Cloning Of Salinity Stress-related Gene In Triticale And Its Function Verification

Object:Saline-alkali in soil is an important factor which influences agricultural environment and restrict the continuable development of agriculture. There are large areas of saline-alkali land in the world. And higher salt concentration affects plants growth even forces them to death. Develop and cultivate salt-tolerant crops is one of ways to solve this problem. Molecular biology, molecular genetics and other related methods have given more light for developing more salt-stress tolerant crops varieties in modern agricultural researches.Methods:In this study, cDNA-AFLP technique was used to screen out salt stress related genes from hexapolid triticale varieties XXHM No.3 and No.5, and RACE technique was used to amply the full-length of genes. Semi-quantitative RT-PCR and enzyme activity were analyzed to confirm the target genes and protein expression for further analysis of plant salt tolerance, the target gene was transformed into arabidopsis by agrobacterium-mediated method to test the gene function.Results:1. Six cDNA fragments related to salt stress, such as phosphoric acid serine decarboxylase, carbonic anhydrase, receptor-like protein kinase, PSⅡ32kD protein, PSⅠhydrophobin protein, differential cDNA fragments of resistance protein, were obtained by cDNA-AFLP technique through 64 pairs of primers.In this research, cDNA-AFLP technique was first used in triticale breeding.2. Triticale carbonic anhydrase No.42 fragment were amplified by RACE, and its full cDNA sequence was obtained, which is 1114bp (accession number:GU145385), including 5'non-coding region of 24bp, an open reading frame of 777bp and 3'untranslated region of 310bp. The CA full cDNA sequence encodes 259 amino acids and the isoelectric point was 8.35 and the molecular weight was 28.076KD. The CA sequence in triticale is 92% homology toβ-CA gene (L36959.1) in barley (Hordeum vulgare) by NCBI sequence analysis. The CA protein in triticale is one of typicalβ-CA genes family by clustal x software analysis, it has zinc atom binding residues which can be initially determined by the small ryeβ-CA gene.3. Expression of CA gene in triticale was analyzed by enzyme activity analysis and semi-quantitative RT-PCR technique. The results indicated that CA enzyme activity was sensitive to the salt stress, however, the response were different in two salt-tolerant varieties, and CA is an important regulated gene respond to salt stress in triticale. This study provides an important theoretical basis for salt-tolerant triticale varieties breeding.4. Agrobacterium-mediated transformation into arabidopsis was conducted, and plants of To generation were successful generated. This work laid a foundation for further study and CA gene functional research.Conclusion:A CA gene was cloned by cDNA-AFLP and RACE technique from hexaploid triticale, and its expression was studied by analyzing CA enzyme activity and semi-quantitative RT-PCR. The result indicated that the cloned CA gene was related to salt stress tolerance in triticale. The study provides important theoretical basis for salt tolerance triticale breeding.