Genome-wide Analysis Of The Ascorbate Peroxidase Gene Family In Taraxacum Kok-saghyz And Function Characterization Of TkAPX3

Taraxacum kok-saghyz Rodin is one of the world’s three major rubber-producing crops,the natural rubber produced at the root has the same structure and performance as the rubber produced by Hevea brasiliensis.It has the advantages of short growth cycle,strong growth adaptability,relatively easy genetic transformation,etc.It is one of the most promising potential alternative plants for natural rubber production.The salinization of cultivated land in my country is serious.Saline-alkali land is about one-fifth of the total cultivated land area,and it is mostly concentrated in the northwest,northeast and north China regions.At the same time,these areas are also the main areas where T.kok-saghyz grows,so improving the salt tolerance of T.kok-saghyz is crucial to its growth and yield.T.kok-saghyz is a kind of cool and cold herb crops.Planting in Hainan has been restricted to a certain degree.Increasing its ability to resist high temperatures can break the limits of temperature on its distribution will benefit the research and commercial planting of T.kok-saghyz.Ascorbate peroxidase（APX）is a key enzyme in the Ascorbic acid-Glutathione cycle.It can remove excess H₂O₂ in plants and plays an important role in the process of plants resisting adversity stress.In this study,the bioinformatics method was used to identify and analyze the T.kok-saghyz APX gene family,and the expression of APX gene family members after stress treatment（cold,heat,salt,drought）and hormones（ethylene,jasmonic acid）were analyzed,the TkAPX3 gene was cloned,and the overexpression TkAPX3 vector was transferred into T.kok-saghyz by agrobacterium transformation method,and the function of the gene was preliminary analyzed.The main results are as follows:（1）In this study,7 TkAPX genes were identified in T.kok-saghyz whole genome using bioinformatics methods.Further analysis revealed that 2 genes（Tk APX4/Tk APX6）show replicant event.According to the results of phylogenetic relationships,7 Tk APX genes were divided into four groups.The chromosomal localization analysis indicated that the 7Tk APX genes were widely distributed and located on 7 different chromosomes.The prediction of subcellular localization indicates that Tk APX1,TkAPX3,Tk APX5 and Tk APX7 were localized in the cytoplasm,Tk APX4 and Tk APX6 were localized on the plasma membrane,and Tk APX2 was localized in the chloroplast.The analysis of the cis-acting elements in the promoter region show that there are a large number of stress response elements in Tk APX sequence,suggesting that the APX gene may have a sensitive stress response mechanism to various external stimuli and stresses.（2）Real-time quantitative PCR analysis showed that the expression levels of 7 TkAPX genes family members in calyxes,petals and pedicels were low and most Tk APXs in roots and leaves was greater than that of flowers and latex.Tk APX1 has the highest expression level in roots and stems,suggesting that it may play an important role in the growth and development of T.kok-saghyz.Furthermore,the expression level of Tk APX gene after stress（cold,hot,salt,drought）and hormone（ethylene,methyl jasmonate）treatment,the expression levels of TkAPX3 in roots and leaves increased significantly compared with the control indicated that TkAPX3 may be involved in the stress response and hormone response in T.kok-saghyz.（3）The cytoplasmic ascorbate peroxidase gene TkAPX3 was isolated from T.kok-saghyz for the first time using PCR technology.The open reading frame is 753 bp,which encodes 250 amino acids.The protein molecular weight is 27.52 k D and the isoelectric point is 5.23.The plant overexpression vector p Super1300-TkAPX3 containing Super promoter was constructed.After transforming agrobacterium GV3101,the T.kok-saghyz was infected by the leaf method,and a transgenic TkAPX3 T.kok-saghyz was obtained.（4）Compared with wild-type T.kok-saghyz,transgenic T.kok-saghyz has certain growth advantages under suitable growth conditions.When under the same salt and high temperature stress,over-expressing TkAPX3 gene significantly increased the T.kok-saghyz APX activity and reduced H₂O₂ accumulation.These results indicate that over-expression of TkAPX3 gene improves T.kok-saghyz tolerance to salt and high temperature stress,protect T.kok-saghyz from oxidative damage caused by salt and high temperature stresses.