扁蓿豆抗寒相关基因的克隆与表达研究-cloning and expression of cold resistance related genes in medicago ruthenica.docxVIP

Cloning and Expression Analysis of the Cold Resistance Related Genes of Medicago ruthenica Abstract Digging deep into cold resistance genes of Medicago and revealing the mechanism can provide theoretical basis for cold resistance breeding on Medicago. In this paper, Medicago ruthenica which has strong cold resistance were taken as materials. cDNA-AFLP and RT-PCR were employed to analyze and to isolate cold stress genes expressed under cold stress using the three-five leaf stage leaves of Medicago ruthenica. The results of qRT- PCR and physiological characteristics were used to analysize the difference between cold resistance gene of Medicago ruthenica and other plants’ related gene and express stage, the correlation on gene express and physiology and biochemistry indexes. The studies can lay the foundation for getting results clearly on cold resistance mechanism. The main results were as follows: The seedling leaf, stem and root of vertical Medicago ruthenica were varying degrees of response to low temperature. Soluble sugar content and POD activity first increased in the root, the highest level was 3.64 and 1.64 times as the control; soluble protein content and SOD activity first increased in leaves, the highest level was 1.52 and times as the control. Soluble protein content and SOD activity had significant positive correlation with POD activity. The results showed that Medicago ruthenica could against the cold by increasing the content of osmotic substances and the activity of anti-oxidative enzyme under low temperature. The mRNA of Actin, CAS15A and CAS15B on Medicago ruthenica were cloned by RT-PCR, and the blast analysis showed that the above genes had 93% nucleotide similarity to the Actin and CAS gene of alfalfa. The MRActin gene expression had no significant differences in leaves, stems and roots under the different temperature treatments, which can be as a reference gene for the quantitative analysis. The MRCAS gene harbored CRT/DRE(TACCGACCA