Cloning And Screening Of ThMYBs Transcript Factor With Stress Response From Tamarix Hispida

Transcription is the first step of the gene expression.And there were many transcription factors(transcription factor,TF)which involved in regulation of transcription.Therefore,cloning and function analysis of transcription factors were very important to understand the gene expression regulation mechanism.MYB transcription factor was one of the largest families of transcription factors in plants,which was widely involved in plant growth and metabolic regulation.In order to clon and screen of resistant excellent MYB genes from Tamarix hispida,a kind of saline woody plant,14 ThMYBs genes were cloned in this study.And the gene expression patterns were analyzed in T.hispida after different abiotic stresses.Moreover,one of the highly induced expression genes was selected to verify the stress resistance function.In the previous study,seven transcriptomes from T hispida were builted,and the unigenese were functional annotation.The keywords "MYB transcription factor" were used as a query to search against the functional annotation of non-redundant unigenes for identifying the MYB genes.A total of 57 unigenes were identified as ThMYBs family genes.And 14 ThMYBs genes were with single clone and full open reading frames.These ThMYBs encode proteins ranging from 284 to 554 amino acids(aa)in length,with predicted sizes from 30.6 to 61.8 kDa and pI values from 5.05 to 9.66.14 ThMYBs class transcription factor mainly divided into two categories through Multiple sequence alignment,Among them,ThMYB2,ThMYB3,ThMYB4,ThMYB8,ThMYB9 and ThMYB13 were closely related,which were typical R2R3-MYB transcription factors.The relative expression patterns of 14 ThMYBs genes were analyzed in roots and leaves of T.hispida under different abiotic stress(high salt,drought)and hormone treatments(ABA,GA3 and JA).The results showed that these genes expressions were changed after stress,suggesting they were the stress response genes,and at least participate in a single stress response of T.hispida.In the roots and leaves,ThMYBl,ThMYB3,ThMYB13 and ThMYB14 were mostly highly up-regulated expression in various stress conditions,which indicated they might play an important role in the abiotic responses of T hispida.Furthermore,the ThMYB13 gene was selected for study whether involved in the resistance stress of T.hispida.The recombinant vector pROKII-ThMYB13 and pFGC5941-ThMYB13 were constructed.And they were transient transformation in T.hispida.At the same time,the pROKII empty vector was also transfered into T.hispida as the control(lab as CON).Expressions of ThMYB13 gene from three kinds of T.hispida were investiged by real-time RT-PCR.The results showed that the expression of ThMYB13 was the highest in overexpression line(OE)and the lowest in the knock-down of ThMYB13 plants(RNAi).Further physiological indexes and physiological staining were measured and compared among the three T.hispida under 150 mM NaCl and 200 mM mannitol stress.The protection enzymes including SOD and POD activities were significantly higher in the OE lines than RNAi and control plants.DAB and NBT Dyeing results showed that the content of O2-.and H2O2 in OE plants was significantly lower than RNAi and control plants.Similarity,the results of Evans blue staining and MDA content showed that the degree of cell damage was lighter and the cell death rate was less in overexpression of ThMYB13 plants than those in CON and RNAi.These results indicated that the overexpression ThMYB13 improved the resistance of salt and drought stress by increasing the activity of protective enzymes and enhancing intracellular scavenging of reactive oxygen species,reducing the accumulation of O2-.and H2O2,thereby,reduced cell damage or cell death and enhanced plant resistance.ThMYB13 was a candidate gene of stress.In the future,its function and the regulation mechanism need to be further studied.