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

Transcription factor(TF)is a type of DNA binding protein that can specifically bind to cis-acting elements on gene promoters.It can regulate a series of downstream genes expression by binding to cis-acting elements on the promoters.So it is extremely important that transcription factors with excellent salt tolerance were screened from stress-resistant plants in the process of genetic engineering breeding.In order to clone and screen CONSTANS-Like genes with excellent salt tolerance from the woody halophyte Tamarix hispida,8 ThCOLs genes were identified in this study.And the expression patterns of these genes under various stress conditions were further analyzed.the genes with highly induced under salt stress were selected to verify their salt tolerance.The specific results were as follows:By searching the 7 T.hispida transcriptome sequences,total of 8 ThCOLs genes(named ThCOLl-8)with complete open reading frames(ORFs)were identidifed.The coding regions lengths of 8 ThCOLs gene were 1089-1467 bp,with the numbers of coding amino acid residues 362-488.The predicted relative molecular weight was 39.6-53.6 kDa,and the theoretical isoelectric point(pI)were 4.83-7.48.Multiple sequence alignment results showed that the 8 ThCOLs transcription factors were mainly divided into three groups,of which ThCOL2,ThCOL5 and ThCOL8 were closely related,which were belonged to the first group,and contained two complete B-BOX domains at the N-terminal.ThCOL3 belonged to the second group,which was a transcription factor with a complete B-BOX domain at the N terminal.ThCOL1,ThCOL4,ThCOL6,and ThCOL7 belonged to the third group,and were transcription factors that contained a complete B-BOX domain and a B-BOX domain with secondary structure changes at the N-terminal.The expression patterns of 8 ThCOLs gene in the roots and leaves of T.hispida treated with abiotic stress(high salt,drought,heavy metal)and ABA treatment showed that the expression levels of 8 ThCOLs gene changed significantly after stress,which indicated that these genes were all stress responsive genes,and may be involved in at least one kind of stress resistant response of T.hispida.Especially,the expression level of ThCOL2 changed significantly at most time points under salt stress in the roots and leaves of T hispida,which may play an important role in the response process of salt stress.In order to further verify whether the ThCOL2 gene was involved in the regulation of salt tolerance response of T.hispida,the recombinant vectors pROK?-ThCOL2 and pFGC5941-ThCOL2 were constructed to carry out the transient transformation of T hispida,and the transient infection of T.hispida by pROKII empty vector was used as the control.The results showed that the transient overexpression(OE)and inhibition expression(IE)transgenic lines were successfully obtained.The results of physiological staining and physiological indexes showed that the overexpression of ThCOL2 gene under 150 mM NaCl stress could enhance the ability to remove reactive oxygen species in transgenic T hispida cells by regulating the activity of protective enzymes,and promote the accumulation of O2-and H2O2 to decrease,thereby reduced cell damage or cell death and enhancing the salt tolerance.It was suggested that ThCOL2 gene may be a candidate gene with excellent salt tolerance.In order to study the salt-tolerant regulation mechanism of ThCOL2 gene in more detail,the subcellular localization and transcription activation activity of ThCOL2 gene were studied.The results showed that ThCOL2 was located in the nucleus and has transcription activation activity.And the transcription activation domain was located at 139-346 aa(Middle region without conserved domain).Furthermore,the TF-Centered Y1H technology(a yeast one-hybrid technology centered on transcription factors)was used to analyze the cis-acting elements recognized by ThCOL2 transcription factors.The results showed that unknown element"ATTGTGAAA" appears most often.Deletion and mutation analysis revealed that"GTGAAA" was a core functional element recognized by the ThCOL2 transcription factor,suggesting that the ThCOL2 gene may regulate downstream genes expression by binding to it.In the future,we will search and identify the downstream genes directly regulated by ThCOL2 transcription factor to analyze the salt tolerance regulatory network of ThCOL2.