Functional Analysis Of Ascorbic Acid Metabolism Gene BcMDHAR2 In Non-heading Chinese Cabbage

Ascorbic acid(AsA)is one of the essential nutrients to maintain the life of animals and plants.Ascorbic acid cannot be synthesized in human body.Therefore,the main source of ascorbic acid for human is from plants.Ascorbic acid can not only maintain the life of animals and plants,but also is an essential substance in the metabolic process.Increasing the content of AsA in plants is beneficial to enhance the ability of resisting external stress and affect their reproductive growth.In addition to the effect of synthesis ability on the accumulation of AsA,cyclic metabolism also has an important effect on the content of AsA in plants.The redox state transition of AsA is realized by AsA-GSH cycle.The expression of related genes in this cycle is closely related to AsA metabolism and accumulation.Therefore,the study of AsA-GSH cycle is helpful to understand the relationship between gene expression and AsA content under the changes of external conditions.It is of great significance to the breeding of non-heading Chinese cabbage[Brassica campestris(syn.Brassica rapa)ssp.chinensis] with high AsA content.The main results are as following:1.The purpose of this study was to verify the function of MDHAR2 in multigene family of MDHAR and to explore its function.The c DNA of BcMDHAR2 was amplified from non-heading Chinese cabbage ‘Suzhouqing’.The bioinformatics of amino acid sequence was analyzed.Subcellular localization was analyzed by transient expression in tobacco,and RT-q PCR was used to detect the expression of the gene under different treatments.The results showed that the gene contained an open reading frame(ORF)of 1308 bp,encoding 435 amino acids.The phylogenetic tree analysis showed that BcMDHAR2 had the closest evolutionary relationship with Brassica rapa.BcMDHAR2 was located in the nucleus,cytoplasm and membrane.RT-q PCR showed that the expression of BcMDHAR2 was up-regulated and then down regulated under low temperature,salt,heavy metal ions,Exogenous hormones.The gene had a certain response to photoperiod,and the expression level increased with the light time.The expression decreased after dark.In conclusion,the expression of BcMDHAR2 changes with the changes of external conditions.The gene can be induced by different hormones,and respond to different stresses.2.In order to further study the function of BcMDHAR2 and its effect on AsA in non-heading Chinese cabbage,the experimental material were transgenic Arabidopsis overexpressing BcMDHAR2 and ’Suzhouqing’ with virus induced gene silencing(VIGS).The differences of phenotype and AsA content between transgenic lines and wild type were observed and analyzed.The expression levels of related genes of AsAGSH cycle in VIGS silenced plants were analyzed.The results showed that the overexpression of BcMDHAR2 resulted in the increase of the number of fibrous roots at A.thaliana seedling stage.Compared with wild type,root length increased by twice.The flowering time of bolting was 1 week ahead of that of wild type,and the redox ratio of AsA increased,which was 1.6～2.2 times of wild type.In ’Suzhouqing’,the content of AsA decreased significantly,which was 0.7 times of the control,and the expression of related genes in AsA cycle changed.In conclusion,BcMDHAR2 is up-regulating the content of AsA,and increasing gene expression affects plant growth.3.In order to explore the mechanism of regulating the expression of ascorbic acid related gene BcMDHAR2 from the level of gene transcription.In the experiment,the cis-acting elements in BcMDHAR2 promoter sequence were predicted and analyzed by using Plant CARE software.The regulatory proteins binding to the promoter were obtained by screening yeast one-hybrid library(Y1H),and the functions of the regulatory proteins were analyzed by sequencing.The prediction of cis-acting elements showed that the promoter region contained typical TATA-box sequences and CAATbox transcription enhancer sequences.In addition,there were many elements related to hormone,stress resistance and light induction.Besides,the results showed that the functions of these candidate transcription factors were related to hormone regulation,root development,antioxidant response and photoperiod,which were consistent with the functions of predictive elements.In conclusion,the promoter binding proteins of the gene are mainly related to stress resistance,hormone and light regulation,which also provides a basis for the study of the function and transcriptional regulation mechanism of the gene.