Study On The Functions Of Two Stress-related HD-ZIP Transcription Factors In Medicago Truncatula

Homeodomain-leucine zipper(HD-ZIP)is a unique transcription factor family in plants,which plays an important role in abiotic stress and morphogenesis.Among them,HD-ZIP I subfamily is mainly related to abiotic stress resistance.Alfalfa is an important forage,which has the advantages of high yield,high nutritional value and strong environmental adaptability.As a model plant of alfalfa,Medicago truncatula is the model plant because it is diploid and has short growth cycle and high seed yield.At present,there are few literatures about HD-ZIP gene in Medicago truncatula.In this paper,the Mt HDZ gene in Medicago truncatula was analyzed by bioinformatics,the gene chip data in the database were used to analyze its expression patterns in different tissues and under drought and salt stress.The functions of Mt HDZ35 and Mt HDZ50 genes,which is belonging to Mt HDZ I subfamily,in response to abiotic stress were studied.Firstly,52 HD-ZIP(Mt HDZ1-Mt HDZ52)genes were identified in Medicago truncatula by bioinformatics.Then,the Mt HDZ protein was divided into four subfamilies named I-IV,by constructing a Neighbor-Joining phylogenetic tree.The analysis of Mt HDZ gene structure and protein conserved motif showed that the structure of subfamily I and II was relatively simple,meanwhile the structure of subfamily III and IV was complex.The gene structure and conserved motif among subfamilies were highly consistent.Through the annotation information of Mt HDZ gene in Phytozome,51 Mt HDZ genes were located on 8chromosomes,and 18 genes(9 pairs of homologous genes)were located on the large segment of chromosome duplication,which indicated that the amplification of HD-ZIP family in alfalfa was mainly related to chromosome duplication.The ratio of synonymous substitution rate to non synonymous substitution rate(Ka/Ks)was 0.096-0.502,indicating that purification selection played a major role in the evolution of HD-ZIP gene family of Medicago truncatula,and the differentiation time of homologous genes was about47.53-90.98 million years ago.Afterwards,the cis-elements in Mt HDZ I and II subfamily promoters were analyzed with PLACE online tool.It was found that most of the cis-elements were related to light,I subfamily identified ci-elements related to drought stress,and II subfamily identified more cis-elements related to polar temperature.Based on the gene chip data of Mt GEA,the expression patterns of HD-ZIP gene in tissues and organs at different development stages and seedlings under drought or salt stress were analyzed.The results showed that most of the Mt HDZ genes showed relatively wideexpression patterns in different tissues,and some of them had tissue-specific expression patterns.After salt stress treatment in different growth stages,20 Mt HDZ genes showed significantly increased or inhibited expression patterns,and 14 Mt HDZ genes showed different increased or decreased expression patterns in the aboveground and underground parts under drought stress.In addition,the expression patterns of many homologous genes in different tissues and under drought and salinity were different,indicating that their functions may have been differentiated.In order to verify the reliability of microarray results,9 Mt HDZ I family genes were selected and their expression patterns were studied by q RT-PCR.The results showed that the expression of 9 Mt HDZ I family genes under salt stress was basically consistent with the results of expression profile analysis.In addition,we studied the function of Mt HDZ35 and Mt HDZ50 in abiotic stress response in Arabidopsis.The results of yeast one hybridization showed that both Mt HDZ35 and Mt HDZ50 had the ability of transcriptional activation,and their transcriptional activation domains were all located at the C-terminal of polypeptide chain.The expression of Mt HDZ35 and Mt HDZ50 genes under drought stress and ABA treatment was analyzed by q RT-PCR.The results showed that the expression of Mt HDZ35 and Mt HDZ50 increased significantly under drought stress,whereas the expression of Mt HDZ35 decreased significantly under ABA treatment,and the expression of Mt HDZ50 increased significantly.The overexpression vector of the two genes was constructed and transformed into Arabidopsis by Agrobacterium mediated transformation.The germination rate and root elongation of Mt HDZ50 overexpression lines under ABA treatment,salt stress and simulated drought stress were analyzed.The results showed that the sensitivity of Mt HDZ50 overexpression lines to exogenous ABA response decreased during seed germination,and the tolerance to simulated drought and Na Cl salt stress increased significantly.This study deepened our understanding of the role of HD-ZIP in resistance to abiotic stress,identified a number of downstream genes in response to drought and salt alkali stress,which is helpful to reveal the function and molecular mechanism of HD-ZIP gene in response to abiotic stress further,and ultimately improve the resistance of Alfalfa through genetic engineering.