| Temperature is one of the necessary conditions for the growth of plant, also is the main limiting factor of plant natural geographical distribution. In plants the low temperature will cause cold injury,In our country and around the world, because of the cold injury resulting in loss of food crops to vegetables, fruit trees and crops every year, therefore, to strengthen the research of plant cold injury and cold resistance, and according to cold resistance of varieties of crop scientifically determine its planting area and seedtime, cultivating strongly cold resistance of new varieties, has become one of the important problems in agricultural production.Recent studies revealed that microRNAs (miRNAs) play important roles in plant responses to a variety of abiotic stresses. Although some miRNAs are identified to be regulated by cold or chilling stress, little information is available about the functions of miRNAs in chilling tolerance of plants.We use the Chorispora bungeana that growning in the high mountain,and it is capable of strong resistance to cold and freezing,we used it as materials, miRNAs could be regulated by low temperature stress was predicted by high-through sequencing in Chorispora bungeana, compared their target gene expression patterns by gene ontology (GO),then tested their characters though real-time fluorescent quantitative PCR, the result showed:(1)We forecasted a total of 46 novel miRNAs, named were cb-m0001, the cb m0002, and so on until thecb-m0046, which includes 10 conserved miRNAs.(2)We obtained 133 conservative miRNAs was identified in the Chorispora bungeana, with 130 in the mRNA transcription group be predicted as target genes. Only nine conservative miRNAs may be controled by low temperature, respectively is miR390?miR164?miR164a?miR168?miR161?miR158?miR169a? miR159 and miR169.(3)Analysis results are verified through the qPCR,we found miR390?miR164? miR164a and miR168 were up-regulated by cold stress, expression of miR161 and miR158 also were up-regulated under cold stress, but there have significant differences, cold treatment 1 and biology repeat also significant difference in cold treatment 2, so we could not accurately judge the expression level under cold stress, expression of miR169a and miR159 under the cold treatment and room temperature controls almost no difference,While the expression of miR169 under cold stress is lower than the room temperature control.(4)Then the target genes CB7248 were cloned, sequenced, according to the results of amplification product length is 400 bp and 3000 bp, respectively and no base mutation in the sequencing results compared with landing on GenBank DNAMAN gene sequence analysis, the results showed the homology of 100%.(5)CB7248 showed important role in disease resistance responses,there had certainly relationship with cold resistance in plants, in this paper we used PstDC3000 bacterium liquid to exhaust cb3?cb6 andcb9 arabidopsis, contrast to wild type col, results show cb3?cb6andcb9 had significantly higher cold resistance than wild-type arabidopsis thaliana by the bacteria liquid exhaust dyeing,and then cold treatment for 24 h.We used high-through sequencng for Chorispora bungeana, and analysised conservated miRNAs and there target genes, verified expression of the miRNAs of regulation by cold stress in the Chorispora bungeana, they provided a large number of gene sequence and functional information for mechanism of cold tolerance of plant as the research, so provid us with abundant resources in order to further study mechanism of chilling stress and freezing tolerance. |
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