Functional Analysis Of Cystatin-associated Gene SRAC1

With the deterioration of the environment,crop growth and development are severely affected by various abiotic stresses in recent years,which result in low yields significantly.For example,high salt can damage the plant cell membrane system,change the cell osmotic pressure and change ion concentration,which result in inactiving various hydrolytic enzymes and protease.Drought can reduce protein biological activities,which result in crop abnormalities and lower yields.Cystatin harbours the typical conserved motifs,namely a Gln-X-Val-X-Gly motif in the central region of the polypeptide chain,a Pro/ Leu-Trp dipeptide motif in the C-terminal region and a conserved Gly residue in the N-terminal region.Cystatin interactes with cysteine protease through a groove structure,which inhibits cysteine protease activity.It helps plants resist to not only low temperature,high temperature,salt and drought but also fungi and insects.It has been found that have an important influences on plant growth and stress resistance,but the function about cystatin related genes and their encoded proteins is almost unknown.Cystatin related genes whether are involved in abiotic stress needs further exploration.This study comprehensively analyzed cystatin related genes in Arabidopsis and functionally characterized SRAC1(salt resistance associated cystatin 1).(1)Bioinformatic analysis revealed the existence of 23 cystatin related genes in Arabidopsis,among them 20 cystatin related genes display similar gene structure compared with known cystatin.(2)Chromosome positioning analysis showed that they distribute at from chromosome 1 to chromosome 5 and close to cystatin genes.(3)Promoter function analysis showed that they not only contain responsing to gibberellic acid,ethylene and light elements,but also contain responsing to a high temperature,low temperature,drought,abscisic acid and jasmonic acid modulated components.(4)Homologous analysis showed that cystatin related genes are roughly divided into two groups,12 genes are classified into the same category with known cystatins and 5 of them have been found to inhibit the activity of cysteine protease.(5)Protein conservative motif analysis showed that they contain relatively conservative Gly and L-E-F-V-V-Y-R-A motif in the N-terminal region and Y-Q-A-K-V motif in the central region,they may form three-dimensional structure similar to known cystatin.(6)After ABA treatment,SRAC1(At5g17090)is significantly up-regulated.Overexpression SRAC1 improve the resistantance to NaCl and educe sensitivity to ABA in transgenic Arabidopsis plants.(7)Enzyme activity analysis showed that SARC1 is a bona fide cystatin,which could effectively inhibit the activity of papain.