Studies On The Differential Expression Of CDNAs Induced By Cold Acclimation In Wheat Seedling Stage

Chilling injury is an important natural disaster in agricultural production, seriously affected crop production and geographical distribution. Therefore, a number of scientists have been regarded plant cold tolerance as an important research subject.In order to look for the available low-temperature-resistant gene, we adopted both modified DDRT-PCR and silver staining techniques to isolate Chinese Spring cDNAs induced by low temperature acclimation in wheat seedling stage in this study. And then do cloning, sequencing and functional analysis of these differentially expressed cDNA. The contents and results are as follows:(1)The total RNA were extracted from the control seedlings and the treated sample, and then synthesized the first strand of cDNA using anchor primers T11A (C, G). PCR amplification was carried out with l0bp random primers, The PCR amplification products were separated by 6% denaturing polyacrylamide gels and visualized by silver staining. Only16 arbitrary primers can amplify different cDNA of 26. Seen from component of bp, the arbitrary primers with 50% content of GC, besides with G or C residues at the 5' end and 3' end were more efficient. T11G is the best efficient anchor primer. We obtained 60 differential displayed cDNA fragments.(2)Among the total 60 cDNA fragments, only 8 positive cDNA fragments have been cloned and sequenced after being testified by Reverse Northern. The GenBank-Accn are ES452725 to ES452732(3)Do comparison with the GenBank EST database, the results showed seven cDNA fragments have high homology with many antiretroviral related gene sequences of wheat, barley, rice, corn and other plants. Obvious, the mechanism of plants response to adversity, such as low temperature, salt, heat etc, is similar at the transcriptional level. This also explains the inherent pertinence of plants response to adversity.(4)ES452726 had highly homologous with LRR14 (Lrr14), TAK14 (TAK14) and LRK14 gene part sequences. LRR14 (Lrr14), TAK14 (TAK14) and LRK14 gained from hexaploid wheat at the course of researching molecular evolution of receptor-like kinase genes. So, ES452726 may represent a receptor-like kinase gene, worked at the low temperature signal transduction process. Be concerned with the process of growth and development, metabolic regulation and adversity stress in plant, ES452727 may belong to a broad regulated gene.(5)We groped a series of wheat RAPD system optimization program that can apply to the mRNA DDRT-PCR through this study.