Cloning And Function Analysis Of ABC Transporter Gene From Tartary Buckwheat

The ATP-binding cassette(ABC)transporter belongs to the membrane integrin superfamily and is distributed throughout prokaryotes and eukaryotes.The energy transport substrate molecules obtained by hydrolysis of ATP enter and leave the cell to function.The multidrug resistance(MDR)in animals is the type of ABC transporter that was first widely recognized by scholars.Studies confirm that in plants,exogenous substances,plant hormones,metal ions,lipids,and secondary metabolites are the major transport substrates for ABC transporters.In this study,we first analyzed the physiological and morphological characteristics of Tartary Buckwheat leaves in the seed germination test and pot experiment under lead treatment,and based on the transcriptome data of the leaves of Tartary Buckwheat under the lead treatment environment,the real-time quantitative verification of differences in lead treatment under heavy metal conditions.The expression characteristics of ABC transporter genes(FtABCB1,FtABCC9,FtABCG11,and FtABCG15)were expressed,and the open reading frames of the differentially expressed ABC transporter genes of Tartary Buckwheat were cloned and expressed by RT-PCR,and the biological sequences of the genes were analyzed;Yeast expression vectors were used to validate the function of each ABC transporter in the heavy metal-sensitive Saccharomyces cerevisiae mutant strain ycf1;a plant expression vector based on pCAMBIA3301 was constructed and overexpressed in Arabidopsis thaliana to further elucidate the ABC transporter in tartary buckwheat plants.The mechanism of enrichment and detoxification provides the basis.The main conclusions of this study are:(1)In the concentration range of 0mg/L-1600mg/L lead nitrate,lead treatment has no significant effect on the germination rate of Tartary Buckwheat seeds;in the range of lead nitrate concentration of 0g/kg-15g/kg,lead treatment on plants The growth and development had no significant effect,but it had an inhibitory effect on the basal pod elongation and plant height at the germination stage of tartary buckwheat;the lead treatment resulted in the decrease of free proline content in the leaves of tartary buckwheat,induction of malondialdehyde content and soluble protein.Content and soluble sugar content(except for reducing sugar)increased significantly.With the increase of lead concentration,SOD and CAT activity increased first and then decreased.These indicators showed that biofilm systemsof Tartary Buckwheat were damaged under certain concentration of lead treatment.However,the good physiological basis of buckwheat makes it possible to cope with stress at low lead concentrations.The bitter buckwheat has a large biomass,a short growth cycle,and good lead resistance,making it a promising bioremediation plant.(2)Analysis of transcriptome data showed that Ft ABCB1,FtABCC9,FtABCG11,and FtABCG15 were upregulated under heavy metal stress conditions;Real-time quantitative analysis showed that Ft ABCC9,FtABCG11,and FtABCG15 all expressed highest in leaves,which were 4.9,23.6,and 3.5 times higher than roots,respectively.The expression of FtABCB1 was similar in roots,stems and leaves.Quantitative analysis under lead treatment showed that four genes were up-regulated in roots,stems and leaves,of which FtABCC9 and FtABCG11 were up-regulated in treatment roots containing 10g/kg lead nitrate and 15g/kg earth,respectively 7.7 and 3.9 times the control.In the 10g/kg lead nitrate treated leaves,FtABCG15 and FtABCB1 were up-regulated,which were 6-fold and 3.7-fold higher than the control,respectively,indicating that the expression characteristics of different ABC genes and the response to lead stress were different.(3)The coding sequences of F1ABCB1,FtABCC9,FtABCG11,and FtABCG15,the ABC transporters of Tartary Buckwheat,are 4047 bp,4476bp,2121 bp,and 2061 bp,encoding proteins of 147.3 kDa,166.6 kDa,78 kDa,and 76 kDa,respectively.Amino acid analysis revealed that it contains the ABC transporter typical WalkerA motif,WalkerB motif,and ABC signature motif.Transmembrane structure analysis showed that Ft ABCB1 and FtABCC9 are positive all-molecule ABC transporters,and FtABCG11 and FtABCG15 are reverse half-molecule ABC transporters.(4)The function complementation of heavy metal-sensitive Saccharomyces cerevisiae ycf1 showed that under the same conditions,the growth rate of the yeasts transformed with pYES2-FtABCB1,pYES2-FtABCG11 and pYES2-FtABCG15 in the experimental group was significantly Slower than that of the control group yeast.It is speculated that the ABC transporter is cytotoxic to yeast and inhibits its growth,and thus the expression system may not be suitable for verifying the function of the ABC gene.(5)The plant expression vector fused with GFP was constructed and subcellular localization experiments of onion epidermis showed that Ft ABCB1,FtABCC9,FtABCG11 and FtABCG15 may all be located in the cytoplasmic membrane.Arabidopsis thaliana plants with T0 generation of transgenic FtABCG11 and FtABCB1 and T2 generation of transgenic FtABCG15 were obtained,respectively,which laid the foundation for further study on the heavy metal resistance function of ABC transporter gene in Tartary Buckwheat.