Cloning Of TtMYB1 Gene In Response To Salt Stress In Tritipyrum And Salt-tolerant Identification Of Overexpression Plants

Wheat is a food crop widely planted all over the world.Soil salinization is one of serious abiotic stresses that threatens the yield.MYBs play important roles in plant response to salt stress.“Y1805”is a salt-tolerant Tritipyrum,which originatedfrom the cross between Triticum aestivum and Thinopyrum elongatum.At present,there are few reports on MYB genes in Tritipyrum.In previous study of our laboratory,we have successfully screened out an“Y1805”-special MYB gene under salt stress.In this experiment,an MYB gene related to salt stress response was cloned from Tritipyrum“Y1805”,and its sequence characteristics,expression pattern under salt stress,subcellular localization and interacting proteins were analyzed.Then,the gene was transformed into common wheat for preliminary function analysis.This experiment might be helpful to understand molecular characteristics and gene function of the MYB gene,which would provide the basis for molecular breeding and germplasm innovation in wheat.The main results were as follows.（1）Using homologous cloning technique,TtMYB1 was successfully cloned from Tritipyrum“Y1805”.TtMYB1 contained a 783 bp open reading frame encoding 259amino acids.TtMYB1 has typical MYB conserved domain.Phylogenetic tree analysis based on amino acid sequences of different species showed that TtMYB1 and Th.elongatum were clustered into the same branch,with the highest homology and the closest genetic relationship.（2）qRT-PCR was used to analyze the expression patterns of TtMYB1 gene under salt stress.The results showed that salt stress could induce the up-regulated expression of TtMYB1 gene.At 5 h（T1 stage）of salt stress,the expression level in roots was significantly higher than that in stems and leaves,which were 1.67-fold and 13.06-fold of that in stems and leaves,respectively.At 1 h（R stage）of recovery,the expression level of TtMYB1 gene in roots decreased to the control level.（3）By constructing the instantaneous expression vector of 1300-TtMYB1-GFP fusion gene,the fusion cells of the target gene and green fluorescent protein were instantly transformed into mesophyll protoplasts by mediation of polyethylene glycol.After co-culture,the subcellular localization of TtMYB1 protein was observed,and the results showed that TtMYB1 was located in the cell nucleus（4）Yeast hybrid library of Tritipyrum“Y1805”was constructed to screen TtMYB1 interacting proteins.Fifty-one proteins interacting with TtMYB1 were confirmed by yeast two-hybrid analysis.Through BLAST comparison,GO annotation and protein-protein interaction analysis,protein-protein interaction happened among 24proteins,most of which were ribosomal proteins.（5）TtMYB1 gene was also transformed into wheat coleoptiles by Agrobacterium tumefaciens.The transgenic lines were successfully identified by green fluorescent protein screening and PCR detection.Salt tolerance of T₃transgenic lines was identified.Salt stress was applied to overexpression wheat and it was found that under normal growth condition,the seedling height of TtMYB1 overexpression wheat lines was significantly higher than that of wild-type（WT）plants.At the T1 stage of salt stress,the degree of leaf wilting in the WT plants was significantly higher than that in the overexpression wheat.In addition,overexpression wheat exhibited stronger root systems than the WT plants.At 24 h（T2 stage）of salt stress and R stage,the root length and seedling height of overexpression lines were significantly higher than those of the WT plants.Therefore,salt treatment had a greater inhibitory effect on the growth of the WT plants than overexpression lines,and a greater inhibitory effect on the root system than on the top.Under salt stress,proline（Pro）content of overexpression and the WT plants significantly increased.With the prolongation of salt stress time,the Pro content in overexpression wheat lines rapidly increased,reached the peak at the T2 stage,and its Pro content was 1.41-fold that of the WT plants.After recovery,the Pro content of overexpression and the WT plants could quickly decrease to the control level in a short time.After salt stress,the soluble sugar content of both overexpression lines and the WT plants significantly increased,reaching the highest at the T2 stage.The soluble sugar content of overexpression lines was 1.26-fold that of the WT plants.After recovery,the soluble sugar content of the overexpression lines decreased faster than that of the WT plants.Compared with the WT plants,the MDA content of overexpression lines significantly decreased under salt stress.At the T2 stage,it reached the peak,with the WT plants being 1.54-fold higher than the overexpression strain.The change in MDA content of overexpression lines was small with the prolongation of salt stress time,which might be because the TtMYB1 gene enhanced the salt tolerance of overexpression lines.Overexpression lines showed stronger salt tolerance than the WT plants in growth and physiological indexes.In conclusion,a new TtMYB1 gene was cloned from salt-tolerant Tritipyrum“Y1805”.Up-regulated expression of TtMYB1 was induced by salt stress.TtMYB1 was located in the cell nucleus.Fifty-one interacting proteins were obtained by yeast two-hybrid analysis.TtMYB1 conferred enhanced salt tolerance in transgenic wheat.This experiment laid an important foundation for understanding salt-tolerant mechanism of TtMYB1 gene and genetics and breeding in crops.