Cloning And Expression Analysis Of The Cold Resistance Related Genes Of Medicago Ruthenica

Digging deep into cold resistance genes of Medicago and revealing the mechanismcan 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 genesexpressed 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 thedifference between cold resistance gene of Medicago ruthenica and other plants’ relatedgene and express stage, the correlation on gene express and physiology and biochemistryindexes. The studies can lay the foundation for getting results clearly on cold resistancemechanism. The main results were as follows:1. The seedling leaf, stem and root of vertical Medicago ruthenica were varyingdegrees of response to low temperature. Soluble sugar content and POD activity firstincreased in the root, the highest level was3.64and1.64times as the control; solubleprotein content and SOD activity first increased in leaves, the highest level was1.52and2.12times as the control. Soluble protein content and SOD activity had significant positivecorrelation with POD activity. The results showed that Medicago ruthenica could againstthe cold by increasing the content of osmotic substances and the activity of anti-oxidativeenzyme under low temperature.2. The mRNA of Actin, CAS15A and CAS15B on Medicago ruthenica were cloned byRT-PCR, and the blast analysis showed that the above genes had93%nucleotide similarityto the Actin and CAS gene of alfalfa. The MRActin gene expression had no significantdifferences in leaves, stems and roots under the different temperature treatments, whichcan be as a reference gene for the quantitative analysis. The MRCAS gene harboredCRT/DRE(TACCGACCA）regulatory motifs, four dehydrin segments and a decapeptidemotif repeated four times similarity with the CAS genes in other plants. MRCAS15genebelonged to the CAS family. Real time PCR ananlysis revealed that the expressions ofMRCAS15A and MRCAS15B were steadily up-regulated under low temperature stress, andthe highest expression level at72h and84h, then the result declined the expression ofCAS15gene could enhance the cold resistance of Medicago ruthenica. 3. The cDNA amplified fragment length polymorphism (cDNA-AFLP) analysissystem was established with the restriction endonuclease EcoRⅠand MseⅠ to analyzegenes differentially expressed under cold stress of Medicago ruthenica.58up-regulatedTDFs involved in photosynthesis, respiration, cell signal transduction, metabolism, defensereaction and many other important process. Blastn and blastx analysis showed that morethan70.79%of the up-regulated TDFs had identity genes with Medicago truncatula. Theresults of quantitative analysis showed that7characteristic genes were involved intemperature regulation.4. The cDNA of MRPP2C, MRHSP70and MRHLH were induced by RACE.Sequence analysis showed that the amino acid residues included conserved domains PP2C,HSP70and HLH, which had high homology with corresponding protein of Medicagotruncatula and many other plants. The qRT-PCR results indicated that the expression levelsof three genes shared sililar tendency and had two high peaks within84h of lowtemperature stress treatments. The expression of MRHLH gene had significant positivecorrelation with MRCAS15gene. The cold-resistant genes MRHLH, MRCAS15A andMRCAS15B had significant positive correlation with SOD activity. The cold ability was acomplex regulatory network of Medicago ruthenica from molecular level and physicallevel.