Cloning And Anti-salt Alkaline Analysis Of Arabidopsis Thaliana AtSRT2 Gene

Histones can be modified under the action of some enzymes,such as phosphorylase/dephosphorylase,acetylated enzyme/deacetylase,methylase/demethylase and so on.These modification can change the binding state of histone and DNA,and then participate in the regulation of gene expression,affecting the growth,development,aging and other physiological processes.Silencing regulators Sirtuin is a special histone deacetylase that regulates cellular energy metabolism and redox status by NAD⁺synthesis and fragmentation.Sirtuin protein is studied in animals and yeats,and is involved in prolonging cell life,DNA damage repair,maintaining genomic stability and energy metabolism.But Sir2 is less in plants,and Arabidopsis contains only AtSRT1 and AtSRT2,the function is not entirely clear.It has been reported that the Sir2 family has a certain relationship with plant development,biological stress and hormone signal transduction,but the relationship with the non-biological stress and the mechanism of action is not reported.In this study,the AtSRT2 gene?AT5G55760?was obtained by RT-PCR.The open reading frame was 1122bp,encoding 373 amino acids.The plant expression vector pGWB-AtSRT2 was constructed by gateway method.The transgenic expression of transgenic T2 was successfully obtained by using Agrobacterium tumefaciens-mediated transformation of plant expression vector into Arabidopsis thaliana.The changes of physiological,biochemical indexes and phenotypes of Arabidopsis thaliana under different salt and alkali stress treatments were analyzed by comparing the obtained AtSRT2 with WT,pGWB2 and atsrt2 groups.The main results summarized as follows:1.The seed germination rate,leaf,root length and fresh weight of four groups were studied,we found that AtSRT2 group had the highest germination rate at 100%,whereas atsrt2 group germination late,germination rate is about 75%under alkaline stress.The root length of AtSRT2 group was 1.25 times,1.19 times,1.56 times higher than WT group,pGWB2 group and atsrt2 group under salt stress,which was 1.35 times,1.28times and 1.64 times higher than that of other three groups under alkaline stress.Fresh weight is also greater than the other three groups.2.Arabidopsis thaliana in the 0,60,120,180m MNaCl and 0,3,5,7mM NaHCO₃stress environment,the control group of Arabidopsis contains osmotic adjustment substances such as proline,soluble protein content no obvious difference.However,with the rising of the concentration of salt,the contents of proline and soluble protein increased.To reach the maximum at 180mmol/L,and the content of proline was 18.6%,28.3%and 100%higher than WT,pGWB2 and atsrt2,respectively.The content of soluble protein was 29.8%,28.3%and 100%higher than that other three groups respectively.The content of proline was 31.6%,20.8%and 45.1%higher than that of WT group,pGWB2 group and atsrt2 group at 7mmol/L,and the soluble protein was41.1%,34.5%and 73.0%higher than WT group,pGWB2 group and atsrt2 group.3.Along with the rising of NaCl and NaHCO₃ concentration,the conductivity also increased.But the overexpression of the transgenic group rose less,the mutation group increased the most.The conductivity of WT group,pGWB2 group and atsrt2 group was26.3%,10.7%and 48.3%higher than AtSRT2 group when treated with 60mmol/L salt stress.At 180 mmo/L,the electrical conductivity of WT group,pGWB2 group and atsrt2group was 8.6%,12.4%and 24.9%higher than AtSRT2 group.The conductivity of WT group,pGWB2 group and atsrt2 group was 26.1%,18.4%and 41.1%higher than that AtSRT2 group under 3 mmol/L alkaline stress treatment,at 7 mM,24.0%,21.8%and54.7%higher than those of AtSRT2 group,respectively.4.The content of chlorophyll decreased with the rising of Na Cl and alkali concentration.However,the AtSRT2 group had the highest chlorophyll content,and the wild type and empty vector group were higher than the mutant.In the treatment of60mM NaCl,the AtSRT2 group was 4%,6.8%and 11.4%higher than the WT group,pGWB2 group and atsrt2 group,respectively.At 180 mM Na Cl,43.8%,31.4%and119%higher than the other three groups,respectively.In the 3mM alkaline stress AtSRT2 group were 11.6%,8.5%and 20.3%higher than those in WT group,pGWB2group and atsrt2 group.The AtSRT2 group was 45.2%,60.7%and 104%higher than the WT group,pGWB2 group and atsrt2 group under 7 mmol/L alkali stress.5.In addition,the effects of stress on the antioxidant system of different Arabidopsis thaliana were also detected.By measuring the enzyme activity of SOD and POD,we found no significant difference in the activity of Arabidopsis thaliana in the control group.However,SOD and POD increased with the rising of NaCl and alkali concentration.The activity of AtSRT2 group was higher than that of the other three groups,and the activity of mutant enzyme was the lowest.When the salt concentration was 120mM,the SOD activity was the most significant difference,and AtSRT2 group were 1.38 times,1.30 times,1.53 times of WT group,pGWB2 group and atsrt2 group.The activity of SOD was the most significant at the alkali concentration of 7mM.The AtSRT2 group was 1.23 times,1.32 times and 1.60 times of WT group,pGWB2 group and atsrt2 group,respectively.The difference of POD activity was most obvious at salt concentration of 180mM.The activity of AtSRT2 group was 1.32,1.26,1.67 times of WT group,pGWB2 group and atsrt2 group.When the alkali concentration reached 7mM in the most significant difference.The activity of AtSRT2 group was 1.23,1.32 and 1.37times in WT group,pGWB2 group and atsrt2 group,respectively.Through the above study,we have a better understanding of the function of AtSRT2 gene and the relationship with abiotic stress,Which provides some basis for further study of the mechanism of action of the gene and how to participate in the response to abiotic stress,which is of great significance to improve plant tolerance under abiotic stress.