Functional Analysis Of OsCatC Gene In Response To Salt Stress In Rice

Rice（Oryza sativa L.）is one of the most important food crops in China and even in world.This crop is extremely sensitive to salt stress,land salinization is therefore a factor that restricts the increase of global rice production and the planting area.When plants suffer from salt stress,the cells will transduce salt stress signals,regulate the expression of related stress resistance genes in plants,thereby slowing down or resisting the damage caused by salt stress.Catalase（CAT）,present in almost all aerobically respiring organisms,plays an important role in defending cells against oxidative damage by degrading the hydrogen peroxide（H₂O₂）to water（H₂O）and oxygen（O₂）.Previously study found that rice CatC was phosphorylated by the receptor-like cytokinase STRK1,which enhanced the activity of CatC,and then improving the salt tolerance of rice.However,there are no experiments to explore the effects of OsCatC overexpression and function loss on salt tolerance in rice.In order to clarify the specific functions of CatC,we constructed OsCatC overexpression lines,and performed experiments on the overexpression lines and functional loss mutant noe1-2 under salt stress conditions in the physiology and biochemistry.The main results are as follows:（1）Bioinformatics analysis showes that,its promoter region contian multiple cis-acting elements related to hormone and stress response,suggesting that OsCatC may be involved in the response to biological and abiotic stresses.OsCatC gene sequence is 1479bp and encodes 492 amino acid residues,and the C-terminal of the CatC polypeptide chain contains peroxisomal targeting signal,which guide CatC protein to locate in the peroxisomal.（2）Phenotypic identification of salt stress at seedling stage showed that OsCatC positively regulates the salt tolerance of rice.There are two OsCatC overexpressing transgenic lines（OE-10,OE-18）were constructed and identified.Through the phenotypic identification of OsCatC overexpressing transgenic lines and the mutant of noe1-2,we found that the tolerance of OE-10 and OE-18 were significantly improved compared with those of Kitaake.Furthermore,the salt tolerance of noe1-2as significantly depressed compared with those of Nip.These results indicated that OsCatC positively regulates the salt tolerance of rice.（3）OsCatC can weaken oxidative damage under salt stress,and positively regulates the tolerance to oxidative stress in rice.Compared with WT,OsCatC overexpressing seedlings（OE-10,OE-18）exhibited higher tolerance to salt stress with lower relative electrolyt eleakage,malondialdehyde（MDA）,H₂O₂,and their catalase activities were markedly increased.The seedlings of mutant exhibited sensitive to salt stress with higher relative electrolyt eleakage,malondialdehyde（MDA）,H₂O₂.And OsCatC overexpressing seedlings length was markedly higher than that of WT after treatment with methylviolet（MV）.These results indicating that the OsCatC overexpressed transgeniclines had a stronger tolerance to oxidativestress.（4）Phenotypic analysis of over-expression of OsCatC^Y210A,over-expression of OsCatC and wild-type seedlings under salt stress indicated that mutation of tyrosine into alanine at site210 of CatC could not enhance the tolerance of rice to salt stress.It was found that the enzyme activity of CatC^Y210Awas higher than that of CatC^Y210D,which speculated that the mutation of tyrosine into alanine at site 210 of CatC could enhance the salt tolerance of rice.Subsequently,there are two OsCatC^Y210Aoverexpressing transgenic lines were constructed and identified.Through the phenotypic identification,it was found that the survival rate of OsCatC^Y210Aoverexpressing transgenic lines under salt stress was slightly higher than WT,but lower than OE-10.To a certain extent,mutation of CatC site 210 tyrosine into alanine can increase CAT activity,but can not enhance the tolerance of rice to salt stress.This study discovered that CatC reduces oxidative damage mainly by degrading the excessive accumulation of H₂O₂under salt stress,there by improved the salt tolerance of rice,and further verified that overexpression of OsCatC^Y210Acan not enhance the tolerance to sal tstress in rice.The study lays a certain foundation for further elucidation of the mechanism of CatC participating in the response to salt stress in rice,and indicates that OsCatC is a promising candidate gene for the cultivation of salt-tolerant rice.