Cloning And Function Analysis Of The Differential Expressed Genes Induced By Salt In Medicago Truncatula

Saline soil distributes abroad in the world and is hard to be meliorated. It is a great block for croppers extending and producing. So it is primary problem needed solved to improve the salt resistance of corppers and utilize the saline soil at agriculture. It was reported that salt resistance in plant is a scalar property manipulated by many genes, which means mechanism of excluding salt is complex. For years many investigations and attentions have been focused on this issue, but the molecular mechanisms, gene systems and expression regulation of salts resistance in plants remain unclear. It has seriously impeded our ability to substantially improve overall resistance to salts through genetic selection schemes and genetic transfection. The aim of the current research was to isolate and clone the salts resistant genes to look into molecular events associated with salts resistance and establish fundamental materials and techniques for further study and genetic selection. The main results obtained are listed bellow.To screen and clone salt resistant genes in Medicago truncatula, differentially expressed cDNA library of Medicago truncatula were constructed using suppression subtractive hybridization(SSH) through following protocol: poly (A)+ RNA were purified using PolyATract?mRNA Isolation SystemsШ(Promega) from Medicago truncatula induced by salt with 6 hours and being normal grow as control; single- and double-stranded cDNA were synthesized from the poly(A)+ RNA using PCR-SelectTM cDNA Subtraction Kit (Clontech) and further digested using Rsa I into cDNA fragments sized from 400 to 600 bp (dscDNA), dscDNAs from the Medicago truncatula induced by salt (as tester) were divided into two portions which were ligated separately with a different cDNA adaptor; the ligated cDNA were hybridized twice with the dscDNA of the normal growing Medicago truncatula at 68℃for 8 h each; the products after double hybridizations were diluted by 200 times and then used for suppression PCR twice; the secondary PCR products was inserted into pMD-18T vector(TaKaLa) and transformed into E. coli DH5αcompetent cells; more than 500 positive clones were obtained; identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments of 250-750 bp in the 29 randomly selected positive clones, which would provide useful baseline for the screening and cloning of specific salt resistant genes...