolerance for maize, cloning of genes involved in chilling tolerance, and breeding by gene engineering to enhance chilling tolerance, is the focus not only in China but also in the world.Maize growing in normal temperature was susceptible to the chlilling, but the chilling resistance of which would greatly increase through chilling acclimation at 14"C. It was the result that many chilling resistant genes in maize expressed so that the chilling resistant mechanism was altered in order to adapt to the chilling stress. The plant will suffer chilling injury if they are pretreated with non-acclimation because of absent chilling resistant mechanism. Genes involved in chilling tolerance were induced by lower temperature. The gene expression of chilling-acclimation and non-acclimation seedlings was different from that of the control, the seedling growing under normal temperature, respectively. Research on differential gene expression plays important roles in understanding mechanism of chilling tolerance and improving chilling tolerance for maize. Chilling resistant maize inbred cheng18 was used to test the difference of mRNA transcription in materials treated with differential temperature by mRNA differential display, and differentially expressed cDNA fragments were cloned, sequenced and analyzed their expression. Seeds were planted in sands and grown at 24 C for 3 days in darkness. The partial seedlings continue to grow at 24 C for 3 days in darkness, other seedlings were then pre-exposed to either 14 C for 3 d in darkness (acclimation period), followed by 3 d in the dark at 4 C (acclimation, chilling period),or directly transferred to 4 C for 3 d in the dark (non-acclimation, chilling period). All the five kinds of materials were harvested to extract total RNA.Thirty primer sets could display abundant differential expression cDNA fragments among the 120 primer sets screen, in which the anchored primers with GA,GC,GG ,CG residues at the 3' end were more efficient, the arbitrary primers with 50%-60% content of GC .besides with G residues at the 5' end and G or C residues at the 3' end were more efficient. 807 cDNA segments were amplified, 95% of which were same at differential temperature. Eleven differentially expressed cDNA fragments with length >300bp weregained. The sequences of DD16 (accession number: AY647239) and DD17 (accession number: AY647238) were submitted to GenBank.Some differentially expressed cDNA fragments were identified by Northern hybridization. The result showed that the transcription of DD16 increased at 14 Cand at non-acclimation treated in 4 C; the transcription of DD21 increased at 14 C. DD16 was homologous to oryza sativa (japonica cultivar-group ) receptor-like protein kinase ARK1. Sequence analysis showed that the nucleotide sequence and amino acid sequence of DD16 shared respectively 85% and 90% similarity with that of ARK1. However, the homology was higher in the catalyze domain of protein kinase. It was speculated that DD16 was candidate gene encoding some protein kinase which played a key role in signal transduction. This suggested that the expression of it was enhanced under the condition of chilling-acclimation and chilling stress. DD21 was homologous to Zea mays malate dehydrogenase with 93% homology. The expression of it was enchanced during acclimation period, because there is resistant mechanism in seedlings to adapt to chilling enviroment and reduce energy stress through producing more ATP. Other differentially expressed cDNA fragments were need to northern hybridization. DD12, DD15 and DD17 were homologous to partial sequences in maize cDNA library established by Dupont. DD19 and DD20 were homologous to partial sequences in maize roots cDNA library of seedlings...