Sugar Beet M14 Lines Cystatin Gene Function Studies

Monosomic addition line M14 carrying an alien chromosome 9 from Beta corolliflora was obtained through hybridization between a wild species B. corolliflora and a cultivated species B.vulgaris. M14 was found having a chromosome transmission frequency 96.5%, showing diphosporic reproduction and stress tolerance. Therefore, M14 has been used as an abundant genetic resource for isolating valuable genes in wild species.Previously, about 298 differentially expressed ESTs sequences in M14 were obtained by SSH and DDRT-PCR experiments. Our colleagues used comparative proteomic method to analyze the flowers of M14 and B. vulgaris using 2-DE and LC-MS/MS. A total of 71 differentially expressed protein spots were successfully identified in the two samples. ESTs information was compared to the protein sequences identified by proteomics using BlastX. Eight proteins which were different in protein expression between M14 and B. vulgaris had been successfully matched. BvM14-Cystatin is one of these proteins.Phytocystatins are plant proteins that inhibit cysteine proteinases. Involvement of phytocystatins in plant defense strategies against various pests and predators, is widely acknowledged. Furthermore, participation of cystatin in response to abiotic stresses has been reported. However, the mechanism by which cystatins respond to abiotic stress was not clear and the reports about over-expression of cystatins on increasing stress-tolerance are few.In this study, a cDNA clone, designated BvM14-Cystatin, encoding a novel phytocystatin was isolated from monosomic addition line M14 using 5'-/3'-RACE extension. The full-length cDNA gene is 690 bp, encodes 104 amino acid residues. It contains some conserved reactive site motifs of cysteine protease inhibitors. Gene expression analysis indicated that BvM14-Cystatin was constitutively expressed in root, stem, leaf and flower. Its expression in root and stem was relative high.Recombinant BvM14-Cystatin was expressed in Escherichia coli and purified. They obviously inhibit the catalytic activity of papain. The remaining hydrolytic activity with 50μg/mL BvM14-Cystatin is 26.09 percent. Plant transformation binary vector pBI121-BvM14-Cystatin was introduced into Agrobacterium tumefaciens strain EHA105. Arabidopsis was transformed by the floral dip method. Arabidopsis over-expressing BvM14-Cystatin (the homozygous lines T2) was generated. Wild-type and the T2 generation of the two transgenic lines were treated with 0 (control),100,200 mmol/L NaCl and 200,350 mmol/L mannitol. Root growth and fresh weight of the transgenic plants were higher by NaCl or mannitol treatment than these of the wild-type. At a NaCl concentration of 175mmol/L, the survival rate of transgenic plants is also obviously higher than wild-type plants. Taken together, these data raise the possibility of using BvM14-Cystatin to improve salt and drought stresses tolerance in plants.This work was supported by the National Science Foundation of China (Project 30871566:Studies on floral organ-specific expressed proteins in sugar beet M14 lines), the National Science Foundation of China (Project 31071473:Studies on the function of the BvM14-cystatin in sugar beet M14 lines)...