The Functions Of Wheat Expansin Gene TaEXPA2 In Cd Stress Tolerance

Cadmium?Cd?is one of the most serious and toxic heavy metal pollutants in the world,which seriously affects plant growth and development.To improve the tolerance of plants to Cd stress is the key to ensure food security and yield.There are many ways to improve Cd tolerance in plants,including altering gene expression.Expansin is a kind of cell wall protein that regulates the elongation and growth of cell wall relaxation cells.It was found in almost all plant cells,tissues and organs.It has been shown that the expansins are involved in the growth and development of many plants and their adaptive responses to stress.Expansins are composed of several subfamilies,including EXPA,EXPB,EXLA and EXLB.EXPA and EXPB are the two largest and most studied families.We previously cloned the EXPA family expansin gene TaEXPA2 from wheat HF9703 in our laboratory.In order to study the function of wheat expansin gene TaEXPA2 under abiotic stress,we transformed dicotyledonous plants tobacco and obtained transgenic plants.In this study,Cd stress tolerance and its mechanism of TaEXPA2 overexpression in transgenic tobacco were analyzed.Its main conclusions are as follows:?1?QRT-PCR analyzed the expression pattern of wheat expansin gene TaEXPA2 in wheat leaves.The results showed the gene was up-regulated under Cd toxicity in wheat leaves.?2?Three positive transgenic tobacco lines was obtained by genomic PCR screening and identification.Different concentrations of CdCl₂ had been selectsd to treat transgenic tobacco on different growth periods.Our results showed that under Cd stress,the germination rate of transgenic tobacco seeds was higher than that of wild type,the main root was longer,the biomass accumulation was obviously increased,and the photosynthetic rate and related parameters of transgenic plants were improved in seedling stage.The results showed that the overexpression of TaEXPA2 enhanced the tolerance of transgenic tobacco to Cd stress.?3?The mechanism of overexpression of TaEXPA2 gene to enhance the tolerance of transgenic plants to cadmium stress was analyzed.At the cellular level,electron microscope analysis showed that the damage of cell structure of transgenic plants was less than that of wild type plants under Cd stress,and the degradation degree of mitochondria and nucleus was lower than that of wild type,and the phenomenon of wall separation was less than that of wild type.On the physiological level,the damage degree of transgenic tobacco cells with TaEXPA2overexpression under Cd stress was significantly lower than that of wild type tobacco cells,and the MDA content and relative conductivity of transgenic tobacco cells were also lower than that of wild type,which indicated that the membrane of over-expressed transgenic tobacco was relatively stable.Compared with wild type,we found that the antioxidant ability of transgenic tobacco with TaEXPA2 overexpression was significantly better than that of wild type tobacco.By comparing perhydrol content and superoxide anion production rate under cadmium stress,we found that transgenic tobacco was significantly lower than wild type,and the activities of superoxide dismutase?SOD?,peroxidase?POD?,catalase?catalase?in transgenic tobacco were significantly higher than those in wild type.It was proved that transgenic tobacco with TaEXPA2 overexpression had good tolerance to Cd stress,which might be related to the improvement of antioxidant ability.?4?We explored the dynamic changes in the accumulation of Cd²⁺in root cells and leaf cells.The results suggested that the Cd²⁺content of transgenic plants was lower than that of WT plants under cadmium stress.The plasma membrane H⁺-ATPase activity of transgenic tobacco plants was higher than that of wild-type tobacco plants.It was helpful to increase the rate of Cd²⁺transport out of plants and reduce the accumulation of Cd²⁺.The activity of V-ATPase and PPase on the plasma membrane of transgenic plants was higher than that of WT,which helps to promote Cd²⁺into the vacuoles and reduce the accumulation of Cd² in the cytoplasm.We further detected the dynamic flow of Cd²⁺within 10 min and found that the Cd²⁺outflow phenomenon was evident in the transgenic tobacco cells.This shows that increasing the cadmium resistance of transgenic tobacco may be related to the decrease of Cd²⁺accumulation in tobacco cells.?5?At the molecular level,the expression levels of genes related to stress response were analyzed,including plasma membrane H⁺transporter gene?NtSOSN,NtHKT521 and NtHKT586?ROS related genes?NTPOD and NTSOD?,proline related gene?NtP5CS?and stress defense related genes?NtERD10C and NtERD10DN?.The results showed that the expression of these genes in transgenic plants was higher than that in wild type plants under Cd stress.?6?Further,the effect of TaEXPA2 homologous gene mutation on Cd stress tolerance was studied using Arabidopsis homolog gene mutant atexpa2.After treatment with 1m M CdCl₂,the growth of all Arabidopsis plants was inhibited,but in the wild type,this degree of inhibition was small compared to that of the mutant plants and was large compared to the transgenic plants.These results indicate that mutations in the TaEXPA2 homologues will increase plant Cd tolerance.In summary,the results presented in this paper suggest that wheat TaEXPA2 was involved in enhancing the tolerance of dicotyledonous plants to Cd toxicity.Overexpression of TaEXPA2 enhanced the tolerance of transgenic tobacco to Cd stress and mutation of its homologous gene enhanced tobacco sensitivity to Cd stress.The enhanced tolerance to Cd stress of transgenic tobacco may be related to the increased of antioxidant capacity and the absorption/transport of Cd²⁺.