Cloning,Expression Analysis And Functional Characterization Of Candidate Oxalate Transporter Genes HbOT1 And HbOT2 From Hevea Brasiliensis

Aluminum toxicity is one of the main limiting factors for crop growth and yield in acidic soil,which mainly inhibits plant growth by endangering plant roots.With the rapid development of industry and the extensive application of chemical fertilizers,the acidity of soil in southern of China and Southeast Asia has decreased significantly,the harm of aluminum toxicity in tropical soil has increased,and the hidden danger of aluminum toxicity in rubber tree and natural rubber industry has become increasingly apparent.In our previous study,it was found that the aluminum tolerance range of rubber tree was 100～200mmol/L,which was highly tolerant to aluminium.Further,the roots of rubber seedlings participated in the detoxification of aluminum by secreting a large number of organic acids under aluminum stress,among which the secretion of oxalic acid was significantly higher than that of other organic acids.The exogenous application of organic acids proved that oxalic acid played a major role in the aluminum detoxification of rubber trees.However,there are few reports in the identification of related oxalate transporters in plants.In this study,two oxalate transporter candidate genes were cloned from rubber tree,named Hb OT1 and Hb OT2,and a homologous gene was cloned from Arabidopsis,named At OT.Through gene cloning,bioinformatics prediction analysis,multiple sequence alignment,evolution and expression analysis,subcellular localization,yeast AD12345678（AD1-8）system,Xenopus oocyte electrophysiological system and Arabidopsis system functional characterization,it was proved that Hb OT1,Hb OT2 and At OT had the function of regulating the transport and efflux of oxalic acid,and played an important role in the mechanism of plant aluminum detoxification.The main results are as follows:1.Using the previously identified Fp OAR protein（Gene Bank:BAJ10704.1）in Fomitopsis palustris as the query sequence,two unidentified rubber tree proteins,XP＿021645179.1 and XP＿021655511.1,and a unidentified Arabidopsis homologous protein,NP＿192696.3,were screened by BLASTP alignment,and their corresponding m RNAs Hb OT1（XM＿021789487.1）,Hb OT2（XM＿021799819.1）and At OT（NM＿117026.5）were obtained.Hb OT1,Hb OT2 and At OT were SNARE＿assoc subfamily proteins of SNARE superfamily which located in the plasma membrane,and were hydrophobic membrane proteins with the basic characteristics of transporters.2.The Hb OT1,Hb OT2 and At OT were expressed differently among different tissues of rubber tree and Arabidopsis by q RT-PCR,with the transcript level of Hb OT1 was the highest in roots,Hb OT2 was the highest in latex and At OT was the highest in roots.Moreover,Hb OT1 and Hb OT2 responded to various metal ion stresses in roots of rubber tree.Hb OT1 mainly responded to Al³⁺and Cu²⁺and Hb OT2 mainly responded to Al³⁺,Cd²⁺and Mn²⁺.In response to aluminum stress,the expression level of Hb OT1 increased with the concentration of Al³⁺,Hb OT2 reached the highest under 50 mmol/L Al³⁺,and At OT reached the highest under 100μmol/L Al³⁺.On the other hand,Hb OT1 and Hb OT2were mainly up-regulated in the short term（≤24 hr）of aluminum stress,while At OT expression increased with the treatment time of aluminum.The above results indicate that Hb OT1,Hb OT2 and At OT genes have the expression characteristics of aluminum detoxify genes.3.Yeast mutant AD1-8 expressing Hb OT1,Hb OT2 and At OT showed stronger oxalic acid resistance and aluminum tolerance.They obtained the ability to regulate oxalate efflux by consuming ATP.Furthermore,the membrane vesicles of Hb OT1-,Hb OT2-and At OT-transformed yeasts were observed to absorb oxalic acid in vitro.4.The channel activity of Hb OT1,Hb OT2 and At OT could not be detected in X.oocytes under normal conditions or aluminum stress whether ATP added or not,indicating that their regulation of oxalate transmembrane transport may not belong to ion channel transport.5.By comparing the root length and oxidative degree of Arabidopsis atot mutant and wild type Col-0 under aluminum stress,the positive regulation of At OT in Arabidopsis root growth and aluminum detoxification mechanism was proved.In conclusion,two oxalic acid transporter candidate genes（Hb OT1,Hb OT2）from rubber tree and one oxalic acid transporter candidate gene（At OT）from Arabidopsis were cloned in this study.The oxalate transport and aluminum detoxify functions of them were preliminary identified by yeast AD1-8 system and Arabidopsis system.It is speculated that they realize the directional transport of oxalic acid by inducing membrane vesicles to absorb oxalic acid and vesicle transport,and realize the chelation of internal oxalic acid to external exchangeable aluminum by regulating the transport of carrier protein that needs to consume ATP.