Functional Analysis And Identification Of Rice OsTMN11 Involved In Manganese And Cadmium Detoxification

In recent years,industrial and agricultural production has developed rapidly,and soil heavy metal pollution has become increasingly serious.Cd,one of the major soil heavy metal pollutants,not only causes damage to plants,but also causes serious harm to human through transmission in the food chain.Experiments have shown that it is feasible to control soil pollution and improve crop quality by cultivating high-absorption Cd plants and low-cumulative Cd crops(Chen et al.,2009).Fully understanding the mechanism of plant absorption and accumulation of heavy metals and metal detoxification are important prerequisites to cultivate and screen new varieties.In addition,the identification of detoxification genes is also heipful.In this paper,the detoxification function of an unknown function rice gene OsTMNll(Transmembrane nine superfamily member 11)was preliminarily explored to provide a theoretical basis for revealing the mechanism of rice tolerance to heavy metals.First,we cloned the full-length sequence of OsTMNll,and detected the spatiotemporal expression pattern of the gene at the transcriptional level.The results showed that this gene was highly expressed in roots at different time points.The expression pattern of OsTMN11 under heavy metal stress was further determined.And the results showed that the gene was induced by Cd,Mn,Fe,Cu and Zn stress,especially Cd and Mn stress,which strongly induced the expression of the gene.The expression of the underground part was higher than that of the aerial part.The experimental results show that OsTMNll is localized to the Golgi apparatus.We speculate that this gene may be associated with stress from Cd and Mn.To verify this hypothesis,we performed a functional identification of the gene.First,we transformed the OsTMN11 heterologous into the manganese sensitive mutant pmrl.The results showed that the gene could compensate for the functional defects of the pmrl(Mn transporter deletion)yeast mutant and reduce the accumulation of Mn in yeast,suggesting that the gene may be involved in the transport of Mn.To identify whether OsTMN11 has a transport function in rice,OsTMN11 RNAi and over expression rice transgenic lines were obtained by Agrobacterium-mediated genetic transformation to obtain homozygous seeds.Different concentrations of Mn and Cd were set for toxicity test.The results showed that under the stress of Mn and Cd,compared with the wild type,the growth of RNAi transgenic rice was poor,which showed that the plant was short,the leaves were yellow and the browning was obvious,however,overexpression lines growth trend is better than wild type.Physiological indicators showed that the dry weight of RNAi transgenic rice was low,the chlorophyll content was decreased.These results indicate that OsTMNll RNAi plants are sensitive to Mn and Cd and overexpressing plants are more tolerant,which proves that this gene is essential for Mn and Cd detoxification.In addition,the Mn and Cd contents of OsTMN11 RNAi plants and wild-type plants under Mn and Cd stress showed that the Mn and Cd contents of OsTMN11 RNAi plants were higher than those of wild type,suggesting that reducing the expression of OsTMN11 would impair and overexpression of OsTMN11 would promote the efflux and detoxification of plants on Mn and Cd.