Analysis Of CdCl₂ Tolerance Function Of ThMYB37 From Tamarix Hispida

MYB transcription factors are widely distributed in eukaryotes and play a key role in cell cycle regulation,anthocyanin accumulation,axillary meristem formation,and adaptation to adverse environments such as low temperature and high salt.In the previous study,a cadmium stress response gene ThMYB37 was cloned from the transcriptome of Tamarix hispida under cadmium stress.In this study,the Cd tolerance function of ThMYB37 gene was further researched,and its Cd tolerance regulation mechanism was preliminarily explored.Following were the specific research results:The cDNA of ThMYB37 was 1191bp and encoded 396aa.Phylogenetic analysis showed that the ThMYB37 protein was belonged to R2R3MYB family.It was closely related to Arabidopsis MYB37 protein,so it was named ThMYB37.The subcellular localization showed that ThMYB37 protein was expressed in the nucleus.The ThMYB37 transcription factor had transcriptional activation activity and the activation domain was located in the C-terminal 197-397aa of the protein.Under CdCl₂ stress,ThMYB37 gene was significantly induced in the roots of T.hispida,but significantly down-regulated in the leaves.In order to analyze the Cd tolerance of ThMYB37 gene,over-expression Arabidopsis with ThMYB37 was obtained by flower immersion infection.And two strains（OE1,OE2）were selected by screening.The growth and physiological indexes of two transgenic lines and WT before and after 150 μM CdCl₂ stress were analyzed and compared.The results showed that the growth status of OE1 and OE2 after Cd stress was better than the control.The staining（DAB,NBT,Evans blue）of overexpressing ThMYB37 gene Arabidopsis was lighter,the contents of malondialdehyde（MDA）,hydrogen peroxide（H2O2）and electrical conductivity of OE strain were significant lower than the over-expressed strain,and the activity of superoxide dismutase（SOD）was obviously higher than the WT.The results indicated that the over-expression plants of ThMYB37 gene could improve the tolerance of Cd stress,because the over-expression plants had higher SOD activity,lower O2-and H2O2,lower membrane lipid degree,better complete cell membrane structure.To further verify the results of heterologous Arabidopsis,a pFGC5941-ThMYB37（1E）inhibitory expression vector was constructed.The pFGC5941-ThMYB37（IE）inhibitory expression vector,overexpression vector pROKII-ThMYB37（OE）and empty pROKII vector（CON）were transferred into T.hispida by transient transformation method.The results showed that under Cd stress,the membrane lipid degree of OE plant cells was lower,and SOD activity was the highest.However,ThMYB37 inhibited expression of plants had the highest H2O2 and MDA content,the highest electrolyte leakage rate and the most serious cell membrane damage.The results indicated that ThMYB37 gene could change the physiological metabolic process of transgenic T.hispida and enhance cadmium tolerance of transient overexpression T.hispida.Furthermore,TF-Centered Y1H technology（yeast one-hybrid technology centered on transcription factors）was used to screen and identify the ThMYB37 transcription factors combined cis-acting elements.Random sequencing results showed that 13 elements were screened out,including 4 known elements and 9 unknown elements.The unknown element"GAGCTTCCAT" had the highest frequency,and "CTTCCA" was identified as its core sequence by deletion and mutation analysis.By searching the promoter elements of Cd stress response gene of T.hispida,it is found that the promoter regions of ThSOD2,ThTPS4,ThSOS1 and ThMIP2 genes contained "CTTCCA".It is speculated that ThMYB37 transcription factor may combine with "CTTCCA" to regulate the expression of ThSOD2,ThTPS4,ThSOS1 and ThMIP2.In the future research,the downstream target genes directly regulated by ThMYB37 transcription factor will be searched and identified.